2° simpósio araucária em biologia celular e molecular · 2° simpósio araucária em biologia...

SUMÁRIO

Cytotoxic effect of a pectin isolated from gabiroba (Campomanesia xanthocarpa Berg) fruit

on T98G glioma cell line ........................................................................................................ 1

Development of superficially modified magnetic nanoparticles with carbohydrates for the

cellular culture of microorganisms ......................................................................................... 3

Evaluation of the cytotoxic and anti-metastatic in vitro effects of Lafoensia pacari in

colorectal cancer cells ........................................................................................................... 5

Quantification of cancer stem cells in melanospheres ........................................................... 6

Evaluation of TCTP and its inhibition in neuroblastoma ......................................................... 7

From human embryonic stem cells to cardiomyocytes: expression profile analysis of

metabolism-related genes ..................................................................................................... 8

Characterization of phenotypic aspects of human glioblastoma cells cultivated in hanging

drops submitted to the combined treatment of Temozolomide and Sinvastatin ................... 10

Correction of the F508del mutation in lung epithelial cells with cystic fibrosis ...................... 12

Circulating microRNAs in cervical carcinogenesis: promising diagnostic biomarkers from

squamous intraepithelial lesions to cervical cancer ............................................................. 14

Analysis of the interaction between stem cells and 3D printed scaffolds for tissue

regeneration ........................................................................................................................ 16

Evaluation of the proliferation of cell lines established in culture treated with polinsaturated

fatty acids ............................................................................................................................ 17

Plasmodium vivax AMA1: Implications of distinct haplotypes to immune response ............. 18

Ribosome-associated lncRNAs in adipose-derived Stem Cells: From RNAs to micropeptides

........................................................................................................................................... 20

Quiescin sulfhydryl oxidase 1 expression in colorectal cancer ............................................ 22

In vitro evaluation of the potential antitumor activity of partially hydrolyzed guar gum and its

chemically sulfated derivative on melanoma cells ............................................................... 24

Investigation of cell death pathways in pemphigus foliaceus ............................................... 26

FOXP3 genetic variants rs3761548 and rs2232365 do not change interleukin-10 plasmatic

and cervical levels in HPV-infection .................................................................................... 27

In vitro evaluation of glucose influence in Plasmodium falciparum growth ........................... 29

Peroxidase Activity of Azolla sp. and Lemna aequinoctialis in effluent contaminated with

nitroaromatic compounds .................................................................................................... 30

Evaluation of the fish oil effect on parameters of obesity and activity of Wistar rats’s

macrophages submitted to metabolic imprinting by small litter ............................................ 32

Morphometric effects of ladder climbing resistance exercise on extensor digitorum longus

muscle of Wistar rats in rheumatoid arthritis model ............................................................. 34

In vivo antitumor activity of low molecular weight heparin in murine melanoma model ........ 36

Determination of gum arabic-gold nanoparticle biological effects in B16-F10 3D model ...... 37

Comparison of biochemical and biological properties of recombinant phospholipases D with

site-directed mutations from venom of spiders Loxosceles gaucho and Loxosceles laeta ... 38

Analysis of histone post-translational modification in Trypanosoma cruzi under nutritional

stress .................................................................................................................................. 40

Evaluation of the internalization pathway and intracellular traffic of the disintegrin and

metalloprotease ADAM23 ................................................................................................... 41

Functional analysis of divergent components for the mRNA export in trypanosomatids ...... 43

Didactic models in the teaching of the cellular morphology of Homo sapiens ...................... 44

Comparative characterization of histone post-translational modifications of the tritryps ....... 45

Development of antibody fragments for selectivity assessment of EGF domain proteins .... 47

Solubilization strategy of protein CD28 ............................................................................... 49

TgHDAC4: a histone deacetylase unique to Apicomplexa ................................................... 51

Development of a model of reconstructed human epidermis (RhE) for in vitro skin irritation

testing ................................................................................................................................. 52

Molecular evolution strategy for optimization of single-chain variable fragments (scFv)

binding to human osteopontin ............................................................................................. 53

Fasting C-peptide / Fasting blood glucose ratio: An auxiliary tool to better understand type 1

diabetes? ............................................................................................................................ 54

Investigation of periphytic community in a water supply river in Umuarama-PR .................. 56

The reduction of walker 256 tumor and wistar rats cakexia promoted by Euphorbia tirucalli

latex .................................................................................................................................... 57

Transcriptional changes in human umbilical vein endothelial cells induced by Toxoplasma

gondii .................................................................................................................................. 58

Large and small extracellular vesicles in Giardia intestinalis: Implications for pathogenesis

and drug treatment .............................................................................................................. 60

Molecular cloning and characterization of a serpin from Loxosceles intermedia venom gland

........................................................................................................................................... 62

Functional characterization of CSDC2 during cardiac differentiation of embryonic stem cells

........................................................................................................................................... 63

Biochemical and functional characterization of a recombinant allergen of Loxosceles

intermedia venom ............................................................................................................... 64

Characterization of porcine heart valve grafts obtained by two different processes of

decellularization and in vitro biocompatibility testing with human cells ................................ 66

Preliminary study about the removal of indoxyl-sulfate, p-cresil sulfate and indole acetic acid

by conventional hemodialysis vs. hemodiafiltration ............................................................. 67

Investigation of biological markers in neurodegenerative diseases and aging ..................... 68

Evaluation of biochemical biomarker responses in Danio rerio larvae in a single and mixture

exposure of Di-n-butyl phthalate (DBP) and Di-iso-pentyl phthalate (DiPeP) ....................... 70

Oxidation of apoptosis inducing factor to disulfide-linked conjugates .................................. 71

Production and evaluation of different constructions of antibody fragments against epiregulin

........................................................................................................................................... 72

Distribution of Merlin transposable elements across eukaryotic groups ............................... 74

Evidence of transposable elements expression in Rhinella marina (bufo toad) specimens

submitted to stress conditions ............................................................................................. 76

Cytotoxicity of bismuth nanoparticles (BiNPs) in the murine macrophage line Raw 264.7 ... 78

In vivo evaluation of the Decabromodiphenyl eter (BDE-209) effects in the darbacazine

treatment on the progression of lung metastasis in mice ..................................................... 79

Effect of TCDD and BDE-209 on cytotoxicity and expression of ATP binding cassette:

MDR1, MDR5, MRP1, MRP2, MRP4 in melanoma murino B16F1 cell line ......................... 81

QSOX1 extracellular interaction with plasma membrane proteins of aortic smooth muscle

cells .................................................................................................................................... 83

Use of Alprazolam during pregnancy: impact on the central nervous system of the offspring

........................................................................................................................................... 85

Cellular and molecular characterization of mesenchymal stem cells derived from pluripotent

cells and prospection of applications in regenerative medicine ........................................... 86

Selective cytostatic effect on human melanoma cell line by a fucose-rich sulfated

polysaccharide from marine algae....................................................................................... 87

Commercial pectin alters viability and cell cycle distribution of glioblastoma cells ............... 88

Founder effect of cancer-associated genetic variants within the Mennonite population

reveals a new cell stress-variant of the ROS-activated ZBTB10 gene ................................. 90

Evaluation of food behavior, serum and biometric mother’s parameters of obese rats puppies

to metabolic imprinting by small litter ................................................................................... 91

The resistance of neuroblastoma cells submitted to different conditions of stress in vitro .... 93

Effects of gradual temperature increase on energetic metabolism of Antarctic fish Notothenia

rossii ................................................................................................................................... 94

Detection of cross-reactivity of anti-IgG from Trypanosomatid-infected rats and cattle by

immunofluorescence ........................................................................................................... 96

Rare endoplasmic reticulum stress variants associated with gastrointestinal disorders in the

brazilian mennonite population ............................................................................................ 98

Evaluation of internalization kinetics and stability of the disintegrin and metalloprotease

ADAM23 ............................................................................................................................. 99

Importance of the familiarization process in the application of the maximum loaded carrying

test in rats ......................................................................................................................... 100

Histone Deacetylase directing sign identification to Toxoplasma gondii apicoplast. .......... 101

Nanotoxicity of silver nanoparticles from biological syntesis .............................................. 102

Exploring the role of a long non-coding RNA during the cardiomyogenic differentiation of

hESC ................................................................................................................................ 104

Protective response against toxic effects of Loxosceles spider venoms using recombinant

mutated phospholipases-D as antigens ............................................................................ 106

Anterior tibialis muscle morphometric changes of obese Wistar rats submitted to whole body

vibration ............................................................................................................................ 108

Correlation between reck isoforms and genes expression in glioblastoma: a bioinformatic

tool .................................................................................................................................... 110

Effects of bismuth nanoparticles produced by lasis on mesenchymal stem cells ............... 112

Kappa carrageenan and its chemically oxidized derivatives: synthesis and cytotoxicity

evaluation on melanoma (B16-F10) and fibroblasts (BALB/3T3) cells ............................... 114

Extracellular vesicles modulate aggressiveness phenotype in human glioblastoma cells .. 116

Analysis of the Keap1 / Nrf2 pathway in cells during uremic toxicity .................................. 118

Aging and resistance training affect the MMP-2 activity in femurs of rats .......................... 119

Understanding the diabetic wound healing by extracellular vesicles perspective ............... 120

Characterization of a potential linker histone in Toxoplasma gondii ................................... 122

Functional characterization of Bar-III, a metalloproteinase from Bothrops barnetti venom . 124

Proteomic prospection to identify plasma biomarkers associated with type 1 diabetes

mellitus ............................................................................................................................. 125

Modulatory effect of vitamins A, D and E on the redox and inflammatory profile in blood cells

of patients with breast cancer ............................................................................................ 127

DNA vacines for cancer treatment..................................................................................... 129

Caracterization of the histone deacetilase 2 of Toxoplasma gondii ................................... 131

Multigenerational exposure to manganese alters the antioxidant homeostasis and functional

conditions of the male reproductive system of mice .......................................................... 132

Determination of the effects of the mesoionic compound 4-phenyl-5- [4-nitrocinamoyl] -1,3,4-

thiadiazolyl-2-phenylamine chloride (MI-D) over tumoral cells (HT29) employing

chorioallantoic membrane (CAM) as a in vivo model ......................................................... 132

Evaluation of decavanadate salts (([V10O28]6-); {(NH4)2[Cu(H2O)6]2[V10O28]}2H2O) in the

development process of HET-CAM vessels ...................................................................... 135

Antioxidant Kraft lignins induced DNA oxidation and increased intracellular reactive oxygen

species in biological system (HepG2 cells) ....................................................................... 137

Evaluation of in vitro influence of Nyssomyia neivai saliva on infective potential of

Leishmania braziliensis stains from the state of Paraná. ................................................... 138

Aerobic physical training associated to omega-3 reduce inflammatory cytokines in the

prostate of Wistar rats submitted to high fat diet ................................................................ 139

Identification and characterization of carbazoles derivatives as new inhibitors of ABCG2

transporter ........................................................................................................................ 141

Characterization of the effects of porphyirinic derivatives in ABCG2 transporter ............... 143

Morphological comparison of the skeletal striated muscle of different experimental designs

after application of a histopathological analysis protocol ................................................... 145

Selection of a more resistant and infective population in Trypanosoma cruzi by high

exposition to normal human serum ................................................................................... 146

1 2° Simpósio Araucária em Biologia Celular e Molecular - Livro de resumos

Cytotoxic effect of a pectin isolated from gabiroba (Campomanesia xanthocarpa Berg)

fruit on T98G glioma cell line

AMARAL, S. C. (1); BARBIERI, S. F. (1); MAZEPA, E. (2); SPISILA, L.J. (1); WINNISCHOFER,

S. M. B. (1,2); SILVEIRA, J. L. M. (1)

1. Federal University of Paraná, Biochemistry and Molecular Biology Department, 81.531-980,

Curitiba-PR, Brazil

2. Federal University of Paraná, Molecular and Cell Biology Department, 81.531-980, Curitiba-

PR, Brazil

E-mail: [email protected] or [email protected]

Introduction: Glioma is one of the most frequent and most lethal malignant primary brain tumors,

associated with poor prognosis. In this way, the search for new therapeutic approaches to improve

treatment becomes relevant. Recently, pectins (complex biopolymers mainly composed of

galacturonic acid residues and a variety of neutral monosaccharides, such as rhamnose, galactose, and

arabinose) have been exploited for their anticancer potential, due to their broad spectrum of

therapeutic properties and low toxicity to healthy cells. However, most biological studies have been

used citrus peel and apple pomace as a source commercial pectins. This work investigated the

antitumor potential of a purified pectin from gabiroba (Campomanesia xanthocarpa Berg; Brazilian

Myrtaceae family species).

Methods: Gabiroba purified pectin (GPP) is composed of galacturonic acid (58.8%), arabinose

(28.5%), galactose (11.3%) and rhamnose (1.1%), comprising 57.7% of homogalacturonans (HG) and

42.0% of type I rhamnogalacturonans (RG-I). GPP was evaluated for it cytotoxic effect by crystal

violet and trypan blue assays. T98G cells were exposed to 25, 100, and 400 µg mL-1 of GPP for 48 h.

The results were expressed as percentage of cells relative to control (100%) ± SD of 3 independent

experiment each one in sextuplicate (p<0.05). Crystal violet is a basic dye with high affinity to nucleic

acids, and it is extensively used to determine cell proliferation through DNA staining of adhered and

fixed cells. In this work, all concentrations of GPP (25, 100 and 400 µg mL-1) were able to decrease

significantly the percentage of adhered cells, reaching a maximal inhibition of 33.62%. Additionally,

the cytotoxic effect of GPP was performed with a second method. Trypan blue assay is based on the

absorption of trypan blue dye by dead cells, since in this stage they are characterized by loss of

membrane permeability. On the other hand, viable cells with intact membrane remain unstained,

therefore the results were expressed as percentage of cells unstained relative to control.

Results and Discussion: The results indicated that all concentrations of GPP analysed (25, 100 and

400 µg mL-1) inhibited significantly cell viability, reaching a maximal inhibition of 44.52%.

Comparing both experiments, GPP seems to be more effective when evaluated by trypan blue instead

of crystal violet. However, this difference between those results can be explained by the fact that

trypan blue counts either adhered as dislodged cells, while crystal violet measures only adhered cells.

In addition, crystal violet may include cells that have already lost membrane permeability but remain

adhered to the plate.

Conclusion: Thus, it was possible to demonstrate that GPP, a pectin isolated from gabiroba fruit

(Campomanesia xanthocarpa Berg), decreased significantly the cell viability of a highly aggressive

human glioblastoma cell line (T98G) through two different assays: crystal violet and trypan blue. The

biological mechanisms behind this effect are under investigation.


Acknowledgement: The authors gratefully acknowledge the following Brazilian agencies for

financial support: National Council for Scientific and Technological Development – CNPq

(309225/2018-3; 141692/2018-9); Coordination of Superior Level Staff Improvement – CAPES

(88887.335103/2019-00) and Federal University of Parana-Brazil-UFPR. The authors would like to

thank the Brazilian Agricultural Research Corporation/Embrapa Forestry, Rossana Catie Bueno de

Godoy and Maria Cristina Medeiros Mazza for provide the gabiroba pulp.


Development of superficially modified magnetic nanoparticles with carbohydrates for

the cellular culture of microorganisms

ARAUJO-NETO, L. A. (1,2); PEREIRA, T. M. (1,3); SILVA, L.P. (1,2,3)

1. Laboratory of Nanobiotechnology (LNANO), Embrapa Genetic Resources and

Biotechnology, Brasília, DF, Brazil.

2. Postgraduate Program in Pharmaceutical Sciences, Department of Pharmacy, Federal

University of Parana, Curitiba, PR, Brazil.

3. Postgraduate in Biological Sciences, University of Brasilia, Institute of Biological Sciences,

Brasilia, DF, Brazil.

Email: [email protected]; [email protected]

Introduction: Magnetic nanoparticles (MNPs) are a specific type of nanomaterial that, when exposed

to a magnetic field, moves toward it. Their applications focus on biomedicine as a strategic targeting

of drugs and as a contrast agent in magnetic resonance imaging as well as in the environmental area

due to its interactions with potentially contaminating molecules. In recent years, the use of this type of

nanomaterial has been demonstrated in the development of three-dimensional cell cultures, mainly for

the formation of tumor mimetics. However, the applications of MNPs to other cell models are still

limited. Parallel to this, planktonic microorganisms when adhered to a surface, perform the

phenomenon of swarming, in order to ensure the expansion of the colony and ensure more niches.

From this, the aim of this study was to produce MNPs with surfaces modified with four types of

carbohydrates, for potential applications in microorganism culture and also aiming at a possible three

dimensional construction.

Methods: By the coprecipitation method, the MNPs were synthesized using a protocol recently

optimized in the Laboratory of Nanobiotechnology of Embrapa Genetic Resources and

Biotechnology. The final volume was separated into five beckers in which the MNPs were

magnetically decanted (MNP-SC) and the other four received 0.3 mM of different carbohydrates

(MNP-Carb1, MNP-Carb2, MNP-Carb3, and MNP-Carb4), with the objective of coating the MNPs,

being decanted magnetically after 20 min exposure to the carbohydrate solutions. These MNPs were

analyzed regarding to hydrodynamic diameters (HD), as well as profiles of chemical groups via

Raman spectroscopy. The minimum inhibitory concentration (MIC) assay against Escherichia coli,

Staphylococcus aureus and Saccharomyces cerevisiae was performed in order to identify the possible

toxicity of MNPs towards microorganisms. Finally, the magnetization of E. coli and S. aureus was

used to investigate the possible interaction of these bacteria with MNPs as well as modulate the

growth and development of swarming.

Results and Discussion: The results showed that MNPs have HD ranging from 100 to 200 nm, with

slightly varied PdIs occurring due to association and dissociation of the nanoparticles, and the Zeta

potential negative. Carbohydrates coatings in the MNPs influenced the profiles obtained by Raman

spectroscopy in different ways, inferring the adhesion of the same to their surfaces. When submitted

to the biological assays to determine their potential toxicity level, it was found that in none of the

concentrations of 1 mM, 800, 600 and 400 μM (of iron) there was altered growth of microorganisms

when observed visually. In addition, the magnetization of the bacteria was promoted in different ways

and the modulation of the swarming formation in E. coli when associated with MNP-Carb2 was

identified, and for S. aureus there was no modification.


Conclusion: The modification in the surface coverage of the MNPs did not infer in the possible

toxicity to the microorganisms. However, it showed differences in magnetizations and modulation of

bacterial swarming formation in E. coli. Therefore, the MNPs synthesized in this work and modified

superficially with carbohydrates, can be used for the culture of microorganism, in order to evaluate

the formation of swarming, as well as the formation of microbial biofilms and future trials with 3D

cultures by magnetization.

Acknowledgement: Capes, CNPq, Embrapa, Federal University of Parana, Araucaria Foundation,

FAPDF, and Nano3D Biosciences.


Evaluation of the cytotoxic and anti-metastatic in vitro effects of Lafoensia pacari in

colorectal cancer cells

ATHERINO, J.C. (1); ATHERINO, M.C. (1); REISHERT, C.L. (1); RABELLO, A.C. (2);

CARVALHO, A. (2); WEFFORT-SANTOS, A.M. (2); SANTOS, C.A.M. (3); SOUZA, W.M. (1,2);

FELIPE, K.B. (2)

1. Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Federal do Paraná

2. Departamento de Análises Clínicas, Universidade Federal do Paraná

3. Departamento de Farmácia, Universidade Federal do Paraná

E-mail: [email protected]

Introduction: Lafoensia pacari has been traditionally used to treat different types of cancer and

inflammatory diseases. Studies performed by our group show that the methanolic extract of this plant

is able to induce apoptosis in the colorectal cancer cell line HRT-18. Furthermore, inflammation is

known to be linked to the resistance of the apoptotic process, as well as to the development of

angiogenesis and metastasis. Thus, the aim of this work is to verify if the cytotoxic and anti-metastatic

in vitro effects of L. pacari are associated with the anti-inflammatory activity of this plant.

Methods: The cytotoxic/antiproliferative effect of 19 extracts of L. pacari stem barks was first

evaluated by the reduction of resazurin in HRT-18 and McCoy cell lines and their effect further

confirmed through the clonogenic assay and the counting of viable cells. HRT-18 cells migration and

invasion were evaluated through the wound healing assay and the transwell cell invasion assay,

respectively. Griess reaction was used to measure the production of nitric oxide (NO) in HRT-18

cells.

Results and discussion: Overall, every tested extract displayed cytotoxicity in HRT-18 cells.

However, extract 14 (E-14) showed higher selectivity to these cells. The cell migration assay showed

that E-14 was able to inhibit HRT-18 cells migration about 64%. Moreover, E-14 was able to reduce

in 73% the invasive capacity of these cells. NO is an endothelial growth factor mediating tumor

vascularization and, in addition to regulating metastasis and tumor onset, it is also a key regulator of

angiogenesis. Treatment with E-14 reduced NO production in the tumor cell line simultaneously with

the induction of cytotoxicity and inhibition of cell migration and invasion.

Conclusion: Results suggest that the cytotoxic activity of E-14 may be related to the anti-

inflammatory activity of the extract. Additional assays are being performed in order to confirm this

link.


Quantification of cancer stem cells in melanospheres

AZEVEDO, D.P. (1); MARIN, M.G.P. (1); ASSUNÇÃO, L.S. (1); RODE, M.P. (1); SILVA, A.H.

(1); CRECZYNSKI-PASA, T.B. (1,3)

1. Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa Catarina,

Florianópolis, SC, Brasil

2. Programa de Pós-Graduação em Química, Universidade Federal de Santa Catarina,

Florianópolis, SC, Brasil

3. Departamento de Ciências Farmacêuticas-CIF, Universidade Federal de Santa Catarina,

Florianópolis, Santa Catarina

Email: [email protected]

Introduction: Melanoma is very aggressive cancer, being responsible for 75% of mortality among

skin cancers. Besides the high mortality, melanoma is highly recurrent and only 1-2% of patients

achieve the cure. The difficulty related to melanoma's treatment is consequent of the tumor

microenvironment and the presence of Cancer Stem Cells (CSC). The best in vitro model for studying

CSC subpopulations is the spheroid model (3D cell culture), in which cells are plated at non-

suspension conditions. To identify CSC is necessary to quantify a number of transmembrane stem

cell-related proteins, such as CD44, CD133, and CD271. The aim of this study was to define the best

cell density to evaluate the CSC population in a B16F10 3D cell culture.

Methods: Melanospheres were obtained by coating a 96 well plate with non-adhesive agarose 2%,

with cells plated at two different densities (100 or 1000 cells/well) and analyzed regarding size,

viability, and percentage of CD44, CD133, and CD271 positive cells.

Results and Discussion: The percentage of CD44 positive cells in 2D culture and in 3D culture (1000

cells/well) was 11% and increased up to 77% in melanospheres formed with 100 cells/well. The

percent of CD133 positive cells was 7% in 3D culture (1000 cell/well), increasing to 77% at a low-

density melanospheres condition. In contrast, CD271 did not statistically differ in any culture

condition. However, there was a tendency to increase for CD271 at low cell density. These results

confirmed that the increase in CD44, CD133, and CD271 is possible in a 3D model at low cell

density. The results also confirmed the relationship between the 3D model at a low density and the

increase in percentages of CSC, in contrast with the 2D model, in which the CSC presence was allays

smaller in all conditions tested. Probably, it is possible because in 3D growing condition and at low

cell density the formation of melanospheres is allowed by clonal replication.

Conclusion: A melanosphere model is an important tool for in vitro studies of the cytotoxic potential

of new antitumoral agents since it can enrich the population of CSC cells when done in an ideal

condition of plating, important drug targets for cancer treatment.

Acknowledgments: Universidade Federal de Santa Catarina (UFSC); Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior (CAPES), Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina

(FAPESC), and Grupo de Estudos da Interação entre Micro e Macromoléculas (GEIMM).


Evaluation of TCTP and its inhibition in neuroblastoma

BALDISSERA, A.B. (1); SEUNARAINE, R. S. (1); MORENO, K.G. (1); ESPOSITO, S.E. (2);

GREMSKI, L.H. (1); VEIGA S.S. (1); SENFF-RIBEIRO, A. (1)

1. Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.

2. Department of Health Science, Pontifical Catholic University of Paraná, Curitiba, Paraná,

Brazil.


Introduction: The TCTP (Translationally Controlled Tumor Protein) plays an important role in

tumor reversion process and is considered a target molecule for cancer treatment. Sertraline, an

antidepressant (SSRI), decreases TCTP intracellular levels and inhibits tumor growth in some animal

models and cells. Neuroblastoma, most common extracranial solid tumor, is a pediatric cancer from

neural crest cells and develops during fetal growth or in the first years of life. There is only one study

in the literature relating neuroblastoma biopsies and TCTP protein levels, specifically phosphorylated

TCTP. Aims: evaluate the TCTP protein levels in neuroblastoma cell lines and the effects of sertraline

treatment.

Methods: CHLA 15 and SH-SY5Y neuroblastoma cell lines were grown in RPMI medium. Cells

were treated with sertraline (0.5 e 1 μM), DMSO or only medium TCTP levels were determined by

immunodetection (western-blotting). Cell viability was assessed by a luminescent assay (cell titer glo

assay, Promega); clonogenicity was performed in semi-solid medium (agar). TCTP content and the

effects of sertraline were also analyzed in a standard protocol of retinoic acid-induced differentiation

using cell lines.

Results and Discussion: Cell lines treated with sertraline decreased TCTP levels, viability and

clonogenicity. Both cell lines were very sensitive to the treatment. Sertraline accelerated the retinoic

acid-induced differentiation and was able to induce the neuroblastoma cell line differentiation in

absence of retinoic acid. These results could be related to decreased levels of TCTP.

Conclusion: Our preliminary results confirm previous data, which suggest TCTP protein as an

interesting target for neuroblastoma. Sertraline treatment was effective and able to difference

neuroblastoma cells suggesting its therapeutic possibility.

Acknowledgement: CAPES-COFECUB, CNPq, Fundação Araucária, UFPR.


From human embryonic stem cells to cardiomyocytes: expression profile analysis of

metabolism-related genes

BARISÓN, M. J. (1); PEREIRA, I. T. (1); ROBERT, A. W. (1); DALLAGIOVANNA, B. (1)

1. Laboratório de Biologia Básica de Células Tronco, Instituto Carlos Chagas, Fiocruz Paraná,

Curitiba, PR, Brazil.


Introduction: Stem cells present a self-renew capacity and, due to their multi or pluripotent nature,

they can differentiate into several specific lineages. These characteristics make stem cells ideal

candidates for use in cell therapy protocols. For instance, has been showed that cell therapy

technologies aim the regeneration of injured heart: transplanted stem cells can contribute with the

regeneration of the damaged cardiac tissue. Therefore, the knowledge of the basic biology of

differentiation process is extremely necessary to develop effective differentiation protocols. The

commitment of stem cells into differentiated ones implicates complex mechanisms involving

signaling and regulatory pathways, and the differential expression of specific genes. Our group has

shown that posttranscriptional regulation mechanisms are central in the determination of the gene

expression profiles of stem cells and its differentiation processes. Using the polysome profiling

technique, polysome-bound RNAs were isolated and high-throughput sequenced, showing that during

cardiomyogenesis post-transcriptional regulation of gene expression contributes to cardiac

differentiation. In this work, we analysed the profile changes in metabolism-related genes along

cardiomyogenesis, using our RNA-seq dataset.

Methods: Protein coding genes included in our polysome-bound RNA-seq data (Fold Change≥2, p-

value≤0.05) were analysed using bioinfomatic softwares as EnrichR, GProfiler and String. For

validation experiments, RNA was isolated using Trizol and cDNA synthetized by in vitro reverse

transcription. Transcript levels quantification was performed using qPCR. Embryonic stem cells

(hESCs) transfection for over-expression experiments was carry-out using Lipofectamine 3000

reagent.

Results and Discussion: Initially, it was performed the gene onthology (GO) analysis of

differentially expressed genes at each time point along cardiomyogenesis (day 0, undifferentiated

hESCs; day 1, embryoid body; day 4, mesoderme; day 9, cardiac progenitor and day 15,

cardiomyocyte). GO terms related to metabolism were evaluated and it was found that some genes

related to lipids, amino acids and carbohydrates metabolism presented a differentially expression

pattern along cardiomyogenesis. Specific genes related to the utilization and storage of lipids and

carbohydrates were found upregulated already in mesoderm-cardiac progenitor transition, as well as

genes involved in regulation between glucose and fatty acid metabolism. Among amino-acids related

genes, IDO1, which codifies to indoleamine 2,3-dioxygenase, the first enzyme of tryptophan

catabolism, presented an interesting profile: its expression is higher in undifferentiated cells

(pluripotent stage), and then decrease, reaching its minimal levels at mesoderm stage. This pattern

was validated by qPCR. To test the hypothesis that HsIDO1 or related metabolites could be involved

in maintenance of pluripotent state, we transfect hESCs with the construction pcDNA3.1/myc-HisB-

HsIDO1 in order to over-express this enzyme and evaluate the resultant phenotype. HsIDO1 was

successfully over-expressed, with transient expression levels of 30-50-fold after 48h of transfection,

when compared to scramble control. After embryoid body formation and induction to mesoderm, cells


over-expressing HsIDO1 also presented a significant decrease in HsIDO1 expression, as observed in

wild-type cells. Levels of pluripotency markers were also measured and no changes were observed.

Conclusion: Metabolism-related genes are regulated along cardiomyogenesis, presenting specific

patterns along differentiation. Between genes encoding amino acids metabolic enzymes, HsIDO1 is

differentially expressed between hESCs and mesoderm cells, presenting a significant decrease in its

expression. The over-expression of this enzyme did not modify this pattern nor pluripotency state of

hESCs. These results suggest that HsIDO1 could be subjected to a tight control of its expression when

cells are induced to exit from pluripotent state.

Acknowledgement: This work was supported by CNPq, CAPES and Araucaria Foundation.


Characterization of phenotypic aspects of human glioblastoma cells cultivated in

hanging drops submitted to the combined treatment of Temozolomide and Sinvastatin

BARK, J. M. (1); SPISILA, L. J. (2); TRINDADE, E. S. (3,4); ULRICH, H. (5); SOGAYAR, M. C.

(5,6); TROMBETTA-LIMA, M. (5,6); WINNISCHOFER, S. M. B. (1,2,3)

1. Postgraduate Program in Biochemistry Sciences, Sector of Biological Sciences, Federal

University of Paraná, Paraná, Brazil.

2. Department of Biochemistry and Molecular Biology, Federal University of Paraná, Paraná,

Brazil.

3. Postgraduate Program in Cell and Molecular Biology, Federal University of Paraná, Paraná,

Brazil.

4. Department of Cell Biology, Federal University of Paraná, Paraná, Brazil.

5. Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo,

Brazil.

6. NUCEL (Cell and Molecular Therapy Center), Medical School, University of São Paulo, São

Paulo, Brazil.


Introduction: Gliomas are the most common neoplasms that affect the central nervous system.

Among them, grade IV astrocytomas, also called glioblastomas (GBMs), are the most aggressive.

Although current therapies involve surgical removal, chemotherapy and radiotherapy, prognosis is

still poor and survival rate ranges from to 15 months. Temozolomide is the standard chemotherapy

currently used, however, several mechanisms of resistance have been described. An example of this is

the presence of tumor stem cells (CSCs) which are less responsive to the administration of the

chemotherapeutic agent. In this context, statins, cholesterol-lowering agents, have been associated

with antiproliferative action in several neoplasias. Studies show that treatment of GBM with

simvastatin promotes an increase in apoptosis, modulation of senescence and autophagic capacity of

tumor cells. The responsiveness of cells to antitumor agents is closely related to the tumor

microenvironment, there by in vitro assays that resemble this microenvironment are important.

Among in vitro 3D-techniques,hanging drops cell culture is an easy and cost-effective method that

has been used in to generate cell spheroids that mimic cell-cell and cell-microenvironment

interactions.

Methods: This project aims to study the potential cytotoxic action of the combined treatment of

temozolomide and simvastatin in human GBM cell lines (U-251MG, U-87MG and T98G) cultured in

hanging drops. Tests were performed using different cell densities at different culture times, as well

as phenotypic analysis of the neurospheres and evaluation of cytotoxicity during spheroid formation

and after spheroid maintenance on a non - adherent substrate of poly-HEMA 1.2%.

Results and Discussion: The results showed that the formation of viable neurospheres with more

homogeneous morphology resulted in the density of 1x103 cells, cultured for 72 hours in suspended

drop followed by 5 days in culture in non-adherent medium (1.2% poly-HEMA). We observed

increased presence of CSC by labeling with CD133. In addition, treatment with temozolomide and

simvastatin during and after spheroid formation demonstrated a change in the size and border of the

neurospheres formed, respectively, suggesting a potential antitumor action. Finally, we sought to

evaluate the maintenance of the spheroids using a known methodology for culture of CSC, using


culture medium defined (NSC), and a significant reduction in size and increase in the rate of cell

death was observed ( by labeling with PI and Anexin-V) of the spheroids submitted to the combined

treatment when compared to the control group.

Conclusions: Thus, from the data of this project, we seek to obtain a differential methodology of in

vitro studies that present low cost, as well as to suggest a new possibility of study of adjuvant therapy

for GBM treatment.


Correction of the F508del mutation in lung epithelial cells with cystic fibrosis

BASSAI, L.W. (1); SHIGUNOV, P. (1)

1 – Instituto Carlos Chagas – Laboratório de Biologia Básica de Células-tronco – Curitiba - PR

E-mail:[email protected]

Introduction: Cystic fibrosis (CF) is a recessive genetic disease autossomic caused by mutations in

the cystic fibrosis transmembrane conductance regulator gene (CFTR). It is estimated more than

70.000 peoples with cystic fibrosis in all the world, but the incidence is different according to country,

for example, in United States the incidence is 1 in 3.500, in Union European 1 in 2.000 and in Brazil 1

in 10.000. The protein CFTR is a glycoprotein present in the membrane of epithelial cells, being

responsible for the conduction of Cl ions and transportation of others substrates through the

membrane. Defects in this protein affects the transport of fluids and electrolytes through cell

membrane, resulting in abnormal viscous secretion in multiple organs, mainly in the lungs and

pancreas. Approximately, there are 2.000 CFTR known mutations that cause CF, but the most

common mutation is F508del, which consists in the deletion of phenylalanine at the position 508.

When this mutation is present CFTR processing is abnormal and the protein ends up being degraded

in the edoplasmatic reticulum. In this ways, the protein concentration on the membrane is reduced.

The disease is incurable and the treatments available consisting in antibiotics, enzymes,

bronchodilators, steroids and drugs to make the mucus thinner only control the symptoms. For this

reason, gene therapy for the edition and correction of the CFTR channel becomes an option for the

treatment and cure of the disease.The CRISPR / Cas9 technique can be applied for gene edition, in the

future maybe to use for correction this mutation in vitro recovering a functional CFTR in a gene

therapy. Objectives: The present work aims to correct F508del in cells of the cystic fibrosis of

pulmonary epithelium (CFBE) with the use of the CRISPR/Cas9 technique. In order to obtain healthy

cells that express the CFTR channel, the objectives of this project is: (1) Establishment of the CFBE

cell line in the laboratory; (2) design of

specific RNA guide (sgRNA) for the CFTR gene and mapping the mutations to the target; (3)

construction of CRISPR expression systems; (4) standardize different transfection methodologies in

cells with an F508del mutation; (5) establish a molecular correction protocol using methods as PCR,

target sequencing and immunofluorescence to confirmation.

Methods: (1) The CFBE cells were selected with puromycin, because in CFBE there are a vector with

resistance gene for it immortalization and the minimum concentration to be used in the selection was

determined for neutral red assay. (2) For design of sgRNA the CRISPOR and CHOCHOP software

were used.

Results and Discussion: The minimum concentration of puromycin was determined by neutral red

assay, where 200ng/mL was sufficient to select the resistant cells, because it maintained the relative

viability less than 50% relative to the control. The CFTR gene sequence (NG_016465.4) was

obtained from the NCBI database and the F508del mutation site was located. About 500 base pairs

around the mutation were selected and with the use of the CHOPCHOP software the possible design

of sgRNA. About ten sgRNAs were selected and the possible off-targets with their location in the

genome were mapped. Based on the location of the off-targets predicted by the CRISPOR software in

the three possible genome sgRNA were selected and synthesized for tests of transfection.


Conclusion: As a work perspective, the selected sgRNA will be test by different transfection methods

in CFBE cells and editing efficiency will be calculate. It is expect to optimize the efficiency of the

repair of cell homology, to minimize the off-targets in correction of the CFTR channel.

Acknowlegment: Inova Fiocruz; CAPES, CNPq.


Circulating microRNAs in cervical carcinogenesis: promising diagnostic biomarkers

from squamous intraepithelial lesions to cervical cancer

BERTI, F. C. B. (1); SALVIANO-SILVA, A. (1); BECKERT, H. C. (1); DE OLIVEIRA, K. B. (2);

CIPOLLA, G. A. (1); MALHEIROS, D. (1)

1. Laboratory of Human Molecular Genetics | Department of Genetics | Federal University of

Paraná | Brazil Av. Cel. Francisco H. dos Santos | 100 | Zip Code 81530-000 | Curitiba | PR |

Brasil | +55 41 3361 1724

2. Laboratory of Molecular Genetics and Immunology | Department of Pathological Sciences |

State University of Londrina | Brazil (Rod. Celso Garcia Cid Pr 445, KM 380, Zip Code

86057-970 | Londrina | PR | Brasil | +55 43 3371 5728


Introduction: Human Papillomavirus (HPV) plays an essential role on cervical carcinogenesis.

Despite that, HPV’s presence and action per se are not sufficient for squamous intraepithelial lesions

(SIL) development, nor progression to cervical cancer (CC). Other factors than HPV are important

players in cervical carcinogenesis, as miRNAs. In spite of their “tiny” size, they are thought to exert

an important impact on cervical microenvironment. MiRNAs are diversely distributed across tissues

(in the cellular compartment) and biological fluids (extracellularly), as cell-free miRNAs or miRNAs

present in extracellular particles, known as extracellular vesicles (EV). After HPV infection, an

interplay between HPV and the miRNA network seems to occur in cervical cells, with HPV

oncoproteins exerting effects over the miRNA transcriptome, while miRNAs seem to influence HPV

global expression profile. Subsequently, as HPV persists and cellular transformation occurs, specific

patterns of miRNA expression are thought to be found in different stages of cervical disease.

Therefore, the aim of this literature review was to present a solid state-of-the-art overview on

circulating miRNAs, from SIL to CC.

Methods: In order to define promising cell-free and EV-derived miRNAs involved in different stages

of cervical carcinogenesis, a search through the literature was performed. Articles had to fulfill some

criteria in order to be included, such as being indexed in PubMed, being written in English, and

matching appropriate keywords (e.g., for cell-free miRNAs some of the predefined terms were

‘circulating’, ‘cell-free‘, ‘serum’, ‘plasma’, ‘miRNA’, ‘microRNA’, ‘LSIL’, ‘HSIL’, ‘CIN’, ‘cervical

cancer’, and the possible combinations among them; for EV-derived miRNAs a similar type of search

was conducted, using proper words for such purpose, as 'extracellular vesicles' and 'exosomal').

Original articles based only on functional experiments and reviews were excluded. Only miRNAs

found deregulated by at least two studies in SIL and/or CC were included.

Results and Discussion: Despite the limited number of studies investigating circulating miRNAs in

distinct biological fluids, accumulating data have pointed some promising circulating miRNAs

involved in SIL and CC, both as cell-free or EV-derived miRNAs. Cell-free miR-9, miR-10a, miR-

20a, miR-196a, for example, were all found upregulated in SIL stages and CC, suggesting their

potential as non-invasive biomarkers for cervical disease. Others as cell-free miR-486 and miR-1246,

and EV-derived miR-21 and miR-146a were found upregulated only in CC, thus being involved in the

late stages of cervical carcinogenesis.

Conclusion: Circulating miRNAs’ role in cervical carcinogenesis is becoming more evident,

especially considering the advantages of liquid biopsy (easily obtained, minimally invasive, allowing


for an early diagnosis). Up to date, a considerable amount of original research reporting the

involvement of several circulating miRNAs in SIL and CC has been reported, highlighting some

promising candidates. Still, this is a research field under recent construction. Gradually, a more solid

overview of circulating miRNAs involved in cervical carcinogenesis is being settled.

Acknowledgement: We acknowledge the Graduate Program of Genetics of the Federal University of

Paraná.


Analysis of the interaction between stem cells and 3D printed scaffolds for tissue

regeneration

BIAGINI, G. (1); PEREIRA, T. (2); LUZ, A.R. (2); BERTI, L.F. (2); CORREA, A. (1); MARCON,

B.H. (1); STIMAMIGLIO, M.A. (1)

1. Instituto Carlos Chagas (FIOCRUZ/PR).

2. Universidade Tecnológica Federal do Paraná (UTFPR).


Introduction: Tissue engineering is a branch of regenerative medicine and is based on the utilization

of biomaterials combined with cells and other bioactive molecules to regenerate functional tissues.

Biomaterials scaffolds are used to reproduce the extracellular matrix and they also act as substrate and

physical support for cells, orienting the formation of the new tissue. Even though the technique is

promising and has already allowed the construction of whole organs (bladder, skin, trachea) in labs,

its use in human patients is still limited. This is due to the challenge of finding materials and

implantation techniques that can substitute living tissues in an adequate manner, giving that the

physical and biological characteristics of living tissues are the result of millions of years of

evolutionary optimization. Currently, several articles have already demonstrated that cells interact

well with different types of biomaterials, but these results are not comparable, given the fact that they

use different cell types, different materials and different analyses’ techniques. Therefore, it is still

necessary to study and analyze how cells and other biological factors involved in the process of tissue

formation interact with the biomaterials in order to obtain a reliable comparison and decide which

biomaterials are more adequate to mimic the intrinsic properties of the living tissue. The choice of

biomaterials also takes into account their biocompatibility, biodegradability and the presence of

physical properties similar to the biological tissue. Other important factors that influence the cells’

response are the porosity of the scaffold, the size and interconnectivity of these pores and the

composition of the scaffold’s surface. In this work, we will initially analyze the interaction between

different types of biomaterials and mesenchymal stem cells considering their adhesion, proliferation

and differentiation in order to understand how the cells behave on the scaffolds.

Methods: Because polylactic acid (PLA) is the most common biomaterial tested for tissue

regeneration, we selected it to perform the initial tests to determine ideal format and pore size and

analyze cell adherence. The scaffolds were fabricated at Universidade Tecnológica Federal do Paraná

(UTFPR) using a 3D printer.

Results and Discussion: For the first tests, scaffold dimensions were set in 14,5mm (d) x 5mm (h),

while pore sizes varied from 430 to 730µm. Electron microscopy was used to observe cell adherence

on the scaffolds after 3 and 8 days in culture. In addition, by also using fluorescence microscopy we

had an indication that cells had not only adhered but had also proliferated. Our perspective is to

determine cell proliferation and analyze the possible effects of different scaffolds in cell behavior

considering its ultrastructure, adherence and proliferation.

Conclusion: Once we determine the parameters for the scaffold manufacture, we will select novel

biomaterials to compare and better understand how they interact with stem cells for future possible

applications in tissue regeneration.

Acknowledgements: CNPQ, Fiocruz, Fundação Araucária, CAPES, Inova, UTFPR, UFPR, PUCPR.


Evaluation of the proliferation of cell lines established in culture treated with

polinsaturated fatty acids

BIALLI, A. P. (1); OLIVEIRA, A.C. R. (1); APPEL, M.H. (2); IAGHER, F. (1)

1. Department of Physiology, Federal University of Paraná.

2. Department of Molecular and Genetic Structural Biology, State University of Ponta Grossa.

E-mail: bialliamanda@gmail.

Introduction: Cancer is the second leading cause of death in humans worldwide and it costs the

world economy just over $ 1 trillion annually. The polyunsaturated fatty acids (PUFA) are

consolidating as important nutraceuticals, adjuvants in various treatments, modulating the immune

system, favoring the restoration of organic functions impaired by chemotherapeutic treatments and

presenting a broad antitumor potential.

Methods: In order to understand the relationship between PUFA and cell proliferation, different cell

lines (SHSY-5Y, B16F10, B16F1, HeLa and MDCK) were cultured in the presence of two

concentrations (1: 100 (T1) and 1: 200 (T2)) of 3 polyunsaturated oils (fish oil (OP), shark liver oil

(OF) and inca gold (OI)). The cell proliferation test was performed using Alamar Blue vital dye (final

concentration 10% v / v) at times 6, 24, 48 and 72 hours.

Results and discussion: For HeLa, when compared to the control, the three oils tested in the T1

concentration and the inca gold in T1 and T2 stimulated the proliferation in the four times tested. OP

and OF in T2 produced significantly greater proliferation after 24h. B16F10 showed increased

proliferation in all treatments and times tested. B16F1 presented a profile similar to HeLa. MDCK

showed increased proliferation rate at all times with the 3 oils tested with T1, and OI at T1 and T2.

The OP in T2 was only able to produce proliferation increase in 72h and OF in the same treatment did

not produce difference. For SHSY-5Y the results were conflicting.

Conclusion: Inca oil showed the highest capacity to induce cell proliferation in all the studied cell

lines, followed by fish oil. The shark liver oil was the treatment with the lowest proliferative rate

among the studied treatments. The treatments had a dose dependent proliferative effect.

Acknowledgments: To the financial support received by the Araucaria Foundation and the technical

support of the State University of Ponta Grossa.


Plasmodium vivax AMA1: Implications of distinct haplotypes to immune response

BITTENCOURT, N.C. (1); DA SILVA, A. B. I. E. (2); VIRGILI, N. S. (1); SCHAPPO, A. P. (2);

GEVÁSIO, J.D.B. (2); PIMENTA, T.S. (3); CASTINEIRAS, C. (1); LOPES, S. (4,5); LACERDA,

M.V.G. (4,5); MACHADO, R.D. (3); COSTA, F.T.M. (1); ALBRECHT, L. (2)

1. Laboratory of Tropical Diseases – Prof. Dr. Luiz Jacintho da Silva, Department of Genetics,

Evolution, Microbiology and Immunology. University of Campinas-UNICAMP. Campinas,

SP, Brazil.

2. Instituto Carlos Chagas, Fundação Oswaldo Cruz – FIOCRUZ. Curitiba, PR, Brazil.

3. Laboratório de Ensaios Clínicos e Imunogenética em Malária, Instituto Evandro

Chagas/SVS/MS, Ananindeua, PA, Brazil.

4. Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Gerência de Malária, Manaus,

AM, Brazil.

5. Instituto Leônidas e Marie Deane, FIOCRUZ-AMAZONAS, Manaus, AM, Brazil.


Introduction: In Brazil, Plasmodium vivax infection accounts for more than 80% of malaria cases

and is the most common species of Plasmodium. This infection has a substantial impact on the

productivity of the local population. The emergence of drug-resistant strains and further complication

of P. vivax infection leads to the intensification of research on alternative control methods. There is

currently no effective vaccine available against malaria. One of the leading candidates is the Apical

Membrane Protein 1 (AMA1). AMA1 is involved in the invasion of Apicomplexa parasites to host

cells. This study looked at the impact of genetic diversity of PvAMA1 into the immune response

towards this antigen in endemic malaria regions. The hypothesis is that genetic polymorphisms might

direct the immune response.

Methods: We extracted P. vivax DNA, amplified, sequenced and aligned 40 P. vivax Brazilian

isolates in order to analyze the genetic diversity. Two distinct PvAMA1 variants were used to

evaluate a naturally acquired immune response in infected and non-infected individuals from endemic

regions by Enzyme Linked Immuno Sorbent Assay (ELISA).

Results and Discussion: Among the analyzed population we found 19 haplotypes. In total, 33 non-

synonymous amino acid sites were present across PvAMA1 whereas 20 (60.6%) are located at B cell

predicted epitopes. Two representative haplotypes were chosen: one haplotype with high frequency

(H5 = 22.5%) and another one with low frequency (H16=2.5%), to analyze the influence of those to

the host immune response. The analysis of immune responses using recombinant proteins

PvAMA1V5 (H5) and PvAMA1V16 (H16) showed that both of them were greatly immunogenic

being the highest antibody response against H5. All samples infected with PvAMA1H5 also had

antibodies towards this sequence. However, only 55% of individuals infected with PvAMA1H5 had

antibodies against PvAMA1V16. The individual infected with PvAMA1 H16 parasite had antibodies

to both variants, nevertheless a higher reactivity was found against PvAMA1V5. Moreover, we

looked at the IgG antibody response to this two PvAMA1 variants in infected and not currently

infected individuals from gold-mining areas and more than 40% of this population had antibodies

against both of them.

Conclusion: Taken together, our findings demonstrate that despite the level of polymorphism,

PvAMA1 can elicit a strong antibody response. Therefore, we suggest that vaccine trials based on


polymorphic antigens should be designed considering strain frequencies at individual study sites. The

high reactivity added to the possible maintenance of the immune response over time make haplotype 5

a potential component of a vaccine formulation based on PvAMA1.

Acknowledgement: We want to express our gratitude to the people that agreed to participate in this

study, to the field team in the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD)

and Instituto Evandro Chagas/SVS/MS. The authors also thank the Program for Technological

Development in Tools for Health-PDTIS FIOCRUZ at the Carlos Chagas Institute, Fiocruz-Paraná,

Brazil. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico

(CNPq), Grant 476904/2013-7 and Fundação de Amparo à Pesquisa do Estado de São Paulo

(FAPESP) Grant 2012/16525-2


Ribosome-associated lncRNAs in adipose-derived Stem Cells: From RNAs to

micropeptides

BONILAURI, B. (1); HOLETZ, F.B. (2); PASSETTI, F. (2); SANTOS, M.D.M. (3); CARVALHO,

P.C. (3); DALLAGIOVANNA, B. (1)

1. Stem Cell Basic Biology Laboratory, Carlos Chagas Institute-Fiocruz/PR.

2. Gene Expression Regulation Laboratory, Carlos Chagas Institute-Fiocruz/PR.

3. Structural and Computational Proteomics Laboratory, Carlos Chagas Institute-Fiocruz/PR.


Introduction: Long non-coding RNAs (lncRNAs) are traditionally classified as transcripts larger than

200 nucleotides and do not have protein coding potential. These RNAs play crucial roles in a wide

variety of biological processes (e.g. transcriptional and posttranscriptional regulation, splicing and

translation). However, little is known about its function in stem cells differentiation and maintenance.

Recently, lncRNAs were observed associated with ribosomes, being ther functions and mechanisms

of action not elucidated yet. Two hypotheses for this association were raised. First, the presence of

small ORFs (sORFs) in lncRNAs, which would have the potential to be translated into functional

micropeptides. Second, a directly translational regulation of several targets (mRNAs/miRNAs).

Therefore, the present work aims to investigate the functions and mechanisms of ribosome-associated

lncRNAs in maintenance and differentiation of human adipose-derived stem cells (hADSC).

Methods: In order to identify the encoding potential of sORFs in lncRNAs we perfomed a Ribosome

Profiling of undifferentiated hADSC (n=3) followed by massive sequencing (Ribo-seq). In parallel, a

peptidomic assay (n=3) was conduct for analysis in mass spectrometry. Briefly, approximately 3x107

cells were lysed with polysomal buffer which divided the sample into citoplasmatic and pellet

fractions. Afterwards, we passed the samples through 10 kDa pore columns and we separated the

peptides in trypsinized and non-trypsinized samples. Several bioinformatics analysis will be

performed to identify these micropeptides-derived from sORFs. For investigate the possible

translational regulation, we selected three polysome-associated lncRNAs from our previous data. We

will amplify and purify them for future cloning using the gateway system. After overexpression, we

will verify the influence of these lncRNAs on the cellular fenotype through functional assays (e.g.

viability, cell cycle, proliferation and adipogenic/osteogenic differentiation) for identify possible

targets and mechanisms. Several bioinformatics analysis will be performed to identify lncRNA-

miRNA-mRNA networks.

Results and Discussion: We perform the Ribosome Profiling and Ribo-seq followed by alignment

against the reference human genome (hg19). We found the presence of ribosomal footprints (RFP) in

lncRNAs and others ncRNAs. Then, the RFP from lncRNAs were translated in silico (forward 3

frames/reverse 3 frames) and a footprint database was constructed. Several others analyses are being

conducted to investigate the protein coding potential of sORF through softwares and own analyses, to

produce another database and compare with peptidomic sequencing. Furthermore, a broad

characterization of these lncRNAs and sORF will be made according to their read counts, length,

exons, GC content, etc. The peptidomic assay were sucessfully conducted and are awaiting analysis

by mass spectrometry. On the other hand, we amplify two of tree lncRNAs selected and we going to

cloning into a overexpression vector (AAVS1-TRE-GW-rtTA). The true association of these


lncRNAs with polysomes was confirmed by RT-qPCR. The in silico interaction network of these

lncRNAs with miRNA/mRNAs was conducted with public databases and our previous results.

Conclusion: Until the moment, we was able to identify the association of lncRNAs with ribosomes in

adipose-derived stem cells. In addition, this association appears to be related to the presence of sORF

in these ncRNAs. As a perspective, we will use our database to identify lncRNAs-derived

micropeptides in the peptidomic assay. We will also overexpress the previously selected lncRNAs to

evaluate possible translational regulation in these cells.

Acknowledgement: Funding agencies (Fiocruz, Fundação Araucária, Capes, Cnpq)


Quiescin sulfhydryl oxidase 1 expression in colorectal cancer

BONILHA, H. (1); YAMOMOTO, J. (1); NAKAO, L. (1)

1. Federal University of Paraná, Basic Pathology Department.


Introduction: Quiescin sulfhydryl oxidase 1 (QSOX1) is a protein found at Golgi apparatus or

secreted from cells, responsible for thiol oxidation during protein folding. Recent studies showed its

important role in extracellular matrix regulation, particularly in laminin incorporation process.

Furthermore, QSOX1 overexpression has been associated with the progression of many tumors, such

as breast, prostate, lung and pancreatic cancers. This series of evidences suggests that QSOX1 could

act as an interesting tumoral prognostic marker. However, until now there is no report in literature

showing its role in colorectal cancer, despite its significant mortality rates worldwide. Therefore, the

aim of this study was to evaluate QSOX1 expression at online public databases, correlating it with

clinical and histopathological factors.

Methods: We have searched for colorectal cancer high-throughput studies available at the online

databases Gene Expression Omnibus (GEO) and R2: Genomics Analysis and Visualization Platform.

Inclusion criteria were: (1) studies that has analyzed colorectal samples collected from patients, (2)

colorectal cancer cell lines or (3) animal models of colorectal cancers that, necessarily, has classified

the samples according to any histopathological or clinical factor. Exclusion criteria were studies that

haven’t informed the applied normalization or the protein expression measurement method. The data

extracted from the databases were then submitted for nonparametric tests (Kruskal–Wallis test when

comparing three groups and Mann–Whitney t-test when comparing two groups) in GraphPad Prism

v.5.0 software.

Results and Discussion: QSOX1 was overexpressed in normal rectum, normal colorectal and, in

normal colon tissues, when compared with retal adenocarcinoma, colorectal cancer and, in primary

tumor site of colon cancer tissues, respectively. Furthermore, QSOX1 was overexpressed in metastatic

site of a tumor, in comparison with its expression in the primary tumor site; in highly metastatic

tissues, QSOX1 expression was increased, when compared with poorly metastatic tissues; and in

metastatic colon cancer, the expression of QSOX1 was higher than in non metastatic colon cancer

tissues. In adenocarcinomas the expression of QSOX1 was higher than in adenomas. The comparison

between well differentiated tumors and poorly differentiated tumors showed that QSOX1 has a higher

expression in well differentiated tumors. In addition, QSOX1 has its expression increased in tumors

larger than 2mm and smaller than 4mm, whilst tumors with more than 4mm showed a smaller

expression of QSOX1. Lastly, QSOX1 demonstrated a correlation with BRAF V600 mutation, once

QSOX1 expression was higher in mice mutated for BRAF V600, when compared with wild type. The

analysis of the results suggests that QSOX1 could have a higher expression in metastatic tumors, in

invasive cancers, in well differentiated tumors, in smaller tumors, and it could indicate correlations

between QSOX1 expression and genetic mutations, whereas the measurement of QSOX1 in normal

samples, showed a higher expression than in primary tumors, suggesting a decrease of QSOX1 on the

start of the tumorigenesis and a posterior increase of its expression in case of invasion, metastasis or

an associated mutation.

Conclusion: The correlations performed in the databases could be useful to guide future studies and

to build hypotheses that should be tested in further studies. Starting from the present search, we


hypothesize that the expression of QSOX1 could be useful to an early diagnosis of metastasis, suitable

treatment and control of the disease and, consequently, a better survival rate of the patients with

colorectal cancer diagnosis.

Acknowledgement: We would like to express our appreciation to the Brazilian free and public higher

education, which is currently under threat by several government cuts.


In vitro evaluation of the potential antitumor activity of partially hydrolyzed guar gum

and its chemically sulfated derivative on melanoma cells

BRAZ JUNIOR, O. (1); BARDDAL, H. (2); ROSSI, G R. (1); RIBEIRO, C. G. (1); FRAGA, A. (1);

BUZZO, J. L. A. (1); BELLAN, D. L. (1); TRINDADE, E. S. (1); CIPRIANI, T. R. (2); SIMAS, F. F.

(1)

1. Cell Biology Department, Federal University of Paraná.

2. Chemistry and Molecular Biology Department, Federal University of Paraná.


Introduction: Although melanoma is accountable for a minority of all skin cancer it is responsible for

most of skin cancer related deaths. Given it is aggressiveness and the lack of effective treatments for

metastatic melanoma, the search for new treatments that have low side effects is mandatory. It is

already known that many types of cancer activate the blood coagulation, and it can lead to a condition

named venous thromboembolism (VTE). VTE is the second major cause of cancer related deaths,

losing only to cancer itself. Heparin and other anticoagulants drugs in clinics showed an increase of

patients’ life expectance. However continuous treatment with heparin can lead to hemorrhage.

Previous studies already described anticoagulant activity of partially hydrolyzed and chemically

sulfated guar gum, thus finding an antitumor activity of this polysaccharide could be promising for

metastatic melanoma treatment.

Methods: Partially hydrolyzed guar gum (HGG) and its chemically sulfated derivative (HGGSL)

were used as treatments in 8 different concentrations (0.0001; 0.001; 0.01; 0.1; 1; 10; 100; 1000

µg/ml). Cytotoxicity and cell proliferation of B16-F10 (murine melanoma cells) and BALB/3T3 clone

A31 (murine fibroblast cells) were analyzed using neutral red and crystal violet methods. Cells were

cultured in 96 well plates and exposed for 72 hours to the treatments. After that, cells were incubated

with neutral red (40 µg/ml) for 2 hours, eluted with a solution composed of 50% ethanol, 49%

distilled water, and 1% acetic acid, absorbance was measured at 540 nm. After washing plates, cells

were incubated with crystal violet (0,25 mg/ml) for 20 minutes, eluted with solution containing 33%

acetic acid in distilled water and absorbance was measured in 570 nm. For apoptosis assay, cells were

cultured in 6 well plate and treated for 72h, then stained with Annexin V/7-AAD and analyzed by

flow cytometry. Clonogenic assay employing B16-F10 cells was done by plating cells in 6 wells plate,

treating for 72h. After this period cells were counted, and 400 cells of each treatment were replated in

6 welll plate for 96h. Colonies were counted and measured with ImageJ.

Results and Discussion: The polysaccharides HGG and HGGSL showed no cytotoxicity both in

B16-F10 and 3t3 cell lines. Despite crystal violet absorbance resulted in no differences between

treatments and control group, it was observed less than half of the cells after 72 h of exposure to 100

and 1000 mg/ml of HGGSL. In order to explain proliferation results, apoptosis analysis was

performed, but there were no differences in cell viability between control and treated groups.

Clonogenic assay showed that HGGSL and HGG at all concentrations have no influence in the

number of colonies, but all concentrations of HGGSL resulted in slightly increase in colonies size. On

the other hand, HGG at 10 and 1000µg/ml HGG decreases the size of colonies.

Conclusion: All concentrations of HGG and HGGSL are not cytotoxic to B16-F10 neither

BALB/3T3 clone A31. For further understanding the proliferation cell results we are performing

mailto:[email protected]


assays to know the influence of polysaccharides in cell cycle. Further investigations in malignancies

parameters will be performed, such as invasion and migration using the B16-F10 cell line.

Acknowledgement: Capes for the Scholarship, Centro de Tecnologias Avançadas em Fluorescência –

UFPR.


Investigation of cell death pathways in pemphigus foliaceus

BUMILLER-BINI, V. (1); SPADONI, M. (1); AUGUSTO, D. G.(1); PETZL-ERLER, M. L. (1);

CIPOLLA, G. A. (1); BELTRAME, M. H. (1); BOLDT, A. B. W. (1)

1. Human Molecular Genetics Laboratory, Department of Genetics, Federal University of

Parana, Curitiba, Brazil.


Introduction: Pemphigus foliaceus (PF) is an autoimmune and endemic disease in Brazil, where it is

also known as “Fogo Selvagem” (wild fire), due to the burning sensation left by painful skin blisters.

Complete loss of desmosomes in the superficial granular layer of the epidermis causes keratinocyte

acantholysis, accompanied by high levels of IgG autoantibodies against epitopes of desmoglein 1.

Despite the pathogenic role proposed for these antibodies, the exact mechanism(s) of keratinocyte

adhesion loss and death remains unknown.

Methods: In this work, we investigated the association of PF susceptibility with 3798 single

nucleotide polymorphisms (SNPs) in 294 genes encoding products implicated in 12 cell death

pathways (intrinsic and extrinsic apoptosis, necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos,

entotic, NETotic, lysosome-dependent, autophagy-dependent and immunogenic pathways). To this

end, we compared microarray hybridization genotyping results (Infinium® CoreExome-24 v1.1

BeadChip - Illumina) of 227 PF patients and 194 unrelated controls. After excluding SNPs with minor

allele frequency lower than 1%, out of Hardy-Weinberg equilibrium in controls, or in strong linkage

disequilibrium (r²≥0.8) with other tested SNPs, we tested 1167 SNPs for genetic association by

logistic regression, correcting for sex distribution and population structure (2 PCAs, using the PLINK

1.9 program).

Results and Discussion: We found ten SNPs associated with PF (p<0.005). Five of them were

associated with PF susceptibility: rs1800630*A of TNF - tumor necrosis factor alpha (OR=1.9;

p=0.00032), rs4112274*A of CD36 - CD36 molecule (OR=2.14; p=0.0015), rs12695175*C of CD47

- CD47 molecule (OR=1.77, p=0.0043), rs7072268*A of HK1 - hexokinase 1 (OR=1.48; p=0.0045)

and rs6075340*A of SIRPA - signal regulatory protein alpha (OR=2.75, p=0.00087). And five were

associated with the protection of PF: rs9355950*G of PARK2 - parkin RBR E3 ubiquitin protein

ligase (OR=0.57, p=0.00036), rs10167879*A of EIF2AK3 - eukaryotic translation initiation factor 2

alpha kinase 3 (OR=0.48, p=0.00068), rs10781522*G of TRAF2 - TNF receptor associated factor 2

(OR=0.64, p=0.0014), rs9325377*A of PAK2 - p21 RAC1 activated kinase 2 (0.48, p=0.0022),

rs10747521*A of RAPGEF3 - Rap guanine nucleotide exchange factor 3 (OR=0.42, p=0.004). These

SNPs occur in ten genes and in six cell death pathways: necroptosis (TNF and TRAF2), apoptosis

(TNF, CD36, PAK2), pyroptosis (PARK2), immunogenic cell death (CD47, SIRPA and EIF2AK3),

parthanatos (HK1) and necrosis (RAPGEF3).

Conclusion: For the first time, SNPs from all cell death pathways were investigated in a unique

disease. The associated variants highlight the interconnectivity between cascades. Functional

validation of these variants will provide a better understanding of the disease. Knowledge of which

pathway(s) cause PF acantholysis will contribute to the understanding of PF etiology and to the

development of new drugs and therapeutic regimens for the disease.

Acknowledgement: CAPES, CNPq, Fundação Araucária


FOXP3 genetic variants rs3761548 and rs2232365 do not change interleukin-10

plasmatic and cervical levels in HPV-infection

CEZAR-DOS-SANTOS, F. (1); TRUGILO, K. P. (1); BERTI, F. C. B. (1); PEREIRA, A. P. L. (1);

OKUYAMA, N. C. M. (1); SENA, M. M. (1); PEREIRA, E. R. (1); FERREIRA, R. S. (1); SINGI, P.

(1); ESPOSITO, A. (1); BONALDO, A. L. L. (1); WATANABE, M. A. E. (1); DE OLIVEIRA K. B.

(1).

1. State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil.


Introduction: Human papillomavirus (HPV), specifically high-risk HPV, is strongly associated with

cervical intraepithelial lesions and cancer. Interleukin-10 (IL-10) influences HPV infection and viral

persistence through viral clearance inhibition and favoring HPV immune escape. CD4+ CD25+

regulatory T cell (Treg) is a great IL-10 producer. Hence, Treg has an important immunosuppressive

activity. Forkhead box P3 (FOXP3) transcription factor regulates CD4+ CD25+ Tregs development

and function. Single nucleotide variants (SNVs) in FOXP3 regulatory regions may alter Treg activity

by regulating FOXP3 gene transcription. Thus, we aim to assess the influence of FOXP3 SNVs

rs3761548 and rs2232365 in intron-1 region on IL-10 plasmatic and cervical levels in an HPV

infection-context.

Methods: The Institutional Ethics Committee Involving Humans of the State University of Londrina

(Londrina, PR, Brazil) has evaluated and approved the project (CEP/UEL 133/2012; CAAE

05505912.0.0000.5231). Our study included 308 women recruited from several health services in

Londrina (Paraná, Southern Brazil), between March 2015 and December 2016. All study subjects

received clear instructions about the project and signed a formal consent, prior to sample collection

(cervical secretion and peripheral blood). Cervical epithelial scrapings were obtained to determine

HPV DNA presence by polymerase chain reaction (PCR). Peripheral blood samples were obtained to

determine FOXP3 SNVs by PCR followed by restriction fragment length polymorphism (RFLP),

while IL-10 levels were assessed by enzyme-linked immunosorbent assay (ELISA). We analyzed

interactions between the IL-10 levels and FOXP3 inheritance models, considering HPV infection, by

Two-Way Analysis of Variance (ANOVA) and Tukey’s post-hoc test. All tests were two-tailed with a

significance level set at 0.05.

Results and Discussion: Women were divided in HPV-infected (n=146) and uninfected (n=162)

groups. IL-10 plasmatic levels were obtained from all women. From these, IL-10 cervical levels were

verified in 30 HPV-infected and 70 uninfected women. Considering the within-subjects analysis of

FOXP3 genotypes and inheritance models, we were not able to detect any significant differences both

in plasmatic and cervical IL-10 levels of HPV-infected and uninfected women. It is important to

highlight that we found IL-10 cervical levels were higher among HPV-infected women than

uninfected (P=0.026). Previously, Maynard et al. showed a dual reporter mouse system of the genes

encoding IL-10 and Foxp3 to track Treg subsets based on coordinate or differential expression of

these genes. They found that a subset of Foxp3-expressing Treg cells might secrete IL-10. It leads us

to suggests that although IL-10 is a putative binding site to FOXP3 transcription factor, IL-10

transcription and translation may occur in a FOXP3-independent manner. This inference would

explain why the FOXP3 SNVs rs3761548 and rs2232365 (whose alleles C and G, respectively, are

known to increase FOXP3 gene expression) do not interfere with IL-10 production.


Conclusion: We infer the IL-10 production is independent of FOXP3 action. Therefore, further

studies are required to better clarify whether FOXP3 transcription factor and IL-10 gene present a

physical interaction and may somehow influence on IL-10 production in different disease contexts.

Acknowledgement: The authors thank the volunteers who participated in the study, the

Intermunicipal Consortium of Health of the Middle Paranapanema, the University Hospital and Clinic

Center of State University of Londrina, the Municipal Health Department of Londrina, Londrina –

PR, Brazil, and Londrina State University Coordination for Postgraduation. This work was supported

by Conselho Nacional de Desenvolvimento científico e Tecnológico (CNPq), Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES), State University of Londrina Coordination

for Post-Graduation (PROPPG-UEL) and Fundação Araucária do Paraná – Programa de Pesquisa para

o SUS.


In vitro evaluation of glucose influence in Plasmodium falciparum growth

CIESLINSKI, J. Z. (1); BITTENCOURT, N. (2); ALBRECHT, L. (1)

1. Laboratory of Apicomplexan Research, Carlos Chagas Institute – FIOCRUZ – PARANÁ,


2. Departamento of Genetics, Evolution, Microbiology and Immunology, Campinas State

University (UNICAMP), Campinas, SP, Brazil.


Introduction: Malaria and diabetes are major public health challenges. Some relationship between

these two conditions has been evaluated 1, but there are still few studies concerning this subject.

Malaria clinical symptoms occur when parasites invade and multiply inside the human red blood cells

(RBCs). During this stage the spontaneous binding of infected erythrocytes to uninfected erythrocytes

may occur forming a rosette. Rosetting is a well-recognized parasite virulence factor which has been

linked to the development of severe disease2. Hyperglycemia also had already associated with severe

and cerebral malaria3,4. In this study, we aimed to evaluate the impact of different concentrations of

glucose in the Plasmodium falciparum growth and rosetting formation.

Methods: P. falciparum of FCR3S1.2 was cultivated according to standard methods5. All parasite

cultures were kept under low CO2 conditions at 37º C, with human A+ erythrocytes at 5% hematocrit

and culture medium RMPI 1640 with glucose and supplemented with 10 % heat-inactivated A+

serum. For the assay, RMPI 1640 without glucose was used to prepare medium with different

concentrations of glucose (5 and 30 mM). Rosette frequency was determined by counting the number

of bound erythrocytes in 100 parasites which was done by staining the parasite culture with acridine

orange and visualized under fluorescence microscopic. Parasitemia was also determined by counting

500 RBCs by Panotic staining.

Results and Discussion: The results showed a slow parasite growth in low glucose concentration

(5mM) and a marked decreased of number rosettes in the second cycle of multiplication in all medium

tested. In the control medium, where the parasite usually was maintained, we observed expected

growth and morphology in all blood stages (4,7% rings and 4,0% trophozoites and schizonts), while

in 5mM glucose was observed deformed rings and just 2,0% of trophozoites. Regarding 30 mM of

glucose, more assays are necessary. Early clinical studies have already shown a relation between

glucose concentration and severity of malaria. This preview assay showed that is possible to evaluated

in in vitro essays some important parameters that indicate a more serious disease, like growth and

formation of rosettes. More assays are needed for understanding how the glucose concentration can

affect the plasmodium growth and its virulence.

Conclusion: The parasite growth was harmed in culture medium with low concentration of glucose.


Peroxidase Activity of Azolla sp. and Lemna aequinoctialis in effluent contaminated with

nitroaromatic compounds

COELHO, B. D. (1); ZANONA, G. H. F. (1); GILONI-LIMA, P.C. (1); LIMA, V. A. (2)

1. Universidade Estadual do Centro-Oeste Unicentro.

2. Universidade Tecnológica Federal do Paraná UTFPR, Campus Pato Branco-PR.


Introduction: Reactive Oxygen Species (ROS) are common in plant cells, as much as a product of

anaerobic metabolism during photosynthesis. This process can be intensified under the effect of

abiotic stress with metals, xenobiotics, etc. The industrial production of explosives generates toxic

effluents whose disposal does not yet have restrictive limits in Brazilian legislation. Thus, the

objectives of the bioassays, in this work were to evaluate the activity of total peroxidases in Azolla sp

and Lemna aequinoctialis Welw. submitted to different concentrations of explosive industry effluent

contaminated with nitroaromatic compounds.

Methods: The species Azolla sp. cultivated in the Ecotoxicology laboratory of Unicentro was

acclimatized in culture medium without nitrogen and submitted to concentrations of 0.0; 0.013; 0.026;

0.050; 0.070 and 0.10% of the effluent contaminated with compounds of the nitroaromatic class. L.

aequinoctialis was grown in L. C. Oligo medium and submitted to concentrations of 0.0; 0.05; 0.10;

0.15; 0.20 and 0.25% of the same effluent. Both bioassays were carried out for 7 days and were

maintained at pH 7.0, with photoperiod 12:12h (light/dark) at 3000 Lux. The total protein content was

estimated by the Bradford method and the total peroxidases activity (POX - E.C 1.11.1.7) was

analyzed by spectrophotometry. The data were used to calculate the lowest concentration of observed

effect (CEO), of effect not observed (CENO) and Toxic Units (UT). The data of the POX activity

were analyzed by Generalized Linear Models (MLG).

Results and Discussion: The POX activity after 7 days of exposure presented a significant increase in

relation to the control at 0.026% (p <0.05). The adjusted dose-response model of the POX activity

was of the Growth/Sigmodal category for the bioassay with Azolla specie sp. The dose-response

relationship (R2 97.98%) showed that the observed effect concentration (CENO) was 0.012% ± 0.02

(UT 8,431.70) and observed effect concentration (CEO): 0.06 ± 0.05 (UT 1,573.81) and the

concentration causing damage to effectively 50% of organisms EC50: 0.03 ± 0.02 (UT 3,642.99). The

concentrations tested in the L. aequinoctialis bioassays were higher and there was a significant

difference in POX activity at concentrations of 0.15 to 0.25%, when compared to the control (p<0.05).

The dose-response relationship was increasing (R2 97.5%), with linear adjustment (POX (uM min-1

mg ptn-1) = 0.9326 + 0.322x) for this specie. The cytotoxic, genotoxic and mutagenic nature of the

effluent has been reported in other host species in organisms of different evolutionary levels, such as

algae, plants and animals. Therefore, it is important to establish restrictive levels for the disposal of

effluent from compounds of the nitroaromatic class, which is currently non-existent in Brazilian

legislation.

Conclusion: There are differences in response pattern between the two receptor species when

submitted to the effluent with nitroaromatic compounds. This can be observed both by the

concentration range tested and by the observed response, by increasing the activity of total

peroxidases in Azolla sp and L. aequinoctialis. The host species Azolla sp was shown to be more

sensitive than L. aequinoctialis. The results reinforce, therefore, the need for changes in Brazilian


legislation regarding the untreated disposal of effluent contaminated with nitroaromatic compounds

that do not affect the plant communities in Brazilian water systems.


Evaluation of the fish oil effect on parameters of obesity and activity of Wistar rats’s

macrophages submitted to metabolic imprinting by small litter

COMOTTI OLIVEIRA, B. (1); FISCHER, S. V. (2); MIYASATO, J. P. (2); RUGINSK, B. (2);

CARVALHAL, S. R. S. (2); LIMA, M. C. A. M. (1); GARCIA, C. M. (2); COUTINHO, D. S. S. (2);

IAGHER, F. (2); APPEL, M. H. (3); FERNANDES, L. C. (2); NALIWAIKO, K. (1)

1. Universidade Federal do Paraná, Departamento de Biologia Celular e Molecular (Paraná,

Brazil).

2. Universidade Federal do Paraná, Departamento de Fisiologia (Paraná, Brazil).

3. Universidade Estadual de Ponta Grossa, Departamento de Biologia Estrutural, Molecular e

Genética (Paraná, Brazil).


Introduction: Obesity is a worldwide public health problem characterized by an intense increase in

adipose tissue due to imbalance of the energy balance. In addition to the increase in fat deposits,

obesity is accompanied by a systemic inflammation, characterizing this tissue as the main site where

beggin inflammation associated with obesity. This inflammation is initiated by the recruitment and

activation of cells of the immune system, primarily macrophages, which secrete proinflammatory

cytokines. In contrast, fish oil is a compound rich in ω-3 PUFAs that has a potential anti-

inflammatory action and may minimize the inflammation.

Methods: Twenty litters were standardized to 10 puppies/motlher. At day 3, litters were adjusted:

Eight litters were adjusted to Normal (NL: normal litter, 10 pups/mother) and twelve litters were

reduced to Small (SL: small litter, 3 pups/mother). After weaning, at day 21 animals were subdivided

into normal (NL,SL) and supplemented groups (NLS,SLS) by providing the latter with 1g/kg/day of

fish oil (180 mg EPA and 120 mg DHA/g). The body mass was measured at 7, 14 and 21 days and

throughout the growth up to 60 days. Food consumption (22-60 days) was measured based on the

subtraction between the feed offered and its surplus. To characterize and evaluate obesity, the Lee

index was calculated at 60 days when the animals were euthanized, the mass of the visceral adipose

(retroperitoneal, mesenteric and perigonadal) tissues. For the evaluation of macrophage functionality,

peritoneal macrophages were isolated for the adhesion, phagocytosis, lysosomal retention and reactive

species production assays.

Results and Discussion: The results suggest that obesity was induced by the reduction of litter during

the lactational period, probably causing an eating disorder in SL animals (SL versus NL) , which led

to higher food consumption that directly reflected the increase in body mass, visceral fat in animals

and a higher Lee index, but without altering the fasting serum parameters. The macrophage

functionality indicated decreased phagocytic activity, increased lysosomal retention, reduction of

superoxide anion and nitric oxide production, and increased production of hydrogen peroxide in SL

animals (SL versus NL), indicating that the functionality of these macrophages was affected by the

onset of obesity and probably due to the greater accumulation of visceral adipose tissue designating

the inflammation. However, supplementation with fish oil started soon after weaning in SLS animals

(SLS versus SL) was effective in decreasing the mass of the retroperitoneal and perigonadal visceral

fats, and in the macrophages showed an improvement in the phagocytosis capacity and an increase in

hydrogen peroxide production, even though there was a decrease in the adhesion capacity, lysosomal

retention and lower production of nitric oxide compared to SL animals.


Conclusion: The obesity model was able to establish obesity in the animals and to cause changes in

the activities of peritoneal macrophages. Fish oil supplementation modulates and improves obesity

parameters by reverting to a lower accumulation of visceral fats and leading to improvement and

protection against obesity and inflammation.

Acknowledgement: CAPES e CNPq.


Morphometric effects of ladder climbing resistance exercise on extensor digitorum

longus muscle of Wistar rats in rheumatoid arthritis model

COSTA, L.N.C. (1); CAMILO, I.R. (2); RETAMEIRO, A.C.B. (1); NEVES, M. (1); STEIN, T. (3);

BERTOLINI, G.R.F. (3); COSTA, R.M.(3); RIBEIRO, L.F.C. (3)

1. Western Paraná State University Center for Biological and Health Sciences / Cascavel/

Master's students of the Master's Program in Biosciences and Health.

2. Western Paraná State University / Center for Biological and Health Sciences / Cascavel/

physiotherapy student.

3. Western Paraná State University / Center for Biological and Health Sciences / Cascavel/

Professors of the Master's Program in Biosciences and Health.


Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory and autoimmune disease which,

besides progressively destroying the structural articular components, affects other tissues, such as

skeletal muscle, on account of its systemic effect. Muscular disorders caused by (RA) imply serious

limitations in the mobility of individuals in productive age, resulting in professional incapacitation,

and impacting the quality of their lives negatively, which also impacts the world economy, given the

early retirements. Physical exercise is a therapeutic alternative, which acts as an attenuator of

inflammatory symptoms, promoting improvement in the functional picture and facilitates disease

control. The premise that this injury afflicts a large portion of the world population, and the

disagreement in treatment protocols justify the present study. Objective: To evaluate the effect of

ladder climbing exercise on the extensor digitorum longus muscle (EDL) of Wistar rats induced to RA

by Freund Complete Adjuvant (CFA).

Methods: Thirty-two male Wistar rats were randomized into four groups (n=8): control (CG), control

+ exercise (CEG), lesion group (LG), and lesion + exercise group (LEG). The animals of the (LG)

were induced to the RA through the process of immunization at the base of the tail and, after seven

days; intra-articular CFA was administered to the right knee. The (CG) animals were given saline

solution to submit them to the same physical stress. For the treatment, the animals of the groups

(CEG) and (LEG) were submitted to 14 days of ladder climbing exercise, with progressive series,

having a weight of 100g adapted to their tail. After the experimental period, the animals were

euthanized by guillotine, the EDL collected, fixed, and processed for morphometric analysis. For this

purpose, the slides stained with Hematoxylin and Eosin were photomicrographs, and 10 fields

increased by 40X per animal, being analyzed by Image-Pro-Plus 6.0 software, totaling 100 fibers per

animal. Quantitative data were analyzed by the Bioestat 5.0 program, tested by ANOVA and Tukey's

test.

Results and Discussion: In relation to the cross-sectional area, when the groups were compared, it

was observed that between (CG) and (GL) there was a significant difference (p<0.01), indicating that

the RA promoted changes in the muscle fibers of the EDL. Between the groups (LG) and (LEG), there

was also a significant difference (p<0.01), indicating that physical exercise promoted variations in

muscle fibers of the exercised group. When the control groups (CG) and (CEG) were compared to the

group (LEG), the result did not imply a significant difference (p>0.05), suggesting that the treatment

of resistance exercise in the injured EDL muscle promoted repair, since its cross-sectional area

approached the control groups.


Conclusion: In this study, RA was found to promote changes in the cross-sectional area of the EDL

muscle. Also, it was observed that the resistance exercise of ladder climbing in Wistar rats contributed

to the repair of the EDL muscle, which, in the long term, may reflect improvements in functionality

and quality of life of individuals with this injury.

Acknowledgement: To the Araucária Foundation, for the financial assistance through the call

016/2016/UNIOESTE/ basic and applied research.


In vivo antitumor activity of low molecular weight heparin in murine melanoma model

CRISTAL, A. M. (1); ARAUJO, C. B. (1); BELLAN, D. L. (1); BISCAIA, S. M. P. (1); ROSSI, G.

R. (1); TRINDADE, E. S. (1)

1. Cell Biology Department / Universidade Federal do Paraná, Curitiba, Paraná, Brazil.


Introduction: Cancer is the second leading causes of global morbidity and mortality. In 2015, this

disease caused more than 8.8 million deaths worldwide. Melanoma is the most aggressive type of skin

cancer because of its high capacity to metastasize. For 2018 more than 287.723 new cases of

melanoma worldwide are expected, with 6260 in Brazil alone. The main risk factors for melanoma

development are exposure to ultraviolet (UV) radiation, family history, immunosuppression, presence

of multiple nevus and fair-skinned color. At the metastatic stage of progression, melanoma has a low

responsiveness to existing treatments, with these generating many collateral effects and diminishing

the patient’s wellbeing. Given that is necessary the search for new therapeutic agents with less

adverse effects and that are able to increase the patient survival. Heparin is a potent anti-coagulant

utilized to treat cancer patients displaying-hypercoagulability which results in markedly increased

thromboembolism. Low molecular weight heparin (LMWH), derived from the partial hydration or

enzymatic action of heparin molecules, possess a reduced anticoagulant activity, and is widely used in

the prevention and treatment of thromboembolic complications in metastatic cancer patients. Studies

have suggested that LMWHs may improve survival in cancer patients with mechanisms that are

different from its antithrombotic effect but are linked to the ability of it influencing directly the tumor

biology. Given previous promising in vitro results using LMWH in the B16-F10 melanoma murine

cell line in our research group, the aim of this work was to investigate if LMWH could result in a

antitumor activity in vivo.

Methods: The experimental metastasis model of lung colonization was performed inoculating B16-

F10 murine melanoma cells intravenously into C57BL/6 mice. After 5 days animals were injected

with LMWH intraperitonially with 1 (n=9); 5 mg/kg(n=9) or vehicle (PBS, n=8), daily for up to 16

days. In the last day of experiment, the animals were anesthetized and euthanized and the organs were

collected and weighed. After fixation, images from lungs dorsal and ventral views were acquired for

analyzes of colonized area using the ImageJ Fiji Software.

Results and Discussion: With respect to lungs weight, there was a reduction between the two

treatment groups with LMHW and the control of 57,96% and 60,94% for the doses of 1 and 5 mg/kg,

respectively. For the analysis of the images, both doses resulted in total area reduction of 33,03% and

28,96% for 1 and 5 mg/kg, with a significant statistical reduction obtained in the analysis of dorsal

colonization area with higher dose. Lungs weight and colonization area showed significant statistical

reduction between treatment groups and control group. This may suggest that LMHW interferes in

colonization process in vivo, reducing some of the steps associated with the metastatic process.

Conclusion: These results suggest a promising in vivo effect using LMWH as therapeutic treatment

approach. This may be due to process of colonization reduction as seen in previous studies. Lungs

histological analyzes are being performed to detect possible alterations in melanoma markers.

Acknowledgement: We thank CAPES for finance this project. We also thank for ICC/Fiocruz and

UFPR animal house that provided mice and where were maintained.


Determination of gum arabic-gold nanoparticle biological effects in B16-F10 3D model

CRUZ, A. F. (1); GONÇALVES, J. P. (1); ROSSI, G. R. (1); VIDOTTI, I. C. R. (2); TRINDADE, E.

S. (1); OLIVEIRA, C. C. (1)

1. Universidade Federal do Paraná (UFPR), Cell Biology Department, Laboratório de Células

Inflamatórias e Neoplásicas, Curitiba-PR, Brazil;

2. UFPR, Chemistry Department, Grupo de Pesquisa em Macromoléculas e Interfaces, Curitiba-

PR, Brazil.


Introduction: Three dimension (3D) in vitro models are increasingly being used for cell biology,

especially in cancer studies. Due to its differential cell interaction characteristics, molecular

expression patterns and tissue morphology, this technique is able to provide results more

representative of the original tissue. Thus the investigation of different compounds with potential

biological activity, as gum arabic gold nanoparticles (GA-AuNS), in such model is favored. GA-

AuNS are biocompatible structures in nanometric scale, chemically synthesized, and affordable to

produce. Previous results showed that these nanoparticles can alter melanoma cells (B16-F10)

malignancy behavior in two dimension (2D) in vitro tests. Considering that, this work aims to

standardize a method for culturing B16-F10 cells in 3D (spheroids) and analyze GA-AuNS effects on

it.

Methods: 20 different B16-F10 cells densities were plated on a 96 well plate with agarose coating

and incubated in DMEM with 10% fetal bovine serum at 37 ºC, 5% CO2, for 10 days, in order to

determine the best cell density. Spheroids were exposed to two different GA-AuNS (0.244 and 2.44

mg/L) concentrations for 7 days. After, spheroids were fixed in paraformaldehyde, imaged to

determine its size, and incubated with Phalloidin Alexa Fluor 647 and propidium iodide for three

dimensional spheroids analysis, via confocal microscopy.

Results and Discussion: We were able to determine that 16x10³ cells per well density produces

spheroids with regular spherical shape and size and a darkened central region demonstrating many

cells, which provides a good analysis point using cryosection. Treatment with GA-AuNS did not

change spheroids area, which was in average 430 µm² of circumference. 0.244 and 2.44 mg/L GA-

AuNS in 2D melanoma model also did not change B16-F10 cell growth, but changed malignancy

features such as invasion ability and clonogenic capacity. Here it was observed that treatment

increased spheroid stability (apparently more compact and adhered cells) compared to control

spheroids, thus it will be further investigated. Confocal microscopy analysis confirmed globular

spheroid shape, showing that central cells are more compact compared to peripheral ones.

Conclusion: B16-F10 melanoma cells at 16x10³ cells density per well on non adherent surfaces

produce regular globular spheroids, with defined three dimensional structure. GA-AuNS tested

concentrations does not change spheroids size, but possibly affect cells adhesion while change cell

compaction when treated with the nanoparticle.

Acknowledgement: CAPES, Fundação Araucária, CME-UFPR, CTAF-UFPR.


Comparison of biochemical and biological properties of recombinant phospholipases D

with site-directed mutations from venom of spiders Loxosceles gaucho and Loxosceles

laeta

DA SILVA, T.P. (1); DE CASTRO, F.J. (1); VUITIKA, L. (1); POLLI, N.L.C. (1); ANTUNES, B.C.

(1,2); MINOZZO, J.C. (2); GREMSKI, L.H. (1); VEIGA, S.S. (1)

1. Cellular Biology Department, Federal University of Paraná, Brazil.

2. Center for Production and Research of Immunobiologicals, Brazil.


Introduction: The spider’s venom of Loxosceles sp. consists in a mixture of different proteins with

toxic effects. The phospholipases D (PLD) present in the loxoscelical venom are toxins of great

medical importance, since they induce local dermonecrosis, hemolysis and systemic damage that can

lead to acute renal failure. The aim of the study is to evaluate the role of specific aminoacids residues

in the biologic activities induced by the toxin making mutations in residues involved in catalytic

activity (H12A-H47A), modulation of the Mg + ion (E32A-D34A) and the putative site of recognition

with the substrate (Y228A).

Methods: In the present study the recombinant isoforms of PLDs from Loxosceles laeta

(AY093599.1) and Loxosceles gaucho (JX866729.1) were subcloned in pET-14b vector and the

mutations were made by QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies)

using primers designed with Agilent’s primers design tool. The constructs were sequenced for

confirmation of the mutations by a sequencing reaction (BigDye Terminator v 3.1 Cycle Sequencing

Kit, Applied Biosystems) using the DNA 3500 Genetic Analyzer automatic sequencer (Applied

Biosystems). PLDs with the H12A-H47A (LlRecDT1H12A-H474 and LgRecDT1H12A-H47A),

E32A-D34A (LgRecDT1E32A-D34A) and Y228A (LlRecDT1Y228A and LgRecDT1Y228A)

mutations were expressed in the prokaryotic system E. coli BL21(DE3)pLysS and purified by affinity

chromatography. The immunological cross-reactivity between the PLDs and rabbit sera raised with

Loxosceles laeta and Loxosceles gaucho venoms were evaluated by Western Blot. The phospholipase

activity was estimated by high performance thin layer chromatography and sphyngomielinase assay

by the Amplex Red fluorescent method. As for the biological activities, dermonecrosis in rabbits and

hemolysis (in vitro) were performed. The wild isoforms LlRecDT1 and LgRecDT1 were used as

positive control in all experiments. The project was aproved by the Ethics Committee – CEUA (n.

1112).

Results and Discussion: PLDs with site-directed mutations were successfully expressed and purified.

All proteins were recognized by sera raised against the whole venom of the same species in Western

Blot. The assays for evaluation of catalytic activity revealed that the mutants LlRecDT1H12A-H474,

LgRecDT1H12A-H47A and LgRecDT1E32A-D34A lost great part of the affinity for the substract

used, the sphingomielin. On the other hand, LlRecDT1Y228A and LgRecDT1Y228A were not able to

dimisih the catalysis as the other mutants. So far, the biological characterization of the PLDs activities

has been investigaded by dermonecrotic activity and this revealed that LlRecDT1H12A-H474,

LgRecDT1H12A-H47A and LgRecDT1E32A-D34A suppressed the necrotic lesion, although the

LlRecDT1Y228A and LgRecDT1Y228A still maintained a residual activity, corroborating with the

previous biochemical tests.These results diverge from the previous data obtained by the research

group with the same mutations done in PLDs of Loxosceles intermedia, in which the mutants H12A-


H47A, E32A-D34A and Y228A reduced all the biochemical and biological effects of phospholipase

D. The results achieved for L. laeta and L. gaucho indicate that there may be some compensatory

effect on the proteins with Y228A mutations by the presence of other residues with aromatic rings in

the surroundings, changes in thermodynamic parameters or energy binding of these proteins.

Conclusion: The site-directed mutations by substitution in residues His12-His47 and Glu32-Asp34

for alanin decreased the activities of PLDs of L. laeta and L. gaucho. Since LlRecDT1Y228A and

LgRecDT1Y228A maintained part of its activities, the next goal is to keep this mutation and produce

two additional mutations in the residues Tyr229 and Trp230, in PLDs of both species, to evaluate if

these residues are somehow involved in the catalysis. Hereafter, we intend to use these and other

mutated PLDs of Loxosceles intermedia to produce a novel anti-loxoscelic serum used to treat victims

of accidents with Loxosceles sp. spiders.

Acknowledgement: The project is supported by UFPR, CAPES, Fundação Araucária and Center for

Production and Research of Immunobiologicals (CPPI).


Analysis of histone post-translational modification in Trypanosoma cruzi under

nutritional stress

DE ALMEIDA, R. F. (1); DE GODOY, L. M. F. (1)

1. Carlos Chagas Institute - Fiocruz


Introduction: In eukaryotes, the addition and removal of histone post-translational modifications

(hPTMs) play an essential role in the remodeling of chromatin and the recruitment of proteins

involved in gene regulation. Large-scale studies have shown that hPTMs are abundant and varied in

Trypanosoma cruzi, etiologic agent of Chagas' disease, suggesting an important epigenetic control.

During the differentiation to the infective form of this parasite, one of the triggers is the nutritional

stress, we investigated the modulation of hPTMs sites in this context.

Methods: We applied mass spectrometry-based proteomics (LC-MS/MS) to evaluate hPTMs

modulation of T. cruzi during nutritional stress.

Results and Discussion: Our proteomic analysis identified more than a hundred new hPTM sites in

T. cruzi histones. The identified modulated sites are being better analyzed by bioinformatics and their

in vitro interaction partners will be investigated later.

Conclusion: The results presented here provide new evidence for a better understanding of epigenetic

mechanisms in T. cruzi and, ultimately, serves as the basis for a selection of candidates for

development of antiparasitic epigenetic drugs.

Acknowledgement: We thank the technical support of mass spectrometry facility team and Carlos

Chagas Institute


Evaluation of the internalization pathway and intracellular traffic of the disintegrin and

metalloprotease ADAM23

DE SOUZA, I.L.M. (1); OLIVEIRA, N.H. (1); HUAMANI, P.A.M. (1); ZANATA, S.M. (1)

1. Departamento de Patologia Básica, UFPR, PR, Brazil.


Introduction: ADAM23 is a transmembrane protein (A Disintegrin and Metalloprotease), expressed

mostly in the central nervous system during development. It is known that ADAM23 plays an

important role in development and maintenance of the nervous system. However, little is known about

its internalization mechanism, endosomal sorting to degradation in lysosomes or recycling back to the

plasma membrane through recycling endosomes. Objective: Evaluate the internalization kinetics and

intracellular recycling traffic of ADAM23.

Methods: Immunofluoresce I: N2A cells were seeded at glass coverslips (5 x103 cells) and cultured

for 48 h. For membrane staining, cells were incubated with Alexa Fluor 594 wheat germ agglutinin

(WGA) in PBS (1:300) and incubated for 1 h at 4 °C. Cells were fixed with 2% PFA in PBS (20 min

25°C) and then blocked with 0.1 M glycine. Non-specific sites were blocked with 2% BSA (1 h).

Cells were incubated with moAb DL11C8 (anti-ADAM23) for 16 h at 4 °C, then incubated with anti-

mouse IgG-Alexa 488 (1:800) for 1 h at room temperature. For actin filaments staining, cells were

permeabilized with 0.1% Triton X-100 in PBS for 20 min, then incubated with Actin-Red for 30 min.

Nuclei were stained with DAPI in PBS (1 mg/ml). Images were acquired with a Nikon A1R MP+

laser confocal microscope. Immunofluorescence II: Transfected N2A cells with pCMV6-ADAM23-

FLAG (Origene) vector were labeled with anti-ADAM23 (DL11C8) for 2h at 4°C and incubated at

37°C for: 15, 30, 45 and 60 min. then fixed, permeabilized, and incubated with anti-Rabs antibodies

(anti-Rab4 (1:50) or anti-Rab11 (1:25)). Anti-adam23 was labeled with secondary anti-mouse 488

(1:400) and anti-Rabs were labeled with secondary anti-rabbit 568 (1:400). Flow cytometry: N2A

cells (1 x 106 cells per condition) were fixed with 2% PFA and incubated with moAb DL11C8 (anti-

ADAM23) or appropriated parallel controls for 14 h at 4 °C. After several PBS washes, cells were

incubated with anti-mouse FITC-conjugated antibody (1:500, Sigma) for 30 min at room temperature,

washed again and analyzed by flow cytometry with a FACSCalibur.

Results & Discussion: ADAM23 appears to be internalized at 15 minutes, recycled back to plasma

membrane at 30 and 60 minutes, indicating that an internalization-recycling cycle occurs in murine

neuroblastomas N2A. ADAM23 co-localizes with fast recycling endosome Rab4, indicating that this

protein is rapidly recycled to plasma membrane until 30 min after beginning of internalization.

ADAM23 also co-localizes with late recycling endosome Rab11, being also recycled back to plasma

membrane within 60 min. The traffic of this protein remains to be further investigated. These findings

are comparable to the ones regarding ADAM12, where internalization seems to occur only for the

mature form of this protein and its recycling process seems to occur at 15 minutes in HEK293VnR,

and ADAM12 co-localizes with Rab4 and Rab11 (STAUTZ et al., 2012).

Conclusions: Regarding endocytosis kinetics, ADAM23 appears to be internalized at 15 minutes.

ADAM23 co-localizes with fast and slow recycling endosomes (Rab4 and Rab11, respectively)

indicating that this protein is rapidly recycled to plasma membrane until 30 min and slowly recycled

back to plasma membrane within 60 min. after the endocytosis initiation. It remains to be elucidated

whether this protein follows to Rab7 and routes to the lysosome to be degraded.


Acknowledgement: CNPq, CAPES


Functional analysis of divergent components for the mRNA export in trypanosomatids

DOMINGUES, P. F. (1); INOUE, A. H. (1); HIRAIWA, P. M. (1); VIDAL, N. M. (2);

GOLDENBERG, S. (1); DE SOUZA, T. A. C. B. (1); FIELD, M. C. (3); ÁVILA, A. R. (1)

1. Instituto Carlos Chagas, ICC, FIOCRUZ, Curitiba, PR, Brazil.

2. National Center for Biotechnology Information, NCBI, National Library of Medicine,

National Institutes of Health, Bethesda, MD, United States of America.

3. Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of

Dundee, Dow Street, Dundee, DD1 5EH, UK.


Introduction: The nucleocytoplasmic RNA export is an essential pathway for gene expression

regulation in eukaryotic cells but it is still poorly understood in protozoan parasites. Orthologs of only

few proteins involved in mRNA export in higher eukaryotes are detectable. We previously described

two conserved DEAD-box RNA helicases, components of the mRNA export pathway in T. cruzi:

TcSub2, an essential for parasite survival and export of mRNA, and Hel45, a shuttling protein and

component of mRNA complexes. Analyses of the interactomes of TcSub2 and Hel45 uncovered

additional components of this system, including lineage-specific proteins of trypanosomatids, named

as TcFOP-like, TcAPI5-like and TcNTF2-like.

Methods: Protein localization of those specific proteins was perfomed by optical and ultrastructural

microscopy assays. The functional analysis has been performed by RNAi in T. brucei to observe the

phenotypic effects. Immunoprecipitation assays followed by mass spectrometry will help on the

identification of additional components involved in mRNA export. Also, crystallography approaches

will be performed in order to obtain the structure of those specific proteins and to provide insights into

mechanisms involved in the mRNA export.

Results and Discussion: We confirmed that TcFOP-like and TcAPI5-like are nuclear proteins and

colocalize with TcSub2, whereas TcNTF2-like is a cytoplasmic protein that co-localize with Hel45

and may migrates between nucleus and cytoplasm. We confirmed the interaction of TcFOP-like,

TcAPI5-like and TcNTF2-like with mRNA export components and immunoprecipitation assays have

been performed to identify new components by proteomic analysis. Knockdown of TcFOP-like,

TcAPI5-like and TcNTF2-like shows that those proteins are not essential for the mRNA export but we

still cannot exclude their role in this pathway. The knockdown of TcAPI5-like and TcFOP-like caused

ultrastructural alterations such as condensation of the chromatin in the perinuclear region,

mitochondrial disruption and multinucleated cell distribution.

Conclusion: The mRNA export machinery in trypanosomes contains conserved and divergent

proteins. Some proteins seem to be lineage-specific innovations and although they are not essential for

parasite survival, their knockdown cause significative morphological effects. We suggest that these

new components are likely part of the evolutionary adaptation to polycistronic transcription/trans-

splicing.

Acknowledgement: CAPES, CNPq, FUNDAÇÃO ARAUCÁRIA, FIOCRUZ


Didactic models in the teaching of the cellular morphology of Homo sapiens

FALLER, A.P.C. (1); LIMA, B. (2); QUADROS, A.A.G. (2); GUEDES, N.L.K.O. (2); BEU, C.C.L.

(2) ; RIBEIRO, L.F.C. (2); COSTA, R.M.(1)

1. Centro de Ciências Biológicas e da Saúde - CCBS/ Universidade Estadual do Oeste do Paraná

– UNIOESTE.

2. Centro de Ciências Médicas e Farmacêuticas – CCMF/ Universidade Estadual do Oeste do

Paraná – UNIOESTE.


Introduction: The human body consists of about thirty trillion cells, with differentiated

morphological and functional characteristics. In cytology teaching, cellular morphophysiology is

usually based on a standard cellular model, which does not offer the student the knowledge of the

actual cellular diversity. The present study aimed to develop didactic models for the teaching of the

cellular morphology of Homo sapiens.

Methods: The models were organized into two-dimensional figures of eukaryotic cells, constituents

of organs of the body, which were schematized based on microscopic morphological information,

covering structure and ultrastructure, obtained from scientific literature in the area.

Results and Discussion: The 16 models developed explore essential morphological aspects of the

size, shape and microscopic appearance of eukaryotic cells in the human body, which represent all

body tissues. Knowledge of cells based on their morphological differences has broad applications in

the sciences. For example, a wide variety of image analysis software packages have been developed to

help convert microscopic images into qualitative and quantitative measurements, a relevant tool in the

processing of cellular images and pattern recognition, fundamental in the identification and

classification of abnormalities, and early detection of cancer. It is also applied in the analysis of

dynamic changes under specific environmental stress, in the understanding of chemotactic response

and drug influences, and in the identification of cellular morphogenesis in different phases of the cell

cycle, - fundamental knowledge in the understanding of current scientific advances. The cells were

grouped according to their functionality in: germ cells, spermatozoa and oocytes; information

transmission and control cells, neurons; support cells, osteoblasts, osteocytes and fibroblasts; coating

cells, such as keratinocytes, absorptive cells and endothelial cells; secretory cells, goblet cells and

hepatocytes; contractile cells, muscle cells; transport cells, red blood cells; and energy reserve cells,

adipocytes. The cellular morphology, together with the knowledge of the functionality, was presented

considering the anatomical position and reinforces that the metabolic performance of the cells

responds to the organic activity. The cells of the models were also plotted in a scale diagram in

micrometers (μm) for a better understanding of their microscopic dimensions.

Conclusion: Thus, by using the human body in cellular exemplification, greater significance is

attached to the object of learning, enabling a progressive awareness of the importance of its formative

microscopic units. The pedagogical proposal presents, therefore, a differentiated, playful and

integrated view of eukaryotic cells, focused on basic education, in addition to the traditional cellular

structure models of animal eukaryotes.

Acknowledgement:A Fundação Araucária, pelo auxílio financeiro por meio do edital 016/2016-

UNIOESTE/ pesquisa básica e aplicada.


Comparative characterization of histone post-translational modifications of the tritryps

FERNANDES, M. (1); GODOY, L. M. F. (1)

1. Instituto Carlos Chagas.


Introduction: In eukaryotes, the epigenetic control of gene expression occurs essentially on the basis

of dynamic changes in the structure and organization of chromatin, among which the so called

"histone code", which includes variant histones and post-translational modifications (PTMs) of

histones is of great importance. Histone PTMs can alter the composition and conformation of the

nucleosome and regulate various aspects of DNA metabolism, such as replication, repair,

transcription, and chromatin condensation. Thus, epigenetic drugs have been of interest for decades

and are already used for the treatment of various diseases, such as cancer. The human parasitic

protozoa Trypanosoma cruzi, Trypanosoma brucei and a Leishmania braziliensis, known as TriTryps,

are responsible for responsible for Chagas disease, sleep disease and leishmaniasis, respectively.

Although their control of gene expression is primarily post-transcriptional, the TriTryps have histone

PTMs and other chromatin modifications. The global map of histone PTMs for T. cruzi was recently

described by our group. However, the number of known histones PTM sites for the other TriTryps is

still very small and very little is known about the functional role of these modifications in

trypanosomatids.

Methods: T. cruzi epimastigote cells were cultured in LIT medium,T. brucei procyclic cells were

cultured in SDM-79 medium and L. braziliensis amastigote cells were cultured in blood agar with

Schneider medium, all of these with 10% FBS at 28°C. Histone-enriched extracts were obtained

through an acid extraction protocol, which lyze the nucleus of the cell and solubilizes all basic nuclear

proteins, including histones. The samples were propionylated, a process that blocks the unmodified or

monomethylated lysines, so the peptides coming from the digestion are of the right size and charge for

analysis in LC-MS/MS. To maximize the coverage of histone sequences, two complimentary

proteomic approaches were used for sample preparation. In the first approach, histone-enriched

protein extracts were digested in solution with trypsin for 16h at 37°C. In the second approach, the

histone-enriched protein extracts were separated by SDS-page 15%, stained with Coomassie blue and

the histone bands were excised, destained and digested in-gel for 16h at 37°C. In both approaches,

peptide digests were desalted using RP-C18 StageTip columns and analyzed by LC-MS/MS.

Results and Discussion: Our data show that histone PTMs are abundant and diverse in both the

flexible tails and globular region of the histones for all the TriTryps. In total, 12 different types of

histone PTMs, 245 sites and 393 PTMs distributed across the TriTryps were found. Among these, we

identified 80 new PTMs for T. cruzi, 130 new PTMs for T. brucei. In addition, this study is the first to

describe PTMs for L. braziliensis, for which 106 PTMs were identified so far. Many of these sites are

conserved between the TriTryps and with other organisms, including humans. In addition, we found

that distinct patterns of PTMs exist in individual histone molecules, suggesting that the TriTryps are

able to regulate histone modifications in a combinatorial way to establish distinct chromatin states,

allowing a possible existence of a sophisticated histone code in trypanosomatids.

Conclusion: With our large-scale proteomic analysis, we have significantly expanded the map of

histone PTMs of T. cruzi and T. brucei and described the first histone PTM profile for L. braziliensis.

Furthermore, show evidences that epigenetic control via histone modifications has the potential to be


as complex and sophisticated in this parasite as it is in other eukaryotes, including humans. Thus, this

work can greatly contribute to improving the knowledge about epigenetic control in TriTryps, paving

the way for further functional studies that can help to understand key regulatory epigenetic

mechanisms on these organisms, at the molecular level.


Development of antibody fragments for selectivity assessment of EGF domain proteins

FERREIRA, A.S. (1,2); LOPACINSKI, A. (1,2); PICCHI, G. (2); GUIMARÃES, B.G. (2);

ZANCHIN, N.I.T (1,2)

1. Cellular and Molecular Biology Post-Graduate Program, UFPR, Curitiba, PR, Brazil.

2. Instituto Carlos Chagas – FIOCRUZ/PR, Curitiba, PR, Brazil.


Introduction: The Epidermal Growth Factor (EGF) family has important roles in cell signaling for

proliferation, differentiation, and survival. Dysregulation of these pathways is associated with

different tumor developments. All members of this family interact with EGFR through their EGF

domain. However, in order to quantify different ligands of this family, robust analytical tools are

necessary to differentiate them. In this sense, there are antibodies and their derivatives that can be

obtained from different organisms or synthetic libraries. Hence, the aim of this project is to develop

antibody fragments to evaluate the selectivity of each front to the members of the EGF family and

EGF-like domains using two main strategies. The first one is to develop two scFv, named MM and

OC, derived respectively from mouse (Mus musculus) and rabbit (Oryctolagus cuniculus) antibodies,

both sequenced by mass spectrometry. The second strategy is to develop sdAf selected from a custom

CDRH3 synthetic library using the yeast two-hybrid method. These fragments will be expressed in

different constructions in order to optimize their yield. The purified recombinant antibody fragments

will be evaluated for binding and specificity to the EGF growth factors. Methods: All EGF family

factors and NRG1𝑎 were cloned into pET32a and expressed using E. coli strain BL21-Star (DE3).

They were purified using affinity chromatography, stored at -20 ºC and their oligomerization analyzed

by size exclusion chromatography. The scFvs were subcloned into pET28a-TEV and pET32a. In

parallel, in silico analyses were performed to evaluate a possible correlation between structure and

yield and new versions of these scFv were generated. For that, all scFv crystal structures from the

Protein Database (PDB) describing soluble E. coli scFv without any special tag were included. The

structures were clustered using structural alignment and structural analysis of CDRL1 and CDRH3.

The custom synthetic library of sdAf was design using a constant framework, CDRH1 and CDRH2.

The place of the CDRH3 was replaced by ccdB with its promoter and terminator. Introduction of the

CDRH3 library can be achieved either by restriction enzyme digestion and ligation or by homologous

recombination.

Results and Discussion: All the EGF factors were obtained in the soluble fraction using an

expression protocol of growth at 37 ºC until optical density of 0.6 at 600 nm followed by IPTG

induction at 16 ºC for 24 h. They were all purified with purity and quantities compatible with the

future assays. AREG, EPR and HB-EGF precipitated after freezing, while EGF and NRG1𝑎 were

purified by exclusion size chromatography and found in monomer and dimer, respectively, even after

long time freezing. The scFv OC was found in the soluble fraction using a 37 ºC expression protocol.

The scFv MM was in inclusion bodies when expressed at 16 ºC, 25 ºC and 37 ºC in the strains BL21

(DE3) ΔSlyD pRARE2 and BL21-Star (DE3). These results are compatible with the structural

analysis where we found a very similar scFv that was expressed in the insoluble fraction.

Solubilization protocols for the inclusion bodies are being tried to obtain soluble MM scFv. The in

silico structural analysis clustered the scFvs into five groups, taking into account their sequence and

CDR length. Different constructions were designed using compatible scaffolds, where the framework


of the scFv from the PDB was used for the OC and MM CDRs insertions. Also, two extra

constructions were designed changing the Vernier zone residues to evaluate the antibody affinity.

Conclusion: The experimental strategy has allowed for the recombinant production of all EGF factors

and at least one scFV fragment. Affinity assays will be performed and the selectivity compared

between the antibody fragments against the different EGF factors.


Solubilization strategy of protein CD28

FERREIRA, S. (1); ZANATA, S. M. (1)

1. Universidade Federal do Paraná


Introduction: CD28 is a coestimulator involved in activation of immature T cells. Orabona et al

(2004) showed the capacity of these protein to also activate the antigen-presenting cells (APCs) which

showed high activity of phagocytosis and production of cytokines. Aiming the study of this signaling

in chickens, the extracellular domain of chicken CD28 protein was expressed in prokariotic system

and presented formation of insoluble aggregates. In many cases, the production of recombinant

proteins in prokariotic systems results in formation of insoluble aggregates. These are known as

inclusion bodies (IBs) and they can accumulate in periplasmic space and/or cytoplasm. The main

advantage of IBs is the high yield because the aggregates appear to form a protective barrier against

proteases. Here, we have shown five strategies to solubilization, of which, one of them was efficient

to keep the protein soluble.

Methods: BL21(DE3) were transformed with plasmid PET28a+CD28, grown and induced with 0.2

mM IPTG for 37 ᴼC 3 h and 220 rpm. Then, bacteria were harvested by centrifugation and suspended

in 50 mM NaH2PO4, 500 mM NaCl, 8 M urea, pH 8,8 1mM PMSF and NEM. Subsequently, cells

were lysed through a French press. Cells were centrifuged, the pellet was dissolved in the same buffer

added of 8 M urea. The protein was purified using the Ni-NTA agarose column under denaturing

conditions. The washes and elution were performed with imidazole. Solubilization of CD28:

Strategies 1 to 4 were performed altering temperature (1); increasing concentration of caothropic

agent (2 to 8M urea) in the washing of inclusion bodies (2); decreasing caothropic agent concentration

(4, 2 and 0 M urea) in the refolding of CD28 through Ni-NTA agarose (3); Renaturation of CD28 by

dilution and concentration by affinity chromatography (4). The strategy 5 involved the dialysis of

CD28 against arginine-buffer where purified samples were subjected to a successive dialysis against

buffer with decreasing chaotropic agent (8M urea) concentration using a methodology of Kakoulidou

et al (2007). In the last step samples were tested against PBS and mix buffer (20 mM Tris / HCl, pH

8.5, 1 mM EDTA, 50 mM glycine-NaOH, pH 10.8, 1 Mm GSH, 0.1 mM oxidized glutathione, 0.4 M

arginine-HCl) containing or not glutathione. We decided to leave protein in mix buffer with

glutathione.

Results and Discussion: The only strategy where the protein remained soluble was 5. Arginine is

extensively used for refolding of IBs and appears to be effective for a variety of proteins differing in

chemical and physical properties (Buchner, 1991). Arakawa et al (2004), suggests that arginine helps

to suppress protein aggregation but is not protein denaturant at 2M. The process of refolding passes

through intermediate structures (autor) ending in a stable structure. The immunity is closely linked to

poultry production, therefore the identification of costimulatory factors has important implications in

this field and is directly related to health and/or avian meat production. Therefore this method of

recombinant production could be implanted in the development of a new biotechnology product

aiming to improve animal health.

Conclusion: To successfully raise commercial poultry it is necessary consider animal health and

welfare and implement an approach that keeps the bird clean from harmful pathogens. The study of

CD28 is important to understand how the APCs could be stimulated and increase the immunity of


birds. The strategy of recombinant protein production and solubilization could be implemented in bird

breeding aiming to increase their immunity and protection against foodborne pathogens.

Acknowledgements: CAPES; CNPq; UFPR, Imunova. and Fundação Araucária.


TgHDAC4: a histone deacetylase unique to Apicomplexa

FRAGOSO, M. S. I. (1); SIQUEIRA, C. M. (1);, SEVERO V. R. (1); DUARTE, E. S. M. (2);

ÁVILA, A. R. (1); NARDELLI, S. C. (1)

1. Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brazil.

2. Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais.


Introduction: Toxoplasma gondii is an obligate intracellular parasite member of the phylum

Apicomplexa and is responsible for toxoplasmosis, a disease that affects a quarter of the world’s

population and which has no effective cure. In order to pass immune barriers, T. gondii differentiates

between different stages, which are tightly regulated by controlling gene expression. Epigenetic

regulation is one of the mechanisms controlling gene expression that seems to play an important role

in T. gondii. One category of enzymes that act as epigenetic regulators are histone deacetylases

(HDACs). T. gondii has seven histone deacetylases (HDACs and Sir2), which are classified into four

classes according to their domains, cofactors and sequence. We are particularly focused in

TgHDAC4, a histone deacetylase of class IV, a class of which the only well characterized member is

HDAC11, found only in metazoans.

Methods: In order to characterize TgHDAC4, we focused on construction of epitope tagged lines that

were used to perform immunofluorescence and immunoprecipitation assays. In addition, for knockout

of the protein of interest, we are currently attempting to replace promoter and the classic knockout.

Finally, TgHDAC4 has two predicted signals common in apicoplast proteins. To verify if these

signals are responsible for directing the protein to the apicoplast we add an YFP tag.

Results and Discussion: Database searches showed that the only region shared with other organisms

is a portion of the typical HDAC domain. The C-terminal of TgHDAC-4 was missanoted in

Toxoplasma database; therefore, the first aim was to obtain the complete gene sequence. By

sequencing the cDNA with the final portion of tghdac4, we confirmed the presence of additional 200

bp that allowed us to construct the endogenous tag line (TgHDAC4-HA). By indirect

immunofluorescence and co-localization assays, we verified that TgHDAC4 is located in the

apicoplast, also co-localizing with TgHU, a histone-like protein. In preliminary data of transmission

electron microscopy, we confirmed the presence of TgHDAC4 at the apicoplast. Western blot assays

identified a protein smaller than expected, of about 100 kDa, common in apicoplast proteins due to

cleavage signal and transit peptides. We added a YFP tagg on the predicted signals and preliminary

data showed a similar label to apicoplast. In addition, we performed an immunoprecipitation assay

followed by mass spectrometry and identify some apicoplast proteins which are possibly targets of

TgHDAC4.

Conclusion: Our data indicate for the first time the presence of a histone deacetylase in the T. gondii

apicoplast, which seems to be unique to Apicomplexa parasites. The apicoplast is an organelle

acquired from endosymbiotic origin, considered the “Achilles’ Heel" of the parasite, due to its

importance for parasite survival. Understanding how this protein works could provide new insights

into the metabolism of the apicoplast and in addition reveal a potential drug target for the effective

treatment of toxoplasmosis.


Development of a model of reconstructed human epidermis (RhE) for in vitro skin

irritation testing

GAGOSIAN, V.S.C. (1,2); SCHWARZER, A.C.A.P. (1,2); OYA-SILVA, L.F. (1); TRINDADE, E.

S. (1); LEME, D.M. (1); PESTANA, C.B. (2)

1. Department of Genetics, Federal University of Paraná (UFPR), Curitiba-PR/Brazil.

2. ALS Laboratórios, São Paulo-SP/Brazil.


Introduction: Over the last 20 years efforts have been made in order to refine, replace and reduce the

number of animals used in toxicological tests. In this context, in vitro methods have been proposed as

an alternative approach for assessing hazard of compounds, especially those applied topically to the

skin. One of the methods recognized by the OECD for the skin irritation test is the use of the

Reconstructed Human Epidermis (RhE). RhE provides a unique tool for testing dermally applied

chemicals in an in-vivo-like system, since this model reproduces key aspects of native epidermis. This

study reports the development of an in-house RhE model meant to be further used for in vitro skin

irritation tests for safety assessment of a variety of chemicals. Our RhE model was developed varying

the techniques described by other authors in order to improve the structure, quality, cost and use of

animal-free reagents.

Methods: RhE construction was tested using primary keratinocytes (KCs) from donors of different

age groups (neonatal or adult). KCs were plated on animal-derived or animal-free coating matrices

(collagen IV or coating matrix kit protein Gibco™, respectively) and initially cultured in submerged

condition for proliferation, followed by an air-liquid interface culture for differentiation and

stratification of RhE. The in-house RhE was characterized regarding the morphological characteristics

by standard histological staining (H&E) and immunohistochemical staining (keratin-10, keratin-14,

involucrin and fillagrin).

Results and Discussion: The animal-free coating matrix did not prove to be effective for in-house

RhE construction, since KC from adults (aKCs) and neonatal (nKCs) were not able to proliferate and

stratify adequately, as this matrix did not provide appropriate cover and did not keep the ideal

conditions for the air-liquid interface. Alternatively, collagen IV coating kept the ideal air-liquid

interface conditions, thus promoting the differentiation of aKCs and nKCs. The age of KC donors

appeared to influence proliferation, since RhE constructed with aKCs did not present the adequate

number of epidermis layers necessary for performing toxicity studies. In contrast, RhEs constructed

with nKCs presented adequate morphological and biochemical structure (≈ six layers of differentiable

viable cells and mature stratum corneum, 64.5 μm thickness), being a suitable model for skin irritation

tests according to parameters stipulated by OECD 439.

Conclusion: In conclusion, our in–house Rhe model constructed with nKCs and Collagen IV showed

morphological and biochemical similarities to human epidermis and therefore can be a promising

model for skin irritation tests.

Acknowledgement: Financial support: FAPESP No. 2016/06985-7 and 2017/12506-7.


Molecular evolution strategy for optimization of single-chain variable fragments (scFv)

binding to human osteopontin

GAITKOSKI, M. B. (1,2); ANDRADE, E. F. (3); ZANCHIN, N. I. T. (1,2,3)


1. Instituto Carlos Chagas, Fiocruz/PR, Brazil.

2. Programa de Pós-Graduação em Biociências e Biotecnologia, ICC-Fiocruz/PR, Brazil.

3. Programa de Pós-Graduação em Biologia Celular e Molecular, UFPR/PR, Brazil.

Introduction: Osteopontin (OPN) is a secreted protein mainly produced by osteoclasts, osteoblasts,

macrophages and lymphocytes. It has important roles in cell-adhesion, tissue growth and

angiogenesis. Osteopontin binds to integrins and CD44 on the cell surface and has been described as a

tumor promoting factor in many types of cancers, such as breast, melanoma, uterus and prostate.

Blockage of osteopontin binding to integrins by monoclonal antibodies was shown to restrain

metastasis and invasion in mice. Therefore, inhibition of osteopontin function by monoclonal

antibodies is considered a promising therapy option, where it would be capable of preventing tumor

progression. AIMINGS: In this work, we aim to improve the affinity of three scFv antibodies

designed to bind to osteopontin and to determine their efficiency to block osteopontin interaction with

its receptors.

Methods: Sequences of three scFv synthetic genes were amplified by error-prone PCR and cloned

into pET-GOF. Escherichia coli BL21-Star (DE3) chemocompetents cells were transformed with the

scFv variant clones, plated onto LB medium with 25 µg/mL kanamycin, 0,5 mM IPTG and the

positive colonies were screened for fluorescent phenotype. Selected colonies will be grown in liquid

LB medium and scFv expression induced by 0,5 mM IPTG. Expression levels will be determined by

fluorescence intensity measurement and the ones presenting higher intensities will be selected for

further scFv yield and affinity assays. Affinity and avidity of the selected scFvs will be assessed by

microscale thermophoresis. The ability of the scFvs to block osteopontin interaction with either CD44

or integrins will be tested by flow cytometry using human Molt-4 and HEK 293 cell lines.

Results and Discussion: The scFv mutated library has been successfully created based on error-prone

PCR and preliminary cloning experiments have demonstrated that E. coli BL21-Star (DE3) is capable

of receiving DNA from ligation reactions. The transformation efficiency, however, is much lower

when compared to strains developed for DNA cloning. Colonies carrying the correct construction did

not displayed the fluorescent phenotype, because most of the scFv+SGFP2 produced were present as

inclusions bodies, as observed by SDS-PAGE analysis. Although some amount of scFv+SGFP2 was

soluble, the green fluorescent phenotype could not be observed on plates after overnight incubation at

37°C.

Conclusion: In the coming weeks, new strategies aiming to improve E. coli BL21-Star (DE3)

transformation efficiency and protein solubility will be tested. At the end of this project, we hope to

obtain scFv variants with a higher affinity to osteopotin than the original clones to show that their

interaction with osteopontin inhibitis binding to their cellular receptors.

Acknowledgements: CAPES, CNPq, Fundação Araucária and FIOCRUZ.


Fasting C-peptide / Fasting blood glucose ratio: An auxiliary tool to better understand

type 1 diabetes?

GALLEGOS, B. (1); ANGHEBEM, M.I. (2); MARTINS, B.R. (1); SILVA, L.P. (1); SOUZA, S.W.

(1); PICHETH, G. (2); REGO, F.G.M. (2); ALBERTON, D. (2)

1. Post Graduate Program in Pharmaceutical Sciences, Federal University of Parana, PR, Brazil.

2. Department of Clinical Analysis, Federal University of Parana, PR, Brazil.


Introduction: Type 1 diabetes (T1D) is characterized by severe insulin deficiency resulting from the

autoimmune destruction of pancreatic β-cells. T1D accounts for 5 to 10% of the total of diabetes

worldwide and its total incidence is increasing at a rate of 3% per year. T1D diagnose and monitoring

is majorly based on glycemic parameters such as fasting blood glucose (FBG), oral glucose tolerance

test and glycated hemoglobin (HbA1c). The continued search for biomarkers is always encouraged,

although, the introduction of a new biomarker in laboratorial routine is often hindered by laborious

process of validation or costs of the new method. Given this, scientists have suggested a new

parameter of remaining β-cell function based on known diabetes markers, fasting c-peptide to FBG

ratio. Therefore, our study aimed to evaluate the c-peptide/FBG ratio applicability from c-peptide and

FBG values of patients with T1D and healthy subjects.

Methods: This study was previously approved by the Ethics Committee on Human Research of the

Federal University of Parana (CAAE: 24676613.6.0000.0102). We analyzed blood samples from

children (0-14 years) and adolescents (>14 and >18 years) with T1D (N=145) and healthy controls

(N=164) paired up by age. Biochemical parameters of FBG and HbA1c were dosed using

turbidimetric methods, while insulin, c-peptide and the inflammatory markers IL-6 and TNF-α were

measured with multiplex technology. Diabetics were classified in 3 groups: low HbA1c (5-6%),

middle HbA1c (7-9%) and high HbA1c (≥10%). The equation used for the ratio of fasting c-

peptide/FBG is defined as: fasting c-peptide/FBG X 100. All Statistic analyses were made using the

software Statistica for Windows 10.0 (StatSoft®, Inc. Tulsa, OK, USA). Kruskal-Wallis test with

Tukey post hoc and Spearman’s Correlation Coefficient were performed, and significance criteria was

set at P<0.05.

Results and Discussion: C-peptide is a biomarker produced in equimolar amounts to endogenous

insulin and has been widely used as a measure of insulin secretion, helping in the estimation of

remaining β-cells function. Healthy subjects had greater fasting c-peptide/FBG ratio of 1.14 (0.65-

1.48) when compared with the low group 0.30 (0.09-0.51) (P≤0.01), middle 0.10 (0.004-0.05)

(P≤0.001) and the high group 0.01 (0.004-0.04) (P≤0.001). When comparing only diabetics, the low

group is statistically different from the high HbA1c group (P≤0.05), but no statistical differences were

observed between low and middle or middle and high group. These data suggest that the high group

has lower secretion of insulin when compared to controls and the low group, even though the non-

significant results may point various pancreatic function and/or dietary control in the groups.

Furthermore, correlations between fasting c-peptide/FBG ratio and HbA1c (R=-0.78 P≤0.001), insulin

(R=0.62 P≤0.001) and IL-6 (R=0.23 P≤0.001) confirmed alteration on glucose metabolism and

chronic inflammation. There was no correlation between fasting c-peptide/FBG ratio and TNF-α.


Conclusion: Mathematical ratios are an easy and cheap way that helps identifying patterns in

diseases. For T1D, fasting c-peptide/FBG ratio showed potential to be used as an auxiliary tool for

monitoring remaining β-cell function, although similar results are appreciable using only c-peptide.

Acknowledgement: We would like to acknowledge Pontifical Catholic University of Parana (PUC-

PR) for granting access to the multiuser technical laboratory, National Council for Scientific and

Technological Development (CNPq) and Araucaria Foundation (PPSUS) for supporting this study.


Investigation of periphytic community in a water supply river in Umuarama-PR

GAMA, R. S. (1); RUGGERI, M. V. R. (1); ANDRADE, J. G. S. (1); RODRIGUES, L. A. P. (1);

SANTOS, M. B. (1); FEITOSA, D. C. (1); TAVARES, L. (1); SCHWIND, L. T. F. (2); GODOY,

R.F.B. (1)

1. Environmental Engineering Department, State University of Maringá, Campus Umuarama.

2. UNINGÁ


Introduction: It is well known that only water qualities parameters evaluation in water bodies do not

provide good information about the ecological aspects of an aquatic ecosystem. These parameters

provide the physical and chemical conditions of that environment at that moment. Therefore, it is

necessary to link the water quality parameters with aquatic organisms communities. Microalgae are

photosynthetic organisms that play important roles on food web and in the reduction of carbon level.

For this research was chosen two points to verify the Periphytic community in a water supply river.

Methods: The collect was done in May, 2019. One point located at upper part of the water body (P1)

and another close to the captation point (P2). Rock and stem were used as natural substrates. The

substrates were scraped and washed with distilled water. Then, it was taken 1mL to visualize in the

optical microscope. P1 presented low turbidity (0.09 NTU), acid pH (value around 5) and smaller

temperature (24.4 ºC).

Results and Discussion: At this point, there were seen only diatoms in the sample and low number of

individuals. A possibly explanation for it could be the smaller intensity of light availability due to

shadow formation by trees. Nutrients availability could be another important factor. Seen that, lower

turbidity is supposed to have lower organic matter contribution. At P2, the turbidity increased to 31.1

NTU while pH stood around 5 and temperature also increased (25.8 ºC). It was seen more

Bacillarophycea individuals (70%) and the appearance of other microalgae classes such as

Cyanophycea (28%) and Zygnemaphycea (2%).

Conclusion: Concluding, periphytic community difference between P1 and P2 seen at this collect

could be related to different environmental conditions, influence of external factors, type of substrate

and nutrients availability. Complementarily, the next studies will include nutrients analysis to

compare with the periphytic community.

Acknowledgment: The authors acknowledge the opportunity to use the biology laboratory and

pollution laboratory at State University of Maringá


The reduction of walker 256 tumor and wistar rats cakexia promoted by Euphorbia

tirucalli latex

GARCIA, C.M. (1); MADALOZO, A.L. (2); COMOTTI OLIVEIRA, B. (3); FISCHER, S.V. (2);

COUTINHO, D.S.S. (2); PEDROSO, I. (2); ZULIN, N. E. (2); BIALLI,P.A. (2); APPEL, M.H. (4);

ADRIANA AYA, Y. (1) ; FERNANDES, L.C. (2); BONATTO, S. J. R. (1); IAGHER, F. (2)

1. Instituto de Pesquisa Pelé Pequeno Príncipe, Paraná, Brazil.

2. Departamento de Fisiologia, Universidade Federal do Paraná, Paraná, Brazil.

3. Departamento de Biologia Celular e Molecular, Universidade Federal do Paraná, Paraná,

Brazil.

4. Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta

Grossa, Paraná, Brazil.


Introduction: Research on medicinal plants for antitumor purposes in recent years has been relevant.

Euphorbia tirucalli latex is used by individuals with different types of cancer, but its efficacy and the

mechanisms involved in fighting tumors are not known. Objectives: To elucidate the effect of oral

supplementation with E. tirucalli latex on tumor growth, cachexia and immune parameters in Wistar

rats bearing Walker's tumor 256.

Methods: Male rats were used, subdivided into 4 groups (n = 20 / group): C, without tumor and

without treatment; W, with tumor and without treatment; SW1, with tumor and treated for 15 days

with 200 ul / ml of latex / day by gavage; SW2, with tumor and treated for 15 days with 500 ul / ml of

latex / day. After euthanasia, tumors were removed and weighed, proliferative capacity of tumor cells,

mesenteric lymph nodes, thymus, ex vivo blood was assessed by the AlamarBlue® technique and

Bcl2 expression in tumor tissue by Western Blot. Biochemical analysis were performed using the

enzymatic / colorimetric method, as well as peritoneal macrophages, glycemia, and triacylglycerol.

Results and discussion: The SW2 group showed a reduction of ~ 60% in tumor mass in relation to W

(p <0.05), ~ 76% in tumor proliferation (p <0.0001) and ~ 50% in Bcl2 expression, In the

triacylglycerol data, it was observed that the treated animals presented a 90% reduction in relation to

C (p <0.05), in the glycemia of the fasted animals, the treated ones in both dosages presented a

reduction of the glycemia

Conclusions: The reduction of tumor mass promoted by latex consumption of E. tirucalli is due, at

least in part, to the reduction of the proliferative capacity of tumor cells, which may have aided in the

reduction of Bcl2 expression in tumor tissue, and may positive contribution to the animal cachexia.

The data obtained by Alamar Blue ® revealed that the T lymphocytes of the supplemented animals

showed viability of, respectively, already in the other tests the lymphocytes obtained from the

mesenteric lymph nodes presented greater viability in the untreated animals. In the cachexia data it

was evident that the supplementation with the latex favors the ability to modulate the glycemia, and

triacylglycerols. By flow cytometry of the tumor tissue no statistical difference was found. These data

suggest that latex has great potential to be used as a source of antitumor substance.

Acknowledgement: To the financial support for the development of this work offered by the organs;

CAPES, CNPq, IPPP. To the collaborators involved in this work.


Transcriptional changes in human umbilical vein endothelial cells induced by

Toxoplasma gondii

GARCIA, L. F. C. (1); ALBRECHT, L. (2)

1. Masters degree student of Fundação Oswaldo Cruz, Instituto Carlos Chagas, Brazil.

2. Researcher of Fundação Oswaldo Cruz, Instituto Carlos Chagas, Brazil.


Introduction: Toxoplasma gondii is a protozoan with a complex life cycle. Its sexual reproduction

occurs in felines while the incomplete reproduction could occur in almost all warm-blooded animals,

including humans. It is known that the parasite is capable of interact with endothelial cells and, thus,

alters its functions. According to it, some important questions involving the commitment of the

endothelial cells in the context of infection by T. gondii could be established.

Methods: Tachyzoites of T. gondii, strain ME-49, were added to culture of Human Umbilical Vein

Endothelial Cells (HUVEC) in a proportion of five tachyzoites per cell (MOI-5) and incubated for

one, two and four hours. After it, mRNA of the endothelial cells was extracted and the cDNA was

used as a template for relative quantification (qPCR). The genes analyzed for the evaluation of cell

activation/dysfunction were separated in four groups: inflammation related genes: IL1A, IL6, IL8,

IL10RA, IL10RB, IL12P40, TNF, INFG and SOCS3; adhesion molecules: ICAM1; coagulation:

ADAMTS13; angiogenesis and vascular permeability: VEGF, ANG1, ANG2, SDC1 and ENOS.

Invasion assay was performed by the inoculation of tachyzoites (MOI-5) in culture of HUVECs and

human fibroblasts (NHDF) and quantified by fluorescence microscopy. The evaluation of T. gondii

extracellular vesicles was done by scanning/transmission electron microscopy, western blotting,

nanoparticle tracking analysis (NTA) and its cytotoxicity level measured by MTT assay.

Results and Discussion: IL6 was increased 3.6 (±0.25) times at hour 4 (p=0.0042). IL8 was

expressed in a similar pattern of non-infected cells at hours 1 and 2 and had no detectable levels at

hour 4. SOCS3 was increased 3.27 (±0,63) times at hour 2 (p=0.0009) and expressed similar levels to

the non-infected cells at hour 4. In a first evaluation, it was not evidenced detectable level of the other

genes. In the infection assay, we observed that at four hours post infection, there were fewer infected

HUVEC compared to NHDF (p=0.0344), however, HUVEC hosted more tachyzoites than the

reference cells (p=0.0026). The extraction of T. gondii extracellular vesicles were accomplished and a

predominant population sized between 100 and 200 nanometers (p<0.01) was observed. The vesicles

are also being identified by western blotting added to classic extracellular vesicle and Toxoplasma

gondii markers and did not present cytotoxicity to HUVEC in concentrations up to 40 μg/ml. The

integrity of vascular endothelium is essential to the maintenance of the volemy, vascular permeability,

angiogenesis, coagulation, migration of leukocytes and prevention against infectious agents. Once

protozoans could alter endothelial functions, a cascade effect could occur in benefit of the infectious

agent, such as enhancing an invasion process. Extracellular vesicles are small particles capable of

transmit information between cells of the same organism or different ones. In the infectious ambit, it

was yet observed that these particles could support the invasion agent.

Conclusion: Such findings reveal a new universe of interaction between parasites and host cells.

Next, the involvement of extracellular vesicles of T. gondii and from infected cells will be evaluated

in the context of endothelial activation or disfunction.


Aknowledgment: CAPES, CNPq and the Program for Technological Development in Tools for

Health-PDTIS-FIOCRUZ for the use of their facilities (Microscopy Facility and Cytotoxicity Facility)

of Instituto Carlos Chagas-FIOCRUZ/PR.


Large and small extracellular vesicles in Giardia intestinalis: Implications for

pathogenesis and drug treatment

GAVINHO, B (1); SABATKE, B. (2); RAMIREZ, M.I. (3,4)

1 – Programa de pós-graduação em Microbiologia, Parasitologia e Patologia, Universidade Federal do

Paraná, Curitiba/PR - Brazil.

2- Programa de pós-graduação em Biologia Celular e Molecular, Universidade Federal do Paraná,

Curitiba/PR - Brazil.

3 – Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba/PR

- Brazil.

4 - Instituto Oswaldo Cruz. Rio de Janeiro/RJ - Brazil.


Introduction: Giardia intestinalis is an anaerobic protozoan that is an important etiologic agent of

inflammation-driven diarrhea worldwide. Although self-limiting, a deep understanding of the factors

involved in the pathogenicity that produces the disruption of the intestinal barrier remains unknown.

There is evidence that under diverse conditions, the parasite is capable of shedding extracellular

vesicles (EVs) which could modulate the physiopathology of giardiasis. In recent years, there has

been a growing interest to improve current knowledge of the nature, constitution and biogenesis of all

secreted EVs. Here we describe new insights of G. intestinalis EV production, revealing its capacity

to shed two different enriched EV populations (large and small extracellular vesicles). Our work also

aimed at assessing the influences of two recently identified inhibitors of EV release in mammalian

cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from

Giardia and their putative effects on host-pathogen interactions.

Methods: The first objective was to obtain different EV populations from G. intestinalis. The

previous protocol (Evans-Osses et al., 2017) was slightly modified to separate putative large

extracellular (LEV) and small extracellular vesicles (SEV) from the total extracellular vesicles. G.

intestinalis trophozoites were treated with PAD-inhibitor Cl-amidine or CBD respectively to

investigate their ability to inhibit EV production. SEVs and LEVs were analyzed for their ability to

interact with host cells. For host uptake of protozoa EVs, the fluorochrome PKH-26 was used for EV

labelling, resulting in a homogeneous staining. Both PKH26-labelled LEVs and SEVs were incubated

with caco-2 monolayers for 1 hour at two concentrations (7 or 14 µg). We also sought to verify if each

EV population have the same phenotype effect after trophozoite post-exposure to PAD-inhibitors on

host cell adhesion. Caco-2 were seeded in 24-well plates and grown to 100% confluence. Inoculations

of 5x105 trophozoites per group were treated with different concentrations of PAD-inhibitors,

submitted to the EV production and then transferred to the cell monolayer for 3 hours (37°C) in a final

volume of 1 mL / well (MOI 10:1). Two concentrations (7 or 14 µg) from both EV subpopulations

were used for treatment of some groups.

Results and discussion: Based on Nanotracking Particle Analysis and protein quantification,it was

possible to recover two different EV populations from our modified method: LEVs at 15,000xg and

SEVs at 100,000xg. PAD-inhibitor Cl-amidine and CBD were both able to effectively reduce EV

shedding, the PAD-inhibitor specifically affecting the release of large extracellular vesicles and

interfering with in vitro host-pathogen interactions. LEVs derived from the protozoa were capable of

restoring the adherence phenotype following treatment with Cl-amidine, in a dose-dependent manner.


In contrast, no effect was observed in the SEVs treated groups. The strong efficacy of the PAD-

inhibitor on Giardia EV release indicates a phylogenetically conserved pathway of PAD-mediated EV

release, most likely affecting the Giardia arginine deiminase (GiADI) homolog of mammalian PADs.

Confocal microscopy revealed punctuated patterns of fluorescence distributed intracellularly. Both

populations appeared to be taken up by the host cells in a dose-dependent manner.

Conclusion: While there is still much to learn about G. intestinalis interaction with its host, like the

role of EVs in drug resistance, our results suggest that large and small EVs may be differently

involved in protozoa communication, and that EV-inhibitor treatment may be a novel strategy for

recurrent giardiasis treatment.

Acknowledgement: We would like to thank Dr. Wanderson Da Rocha for sharing his laboratory at

the Universidade Federal do Parana. Finally, this study has received support from FIOCRUZ, CNPq,

CAPES and Programa Basico de Parasitologia AUXPE 2041/2011 (CAPES), Brazil. M.R is currently

fellow from CNPq-Brazil.


Molecular cloning and characterization of a serpin from Loxosceles intermedia venom

gland

GRAEFF, S. Z. (1); NOWATZKI J. (2); DA JUSTA C. H. (1); DE BONA, E. (1); GREMSKI, H. L.

(1);VEIGA, S. S. (1)

1. Department of Cell Biology. Federal University of Paraná, (UFPR).

2. Institute of Medical Biochemistry. Federal University of Rio de Janeiro, (UFRJ).


Introduction: Serpins are molecules that act as inhibitors of serine proteases, which are enzymes that

participate in important physiological processes. Unregulated proteolysis can lead to diseases and

functional problems. A sequence encoding a serpin was identified in the venom gland of the brown

spider Loxosceles intermedia, with approximately 1200 bp and calculated molecular mass of ≅40

kDa. We aimed to produce the L. intermedia venom serpin as a recombinant protein in a eukaryotic

expression system using insect cells, which promotes post translational modifications, yielding the

protein in its soluble and active form.

Methods: The sequence, which was first described with the transcriptome analysis of L. intermedia

venom glands, was subjected to a similarity analysis. The sequence was inserted into pENTR/D-

TOPO vector, confirmed, and then recombined to the linear baculovirus DNA through the Gateway®

system. SF9 insect cells were then transfected to obtain the first viral stock from which new viral

stocks were made in order to obtain viral titration staggering. The confirmation of viral titration was

obtained by qPCR. After expression, two specific bands were detected by immunoblotting assay using

anti-venom polyclonal antibodies. For biological evaluation the test of inhibitory activity of the

enzymatic activity of trypsin was performed, by reverse zymography test. This research was approved

by UFPR animal ethic’s committee under the number 1146.

Results and Discussion: Results showed homology with other serine protease inhibitors belonging to

the serpin family. Molecular modeling and similarity analysis were performed for structural analysis.

Two specific bands were detected by immunoblotting assay using anti-venom polyclonal antibodies:

One band at the expected mobility for the monomer serpin ≅44 kDa and another one at ≅72 kDa,

which probably represents the dimeric form. Reverse zymography test revealed inhibitory activity

upon trypsin in ≅72 kDa, showing that the protein in the dimeric form is active.

Conclusion: Data obtained here will contribute either to understand the serpin biological activity and

detection of its possible applications, since the serpins already described are involved in many

physiological processes such as blood coagulation, inflammation, fibrinolysis and antitumor action.

Acknowledgement: UFPR, LME, CAPES; Fundação Araucária; CNPQ.


Functional characterization of CSDC2 during cardiac differentiation of embryonic stem

cells

GOMES JÚNIOR, R. (1); PEREIRA, I. T. (1); DALLAGIOVANNA, B. (1)

1. Instituto Carlos Chagas (FIOCRUZ / Paraná).


Introduction: Heart diseases are the main cause of death in the world, being responsible for 31% of

global mortality. Hole organ transplant is the most efficient treatment, however, it is limited by

shortage of donors. In this context, alternative therapies are required and the clinical potential of

embryonic stem cell (ESC), due to their ability to differentiate into several cell types, have stimulated

the development and improvement of methods that promote the differentiation of functional cells,

including cardiomyocytes. Thus, understanding the regulation mechanism of this process is essential

to optimize protocols of ESC cardiomyogenic differentiation. Studies have shown low correlation

between mRNA and protein expression, reinforcing the role of post-transcriptional regulation in

control of protein levels. RNA binding proteins (RBPs) are the core of post-transcriptional regulation

since they influence the structure and interaction of mRNA, and participate of its biogenesis, stability,

function, transport and localization. Considering that, the characterization of RBPs is essential to

understand this regulation network. Cold shock domain containing protein 2 (CSDC2), also called

PIPPin, is a RBP expressed during rat brain development, which participates in the regulation of

histones expression during the pyramid neurons differentiation. Preliminary data from our group has

shown an increased expression of the CSDC2 gene during ESC differentiation into cardiomyocytes,

rising the hypothesis that CSDC2 could have a key role as a regulator during this process. Therefore,

this study aims to investigate the role of CSDC2 during human cardiomyogenic differentiation and to

identify targets regulated by this protein.

Methods: Human ESCs (H1 cell line) were used to derivate a new cell line carrying an inducible

overexpression system containing the encoding sequence of CSDC2 (H1 iCSDC2). CSDC2 gene

expression was analyzed by qPCR after induction with 500 ng/ml of doxycycline for 24 hours and the

protein expression by immunofluorescence after 72 hours using anti-CSDC2. Also, E. coli strain

BL21(DE3)pLysS was used to express recombinant CSDC2 (cloned into pET28b) by induction with

IPTG 0,5mM for 5 hours at 30°C. Bacteria lysate was purified at AKTA using Ni-NTA affinity

column and the purification efficiency was analyzed by SDS-PAGE.

Results and Discussion: Doxycycline induction in H1 iCSDC2 cells showed 500-fold CSDC2

expression by qPCR, when compared to the non-induced cells, and corroborated the

immunofluorescence positive CSDC2 expression. SDS-PAGE of the recombinant protein showed that

6His-CSDC2 was expressed mainly in the soluble fraction with a high degree of purity.

Conclusion: For the next steps, we aim to establish a CSDC2 knock-out cell line and analyze the

efficiency of cardiac differentiation protocol in both mutants, positive (iCSDC2) and negative

(CSDC2 KO). In addition, we aim to obtain a homemade antibody against CSDC2 and perform in

vitro assays, such as pull-down and immunoprecipitation, to identify potential targets of this protein.

Acknowledgement: FIOCRUZ; Capes; CNPq; Fundação Araucária.


Biochemical and functional characterization of a recombinant allergen of Loxosceles

intermedia venom

JUSTA, H.C (1); DE BONA, E (1); SCHEMCZSSEN, Z (1); GREMSKI, L.H (1); VEIGA, S.S. (1)

1. Laboratório de Matriz Extracelular e Biotecnologia de Venenos. Universidade Federal do

Paraná – Programa de pós-graduação em Biologia Celular e Molecular, Curitiba – PR.


Introduction: Spiders of the Loxosceles genus are among the clinically relevant Brazilian spiders.

The state of Paraná registered in 2017, 4.198 accidents evolving these animals, and in 2018 4.098.

The bite of these spider triggers a set of typical signs and clinical symptoms that are called

Loxoscelism. Among those clinical manifestations are: a serious dermonecrotic lesion, inflammatory

events and allergic reactions like erythema and exanthema. These effects are due to the synergic

effects of toxins found in the venom of Loxosceles spiders. The transcriptome of the venom gland of

Loxosceles intermedia showed, among these toxins, a sequence similar to allergens (named LiALT).

This molecule can be a key to better understand the mechanisms of hypersensitivity triggered by this

venom and it was not yet expressed. Moreover, allergy is the 4º most common chronical disease in

Brazil, whether the allergy is due to food, dust or the bite of insects. Since the prevalence of allergies

increases and the allergic reactions resulting from the bite of Loxosceles spiders have not yet been

elucidated, the aim of this work is to clone and express the LiALT molecule and to understand

whether it is related to these hypersensitivity events.

Methods: The coding sequence of LiALT was cloned in pENTR™/D-TOPO® vector using the

Gateway System™. The hosts of Baculovirus were Spodoptera frugiperda (Sf9) cells. Real time PCR

(qPCR) was used to detect and quantify virus and a Western Blotting (W.B.) was performed to detect

expressed proteins. Large scale expression was carried out during 144h using 5% of viral stock. The

supernatant of expression was purified in affinity chromatography (HisTrap Excel) associated with an

AKTA equipment. Purity of the sample was confirmed. The resulting protein was recognized by α-

venom antibodies of L. intermedia. Detection of vascular permeability increase using Evans Blue was

performed in mice.

Results and Discussion The transfection was successful, since morphologic differences between the

Sf9 control cell and the infected cells were observed. Corroborating this result, qPCR amplified the

target gene (gp64) that encodes a glycoprotein present in the viral capsids of Baculovirus. The

expression tests were realized from 0-9 days with 0%, 5% and 20% of Baculovirus. Antibodies that

recognize L. intermedia venom toxins revealed an unspecific profile by the time of 144h, but by the

time we observed an increasing band of 45 kDa - a molecular mass that corroborates with the

predicted molecular mass of LiALT. After the purification step, LiALT was detected by the same

antibodies as a 45 kDa band. The vascular permeability experiment in mice showed that this protein

increases the vascular permeability in a dose dependent manner (5 μg and 10 μg). Tests will be made

with basophils to evaluate if this molecule is able to induce degranulation and the release of

inflammatory mediators.

Conclusion: This molecule was recognized by the serum of rabbits immunized with total venom of L.

intermedia, suggesting that LiALT is a toxin present in the venom of those spiders. In addition, the

vascular permeability experiment showed that LiALT increases plasma extravasation, which is a


symptom triggered by allergens. This recombinant protein will help to understand the mechanisms of

allergy due to the bite of Loxosceles spiders and also may be used has a bio tool to identify individuals

that are allergic to the bite of these animals.

Acknowledgment: CAPES, CNPq, Fundação Araucária, UFPR


Characterization of porcine heart valve grafts obtained by two different processes of

decellularization and in vitro biocompatibility testing with human cells

LEITOLIS, A. (1); HEUSCHKEL, M.A. (1); RODERJAN, J. G. (3); SUSS, P.H. (2); DA COSTA,

F.D.A. (2); STIMAMIGLIO, M.A. (1); CORREA, A. (1)

1. Laboratory of Basic Biology of Stem Cells, Carlos Chagas Institute, Fiocruz-Paraná, Curitiba

81350-010, Brazil.

2. Pontifical Catholic University of Paraná—PUCPR, Curitiba 80215-901, Brazil.

3. Technological Federal University of Paraná—UTFPR, Curitiba 80230-901, Brazil;


Introduction: Heart valves are structures responsible for unidirectional blood flow maintenance into

the heart. Changes in valvar functionality may occur after pathological conditions and aging resulting

in heart valve disease. The main clinical therapy for this disorder is valve replacement; a procedure

performed almost 300.000 times each year. The prosthetic replacement can be performed with

biological models (porcine, bovine and human); however those tissues may induce immune responses

due to cellular content. Thereby, decellularization is a tissue engineering approach to eliminate

allogeneic cells. This method has been tested to improve the biocompatibility of biological prostheses.

The present study evaluated the decellularization efficiency of two protocols performed in porcine

pulmonary valves and analyzed the in vitro biocompatibility of these grafts.

Methods: Porcine pulmonary valves obtained from a slaughterhouse were decellularized with 0.1%

SDS solution (Group 1) or 0.1% SDS solution followed by incubation with hypertonic buffer (10 mM

Tris-HCl and 2.5 M NaCl) and 20 IU/ml Benzonase (Group 2). The treated heart valves then were

subjected to histological, cytotoxicity, DNA and scanning electron microscopic analyses to confirm

decellularization and extracellular matrix (ECM) structure. The biocompatibility was verified through

the co-culture of porcine tissue with mesenchymal stem cells and valvular interstitial cells (VIC).

Results and Discussion: The decellularized tissues showed no histological evidence of cells in the

cusps and conduit regions, however, DNA remnants were verified mainly in tissues from group 1. The

content and arrangement of elastin, collagen and GAG were not altered in group 1, but a marked

reduction in collagen and GAG was observed in group 2. Tissues from both groups were non-

cytotoxic. The biocompatibility assays confirmed that cusps from both groups worked as a scaffold

for stem cells and VIC. However, cell repopulation into decellularized valve fragments was more

effective in scaffolds from group 1.

Conclusion: The protocol used in group 2 resulted in a greater decellularization of the porcine heart

valves than the protocol of group 1. Nevertheless, protocol 2 was accompanied by ECM impairment

and a less effective cell repopulation. Further analyses of immunological responses induced by treated

tissues are being conducted to verify which protocol generated a more advantageous graft.

Acknowledgement: This study was supported by the National Health Fund, Brazilian Ministry of

Health (Grant number 814611/2014) and Coordenação de Aperfeiçoamento de Pessoal de Nível

Superior, Brasil (CAPES)–Finance Code 001. The authors thank the staff of Carlos Chagas Institute

(Fiocruz-PR) for laboratory and administrative support.


Preliminary study about the removal of indoxyl-sulfate, p-cresil sulfate and indole acetic

acid by conventional hemodialysis vs. hemodiafiltration

LIMA, J.D. (1); PECOITS-FILHO, R. (2); RODRIGUES, S.D. (1); NAKAO, L.S. (1)

1. Universidade Federal do Paraná.

2. Pontíficia Universidade Católica do Paraná.


Introduction: Chronic kidney disease (CKD) is characterized by the progressive and irreversible

failure of kidney function. One of the consequences of the kidney failure is the development of uremic

syndrome, in which there is the accumulation of toxins in the bloodstream. Some toxins bind to

plasma proteins (PBUT), making their removal inefficient by diffusion through the small pores in the

dialyser membrane in hemodialysis (HD). Some clinically relevant PBUT include: indoxyl-sulfate

(IS), p-cresil-sulfate (PCS) and indole acetic acid (IAA). An alternative method is hemodiafiltration

(HDF), in which along with the diffusion, it also uses ultrafiltration, that rises the pression inside the

dialyser, catalyzing the removal of higher molecular weight molecules. Furthermore, it has been

demonstrated that HDF treatment decreases the mortality in about 30%. However, the levels of IS,

PCS and IAA has not been compared between those two treatments. Then, our aim was to compare

the plasma concentration of IS, PCS and IAA in patients submitted to HD and HDF.

Methods: In a preliminary setting, thirty-two pre-dialysis plasma samples of CKD patients (HD=15;

HDF=17) were analysed by High Performance Liquid Chromatography (HPLC). A total of 196

patients, randomized in the 2 dialytic methods has been recruited to participate of the HDFit project.

Samples were collected in three different times during both treatments, T=0, T=3 and T=6 months.

Results and Discussion: The results showed that IS concentration was significantly decreased in

HDF treatment, compared with HD, at T=3 months (63 vs 91 uM, p<0.05). Also, significant changes

were observed along time in each treatment: IAA levels decreased from T=3 to T=6 in HD (15 vs 11

uM, p<0.05), while they decreased from T=0 to T=3 in HDF (11 vs 9 uM), p<0.05). Conclusion:

Although no other comparison reached significant difference, these preliminary data suggest that HDF

might have improved results than HD regarding IS, pCS and IAA removal.

Acknowledgment: We would like to thank Dr. Pecoits-Filho by sharing HDFit samples and CAPES

for the financial support.


Investigation of biological markers in neurodegenerative diseases and aging

LIMA, M. R. (1); PESTANA, C. R. (1)

1. Instituto de Ciências da Vida e da Natureza. Universidade Federal da Integração Latino-

Americana (UNILA)


Introduction: Aging is a common hallmark of all living organisms and characterized by a

progressive loss of physiological function. In the nervous system, changes in cell homeostasis lead to

the death of neuronal cells contributing to the development of neurodegeneration diseases such as

Amyotrophic Lateral Sclerosis, Huntington's Disease, Parkinson's and Alzheimer's Diseases. The

investigation of blood peripheral biomarkers is a low invasive alternative approach for tracking

changes in energy metabolism and immune response with age. This work reports a cross-sectional

analysis aimed to determine potential changes biomarkers levels and its relation to cognitive and

motor impairment. Such findings can contribute to the better understanding of biomarkers in the early

diagnosis of age-related diseases.

Methods: A retrospective clinical sample database analysis was previously performed to select

potential aging biomarkers. Thus, one hundred volunteers were also selected with 50 adults (age

between 20 and 59 years) and 50 elderlies (aged over 60 years). By signing the term free and

informed consent (TCLE), all volunteers were submitted to the cognitive function test by Mini Mental

Status Examination (MMSE) and motor function test by Unified Parkinson's Disease Rating Scale

(UPDRS). Subsequently, peripheral blood samples were collected for the determination of markers

associated with the lipid oxidative profile (including enzymes and trace metals).

Results and Discussion: Scores of the cognitive and motor tests revealed a progressive decline in

performance with age. Significant differences were observed between the lipid profile of adults and

the elderly people by an increase in total cholesterol, Low Density Lipoproteins cholesterol (LDL),

Very Low Density Lipoproteins cholesterol (VLDL), triglycerides and a decrease in High Density

Lipoproteins cholesterol (HDL) as age increased. It was also observed the decrease of trace metals

important contribution antioxidant such as iron, selenium and manganese in elderly volunteers in

relation to adult volunteers. In addition, these values were even lower in the elderly volunteers with

worse than elderly volunteers with normal motor and cognitive test scores. Lower levels of selenium,

iron, zinc, copper and rmanganese were observed in elderly volunteers with some type of impairment

due to neurodegeneration, and the manganese looks more associated with cognitive declines. Higher

levels of aluminum were also observed in elderly volunteers. There were significant changes in the

lipid profile and trace metals between adult and elderly volunteers, and significant reductions in the

concentration of these trace metals in elderly individuals with different levels of impairment.

Conclusion: Metabolic markers are described as one of potential risk factors for the cognitive and

motor decline with age. In this context, changes in the lipid profile are related to the pathophysiology

of neurodegeneration, as well as alterations in the cellular antioxidant pattern (oxidative stress). There

are multiple simultaneous changes with the advancement of the age from the physiological, cellular

and molecular mechanisms. The onset of pathologies associated with aging is the result of a set of

synchronic biological variations that can be partially identified through serum markers like circulating

lipids and trace metals levels with antioxidant activity. Therefore, these results may contribute to the

understanding how biomarkers can be used for the diagnosis and treatment of aging-related diseases.


Acknowledgement: We would like to thank UNILA for the facilities and reagents, the Master Lab

laboratory for the partnership and integral support and CAPES for the financial support


Evaluation of biochemical biomarker responses in Danio rerio larvae in a single and

mixture exposure of Di-n-butyl phthalate (DBP) and Di-iso-pentyl phthalate (DiPeP)

LIROLA, J.R. (1); GUILOSKI, I.C. (1); PIAU, T.B. (2); GONÇALVES, L.P. (2); GRISOLIA, C.K.

(2); MARTINO-ANDRADE, A.J.(1); SILVA DE ASSIS, H.C. (1); LEME, D.M. (1); CESTARI,

M.M. (1)

1. Federal University of Paraná – UFPR, Curitiba, P.R.

2. Institute of Biological Sciences, University of Brasilia – UnB, Brasília, Distrito Federal.


Introduction: Phthalates are plastic additives used to give malleability to plastic products and are

easily released into the environment. Di-n-butyl phthalate (DBP) is known to be toxic due its

antiandrogenic action in mammals. Di-iso-pentyl phthalate (DiPeP) appears in fuel production and is

widely used in Brazil. Few studies bring the action of phthalates on fish, especially in the early stages

of life of these animals. The objective of this study was to evaluate changes in the activity of

glutathione S-transferase (GST), acetylcholinesterase (AChE) and lipoperoxidation (LPO) in Danio

rerio larvae exposed to DBP and DiPeP, isolated and in mixtures.

Methods: According to OECD 236/2013 (OECD Guidelines for the Testing of Chemicals), the

exposure was performed in 24-well plates containing 2.0 mL of contaminant for 96-hours. The

experimental groups were: negative control; solvent control (0.1% methanol v/v); 0.001; 0.003; 0.007;

0.015; 0.031; 0.062; 0.125 mg/L DBP; 0.001; 0.003; 0.007; 0.015; 0.031; 0.062; 0.125 mg/L DiPeP;

and mixtures groups (DBP+DiPeP = MIX) at the concentrations of 0.001; 0.003; 0.007; 0.015; 0.031

mg/L MIX. For biomarker analyses (GST, AChE and LPO), ten 10 larvae per replicate (n=15) in each

group.

Results and Discussion: DBP and DiPeP did not alter GST activity. However, an increase in GST

activity occurred at the concentration 0.031 mg/L of the phthalates mixture. GST plays an important

role in biotransformation (Allocati et al., 2018). Isolated phthalates (DBP at 0.007 and 0.015 mg/L,

and DiPeP at 0.003 mg/L) and mixture (at 0.007 and 0.015 mg/L) caused an increase in LPO levels.

Acetylcholinesterase is used as biomarkers of neurotoxicity (Van Der Oost et al., 2003). AChE

activity was increased on exposure to both phthalates isolated and in mixture. This increase was

observed in the groups 0.007 mg/L DBP, 0.003 mg/L DiPeP, 0.003 mg/L MIX and 0.007 mg/L MIX.

In fish, little is known about the mechanisms of AChE activation.

Conclusion: In this work it was possible to verify that phthalates isolated and in mixture caused

toxicity in Danio rerio larvae LPO increase and activivation of AChE activity were observed on

exposure to DBP, DiPeP and MIX. However, only the mixture caused an increase in GST activity.

Thus, we can conclude that studies with phthalates mixtures are necessary.

Acknowledgement: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq


Oxidation of apoptosis inducing factor to disulfide-linked conjugates

LO, S.M. (1); HUAMANÍ, P.A.M. (1); VALDAMERI, G. (2); ZANATA, S.M. (1); NAKAO, L.S. (1)

1. Department of Basic Pathology, Universidade Federal do Paraná, Centro Politécnico, Jardim

das Américas, 81531-980, Curitiba, Paraná Brazil.

2. Department of Clinical Analysis, Universidade Federal do Paraná, Campus Jardim Botânico,

Jardim Botânico, 80210-170 Curitiba, Paraná, Brazil.


Introduction: Apoptosis-inducing fator (AIF) is a mitochondrial inner membrane flavoprotein, with

the majority of its sequence facing the intermembrane space. AIF was first described as an apoptotic

protein, but AIF-deficient cells have decreased expression of respiratory complex I, impaired

oxidative phosphorylation and aberrant mitochondrial morphology. In addition, these cells present

more oxidative damage and are more susceptible to peroxide-induced death. AIF has three cysteine

residues, which have not been considered reactive, however thiol reagent para-

chloromercuriphenylsulfonic acid (pCMPS) could diminish its in vitro activity. Therefore, here we

investigated whether the cysteine residues of AIF could have a role in such antioxidant activity.

Methods: The recombinant protein hAIF Δ1-120 and its cysteine mutants (10 μM) were oxidized

with 2 mM hydrogen peroxide or with 0.5 mM diamide for 15 minutes at 37°C in PBS. These samples

were submitted to a 10% SDS-PAGE in non-reducing or reducing conditions, and the gel was stained

with Coomassie Blue. To investigate whether thioredoxin (Trx1) could be responsible for AIF

reduction, hAIF Δ1-120 oxidized with diamide was incubated with reduced recombinant Trx1 for 15

minutes at 37 °C in 50 mM Tris pH 7.2. HEK293T cells were transfected, using a calcium phosphate-

based method, with AIF-HA, AIF C256S-HA, AIF C317S-HA and AIF C441S-HA pCIneo vectors or

with the empty vector. After 48 h, cells were treated in PBS with 0.5 mM hydrogen peroxide for 2 h

or 75 μM diamide for 10 minutes at 37°C. Cell extracts were analyzed by Western Blot with anti-HA,

anti-AIF and anti-beta actin antibodies. Knockdown of Trx1 was perfomed co-transfecting siTrx1

with AIF vectors and same oxidation conditions was done as described above.

Results and Discussion: Our data showed that recombinant human AIF, as well as overexpressed

AIF in HEK293T cells, can be oxidized to disulfide-linked conjugates (DLC) under oxidative

conditions. In cells, substitution of cysteine residues 317 or 441 severely affects the amount of the

DLC formed, indicating their involvement in disulfide bonds, whereas in recombinant AIF, DLC are

not produced in the absence of cysteine residues 256 or 441. This discrepancy could be explained by

the models used, but this suggests that more than one cysteine is involved in the DLC formation.

Moreover, recombinant human Trx1 was able to disrupt the DLC formed in vitro and knockdown of

Trx1 in cells produced more DLC after oxidative stimulus. Interestingly, AIF-Trx1 interaction was

found to occur in mitochondria.

Conclusion: Overall, these findings demonstrate that AIF can suffer redox modifications, which can

be regulated by thioredoxin 1 system.


Production and evaluation of different constructions of antibody fragments against

epiregulin

LOPACINSKI, A. (1,2); FERREIRA, A. S. (1,2); PICCHI, G. F. A. (1); GUIMARÃES, B.

G. (1); ZANCHIN, N. I. T. (1)

1. Laboratório de Biologia Estrutural e Engenharia de Proteínas, Instituto Carlos Chagas,

FIOCRUZ, Curitiba, Paraná, Brazil.

2. Programa de Pós-graduação em Biologia Celular e Molecular, Universidade Federal

do Paraná, Curitiba, Paraná, Brazil.


Introduction: Epiregulin (EPR) is a member of the epidermal growth factor (EGF) family.

Increasing evidence implicates EPR in cancer development and it has already been

demonstrated that there is a change in its serologic levels in some types of cancer, especially

lung cancer, which makes this protein a potential biomarker to be used in the diagnosis and

follow-up of these patients. Despite these evidences, studies evaluating the role of EPR as

such tool are still scarce. A fundamental step for its validation, consists in improving the

methods used for quantification of its levels. The existing methods are based on the

recognition of this protein by antibodies. However, due to the high structural similarity

between all members of the EGF family, the available antibodies used for quantification of

EPR serological levels may not present the required specificity and sensibility for its

validation as a biomarker for cancer. Thus, the purpose of this work is to produce an anti-

EPR scFv antibody fragment in Escherichia coli, in order to test its affinity and selectivity for

EPR in comparison with the other members of the EGF family. As complete antibodies are

complex molecules difficult to produce, single chain antibody fragments (scFv) emerge as an

attractive alternative since they can be produced in systems of easier genetic manipulation,

such as bacteria. Even though the scFv structure is simpler relative to full-length antibodies,

they still require optimization to increase expression levels and solubility. The utilization of

fusion partners is one of the most common strategies to improve expression levels. Therefore,

the objective of this work is to produce different constructions of antibody fragments against

epiregulin to compare their expression levels and solubility.

Methods: The scFv fragments were amplified by PCR and cloned into different expression

vectors for expression in fusion with a hexa-histidine tag and with either Trx or GFP.

Synthetic genes of the EGF family factors were acquired from a gene synthesis supplier. All

of them contain a Trx-tag and were expressed and purified to be used in further experiments

of interaction with scFv.

Result And Discussion: The light and heavy chains variable regions were amplified by PCR

using an anti-EPR Fab as template. The primers contained the sequence of a glycine and

serine linker used to link both chains, giving rise to a fragment of the scFv type. Then, the

amplicons were cloned into pET28a-TEV, pET28a-TEV-GFP and pET32a-Trx vectors.


Expression tests in the strain of E. coli BL21-Star (DE3) were performed to determine the

optimal conditions for expression of the antibody. The best condition was incubation at 37 ℃

for 4 hours. The scFv expression was efficient for all three constructs: containing a C-

terminal histidine tag, fused to GFP in the C-terminal and fused to Trx in the N-terminal.

None of the constructs produced a soluble antibody and the protein was mostly present in

inclusion bodies. An attempt to solubilize the scFv was performed in the following manner:

the three constructions were submitted to a cycle of sonication and after the process a fraction

of all of them was found in the soluble fraction, showing that sonication was sufficient to

remove the scFvs from inclusion bodies. Members of the EGF family were also cloned in

pET32a, expressed and purified. Once the purification protocol of scFvs is standardized,

interaction tests between the antibody constructs and the ligands will be performed.

Conclusions: scFv fragments were produced as inclusion bodies and were partially

solubilized by a sonication step. The use of thioredoxin as a fusion partner to all of EGF

family members has been a successful strategy to express soluble and stable EGF factors.


Distribution of Merlin transposable elements across eukaryotic groups

LOPES, A.L.K. (1); LUDWIG, A (2); KRIEGER, M.A. (1,2)

1. Universidade Federal do Paraná, PR, Brazil.

2. Instituto Carlos Chagas, Fundação Oswaldo Cruz-Fiocruz, Curitiba, PR, Brazil.


Introduction: Transposable elements (TEs) are defined as repeated DNA sequences that can move in

the host genome. TEs exhibit a broad diversity in their structure and transposition mechanism and

were classified into two classes according to these characteristics. Class II elements, or DNA

transposons, move by “cut and paste” mechanism involving the excision and reinsertion of the DNA

copy of the element. DNA transposons can be divided into 7 main superfamilies: Tc1/mariner, hAT,

PiggyBac, Mutador, Merlin, Transib, P. The Merlin superfamily was first described in 2004 and were

detected by computational analysis in a wide range of animal genomes. However, in the last years, a

massive number of new genomes have been sequenced, allowing the identification of transposable

elements. Here, we described a global analysis of Merlin elements in Eukaryote.

Methods: We performed a search for the Merlin elements deposited in the Repbase, a database of

repetitive DNA elements. Additionally, in order to carry out a global search of these elements, we

selected the sequence MERLIN1SM (Repbase nomenclature) of the planarian Schmidtea

mediterranea to be used as a query in NCBI server BLASTp searches against eukaryotes. A hit was

considered significant when the e-value was lower than 10-4 with at least 80% of coverage. Sequences

from the same species were filtered with cd-hit-est tool (80%). Moreover, these sequences were

evaluated for the presence of conserved domains in CD-search from NCBI server. The selected

sequences were used for multiple sequence alignment using ClustalW on the platform Galaxy and

edited using GeneDoc tool. Phylogeny of the elements was inferred by the Neighbor-Joining method

using the MEGA7 software and Maximum likelihood as implemented on CIPRES.

Results and Discussion: We identified 31 sequences with DDE motif in Repbase and, also selected

other 15 sequences quoted in literature. In Blast search, we find an additional of 273 sequences after

filtering. A total of 322 sequences were initially retrieved. Our results show a wide distribution of

Merlin in eukaryotes, including animals, fungi, plants and some protozoa. About 100 species

presented Merlin elements that had not been previously identified. In some species, Merlin was more

representative like Anncaliia algerae (fungi) with 338 sequences, Octopus bimaculoides (octopus)

with 199, Trichuris suis (nematode) with 104 and Centruroides sculpturatus (scorpion) with 54.

Merlin elements were more widely distributed in animals with 91 species identified. These species

include 44 arthropods, 14 nematodes, 6 fish, 6 cnidarians, 4 molluscs, 3 annelids, 3 flat worms, 1

amphibian, 1 ascidian and 1 echinoderm. A single Merlin element was identified in plants, Selaginella

moellendorffii, which is deposited in the Repbase database. Also, we identified 7 species of fungus

and 4 species of oomycetes. In the previous work, no Merlin element was identified in fungi and

plants. The phylogenetic trees showed no resolution for the more basal relationships. This may be a

result of a great time of divergence of these sequences.

Conclusion: Although we observed a more extensive distribution of Merlin than it was observed

before, it is rather discontinuous. This may indicate the Merlin is an ancient element in eukaryotes

with the possibility of past occurrence of horizontal transfer of the element between species of distinct

groups and/or loss of the element in some lineages.


Acknowledgment: CAPES, Fiocruz, UFPR


Evidence of transposable elements expression in Rhinella marina (bufo toad) specimens

submitted to stress conditions

LUDWIG, A. (1); SCHEMBERGER, M.O. (2); GAMA, J.M. (3); DUARTE, I. (3); GAZOLLA, C.B.

(3); LOPES, A.L.K. (3); MATHIAS, C. (3); PETTERS, D. (3); ZATTERA, M. L. (3) & BRUSCHI,

D.P. (3)

1. Instituto Carlos Chagas – Fiocruz-PR.

2. Universidade Estadual de Ponta Grossa (UEPG).

3. Universidade Federal do Paraná (UFPR).


Introduction: Transposable elements (TEs) are mobile components of eukaryotic genomes and are an

essential source of genetic variability being able to act as drivers in adaptation and evolution. Usually,

the response of a species to a novel environmental challenge is rapid and plastic and, in this sense, the

TEs activation can generate rapid genomic and functional diversification. Rhinella marina toad is an

invasive species well adapted to diverse environments in different parts of the world, and the TEs

compose 30% of its genome.

Methods: Based on this premise, we evaluated the transcriptional activity of TEs of R. marina. We

analyzed the RNA sequencing data, published by Gardner et al. (Dev Comp Immunol. 2018;88:114-

123.), from spleen tissues under two conditions: (1) specimens submitted to an acute immune

challenge of lipopolysaccharide (LPS) and (2) specimens submitted to LPS plus corticosterone, the

main glucocorticoid produced in amphibians under stress. The authors found 277 differentially

expressed genes (DEGs) from a transcriptome of 63,366 transcripts. We performed the detection of

TEs in the basic transcriptome and the DEGs sequences using the pipeline PiRATE. TE classification

was performed with PASTEC. TEannot was used to estimate the total proportion of TEs in the

transcriptome. Differentially Expressed TEs were used as queries in local blastn searches against the

R. marina genome. TEs copies were retrieved for basic characterization looking for the presence of

ORFs, expected protein domains and terminal repeats.

Results and Discussion: A total of 16,687 fragments of repeats were initially detected in the

transcripts (26.1% retrotransposons, 8.56% DNA transposons, 65.34% others). Potential host genes,

uncategorized and chimeric sequences were eliminated resulting in a library of 3,071 TE fragments

after clustering sequences by families. The cumulative coverage of TEs in the transcriptome was

17,128,877 bp (13.8%), and the most abundant TEs were from order LINE (4.18%) and DIRS

(3.26%). TIRs DNA transposons represented only 2.16% of transcriptome contrasting with their

genome coverage of 19.17%. From the DEGs, we observed one DIRS element upregulated in

treatment 2. Also, we found two LINE and two Penelope families differentially expressed in the

treatment 2 in relation to the treatment 1. Analysis of the genomic copies revealed that most of these

TEs are potentially active. Further study should be carried out to better understand the significance of

the gene expression; however, we can suggest the activation of these TEs in response to treatments.

Although the TE transcription was found in somatic tissue, it means they are potentially encoding and

could also be activated in the germline.

Conclusion: Our work indicates that several distinct families of TEs are transcriptionally expressed in

R. marina and could help to generate a rapid response of specimens to the environment. Also, we can

suggest that these TEs could be activated in the germinative cells producing raw material to be

mailto:[email protected]


selected and shaped by the evolutionary processes behind the success of this invasive species. Thus,

the TEs are important targets for investigation in the context of species adaptation.

Acknowledgment: This work is a result of practical activities developed together with the students

during the discipline “Tópicos especiais em genética III (Elementos Genéticos Móveis)” of the

Graduate Program in Genetics of the UFPR.


Cytotoxicity of bismuth nanoparticles (BiNPs) in the murine macrophage line

Raw 264.7

LUZ, J.Z. (1); MELA, M. (1); BEZERRA, A.G. (2); MACHADO, T. N. (2), OLIVEIRA RIBEIRO,

C.A. (1); FILIPAK NETO, F.F. (1)

1. Department of Cell Biology, Federal University of Parana, Curitiba, Brazil.

2. Department of Physics, Federal University of Technology-Parana, Curitiba, Brazil.


Introduction: Nanoparticles (NPs) have been widely used due to advance of nanotechnology in the

last two decades. Among metal NPs, bismuth NPs (BiNPs) are promising for different biomedical

applications. Toxicological investigation is a crucial step before application of NPs and although the

understanding of possible toxicological consequences of BiNPs is at early stage, studies indicated that

these particles can initiate subcellular, cellular and tissue toxicity in several experimental models.

Possible toxic effects of BiNPs may be related to the immune system, since immunomodulatory

effects of NPs have already been described. The murine macrophage Raw 264.7 cell line was used in

this study to evaluate the cytotoxicity of BiNPs.

Methods: The BiNPs were synthesized by laser ablation in solution (LASiS) and then characterized

by the ultraviolet-visible (UV-Vis) spectroscopy and dynamic light scattering (DSL). The cells were

cultured in high glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and

antibiotics. The assays to evaluate the cytotoxicity of BiNPs were divided into 3 categories:

investigation of non-specific cytotoxicity (MTT, crystal violet and trypan blue), investigation of

specific cytotoxicity (phagocytic activity, production of reactive oxygen (ROS) and nitrogen (RNS)

species) and related to the cellular mechanisms (DNA damage, incubation with phosphohistone

H2AX antibodies). The Raw 264.7 cells were exposed to BiNPs at concentrations of 0.01-50 mg.mL-

1.

Results and Discussion: The BiNPs were spherical in shape and had wide size distribution range

(higher prevalence between 25 nm and 60 nm). When exposed to 50 mg.mL-1 of BiNPs, cells had

altered parameters, such as reduction of conversion of MTT to formazan, decrease of cell adhesion

and increase of phagocytic capacity. However, in general there were no significant decreases of cell

viability (trypan blue), and of proliferation or changes in the levels of ROS and RNS in the exposed

cells.

Conclusion: Due to the considerable increase in phagocytic activity in both 24 and 48 h of exposure

to 50 mg.mL-1, this we suggest that BiNPs may cause immunotoxicity by influencing

proinflammatory responses, which reinforces the importance of research for the understanding of the

toxicological mechanisms before the biomedical application of these NPs.

Acknowledgement: We acknowledge the graduate program in cell and molecular biology of the

Federal University of Parana. This research was supported by CNPq (Brazilian Agency for Science

and Technology, scholarship) and Fundação Araucária de apoio ao desenvolvimento científico e

tecnológico do Paraná (Research Foundation of Parana State, research funding).


In vivo evaluation of the Decabromodiphenyl eter (BDE-209) effects in the

darbacazine treatment on the progression of lung metastasis in mice

MANUITT, P. (1); BISCAIA, S. (1); LIMA, T. (1); ALMEIDA, G. (2); LEÃO-BUCHIR, J. (1);

ROQUE, A. A. (1); OLIVEIRA, E. C. (2); TRINDADE, E. (1); OLIVEIRA C. (1).

1. Postgraduate Program in Cell and Molecular Biology, UFPR, Paraná, Brazil.

2. Graduate Program, University Uniandrade, Paraná, Brazil.

3. Universidade Tecnológica Federal do Paraná – Campus Dois Vizinhos, Paraná,

Brazil.


Introduction: In 2018 approximately 18.1 million cases of cancer were reported and are estimated

9.6 million cancer deaths. Two-thirds of cases can be related with some environmental factor as the

exposure to pollutants. Polybrominated diphenyl ethers (PBDEs) are brominated flame retardant,

hydrophobic, toxic to cells and bioaccumulative, and biodegradable into brominated compounds.

PBDEs are compounds described as "emerging pollutants" where the toxicity and persistence in the

environment are not completely understood. Decabromodiphenyl eter (BDE-209) is the most

prevalent PBDE found in the atmosphere and is detected in blood, semen, adipose tissue, breast milk,

umbilical cord blood and placental tissues of humans. Studies have shown that BDE-209 interferes

with tumor development in vitro increasing adenomas and follicular cell carcinomas of the thyroid

gland or breast cancer. However, nothing has been investigated regarding the possibility of exposure

to these compounds favor the cancer development interfering with cancer treatment as chemotherapy

with Dacarbazine 5- (3,3-Dimethyl-1-triazenyl) imidazole-4-carboxamide. This compound remains

the most widely used treatment for metastatic melanoma treatment. The aims of the present study was

to investigate if the previous exposure to BDE-209 alters negatively the effects of the chemotherapy

treatment.

Methods: 100 mice (50 males and 50 females) of 7 weeks old were separated in 5 groups and

subjected to five treatments as following: control, vehicle (corn oil), dacarbazine (80 μg / kg), BDE-

209 (1 nM) and BDE- 209 (1 nM) + dacarbazine (80 μg / kg). Treatment with BDE-209 (ten doses)

was conduct by gavage every 5 days. After treatment, murine melanoma B16F10 cells were injected

into the caudal vein. The groups treated with BDE-209 and vehicle were still exposed each 5 days.

The dacarbazine treatment was performed with intraperitoneal injection every 3 days. After 21 days of

cell inoculation, all animals were euthanized and the colonization of tumor cells in the lungs and the

general health condition of the animals were evaluated.

Results and Discussion: In the murine model of lung tumor colonization, each melanoma cell

generates an independent tumor in the lungs that could be identified and quantified. Significant

differences were observed in animals treated with dacarbazine were a lower tumor cell colonization

was found (20,11 ± 10,33 tumors), evidencing the effectiveness of treatment, but individuals

previously treated with BDE-209 showed a higher colonization of tumor cells including in animals

treated with dacarbazine (76,6 ± 20,68 and 80,63 ± 20,52 respectively). The exposure to BDE-209

lead to an increase in the number of cancer cells in the lung of the studied animals. This results

demonstrated the interference of environmental pollutants altering the prognosis of the disease.


Conclusion: The exposure to BDE-209 can potentially decrease the efficiency of Dacarbazine

treatment of melanoma in mice. This study show how pollutants described as not carcinogenic can

aggravate the prognosis of cancer.

Acknowledgement: This work was support by CNPq and CAPES.


Effect of TCDD and BDE-209 on cytotoxicity and expression of ATP binding

cassette: MDR1, MDR5, MRP1, MRP2, MRP4 in melanoma murino B16F1 cell line

MARCHI, M (1); MOGGIO, L.E (1); NETO, F.F.(1); SILVA FILHO, B.F. (1); OLIVEIRA

RIBEIRO, C.A. (1)

1. Universidade Federal do Paraná, Curitiba, Paraná, Brazil.


Introduction: Environmental contaminants 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 2,2 ',

3,3', 4,4 ', 5,5', 6,6'-decabromodiphenyl ether (BDE- 209), are denominated persistent organic

pollutants generated primarily during anthropic activities and have been constantly released into the

environment. Due to their lipophilic properties the exposure to these contaminants is frequent, starting

in the fetal phase and extending after birth and through adulthood. Melanoma is a type of aggressive

skin cancer, featuring metastatic ability and rapid progression. It is known that during chemotherapy

treatment there may be a decrease in the drugs effects due to increased expression and/or activation of

ATP binding cassette proteins. These membrane proteins act by recognizing different compounds and

promote the efflux of cellular metabolites, secretion of hormones, as well as efflux of xenobiotics out

of the cell. This property makes the drugs present in the treatment their effects minimized exactly by

the action of these carrier proteins. In the present study, we used the B16F1 murine melanoma line as

an experimental model to evaluate the role of the xenobiotics BDE-209 and TCDD in the cellular

efflux proteins.

Methods: The cells were cultured in high glucose DMEM medium supplemented with 10% fetal

bovine serum (FBS) and antibiotic and incubated at 37 ° C and 5% CO2 in a humidified atmosphere.

The assays for the cytotoxicity investigation of environmental contaminants were MTT, neutral red,

crystal violet and clonogenic. To evaluate the activity of the efflux proteins were performed the assay

MXR and analysis of gene expression the MDR1; MDR5; MRP1; MRP2 and MRP4, ABC carrier

protein genes. For the experiments, the cells were exposed for 24 hours and 15 days with BDE-209

and TCDD, at concentrations of 0.01; 0.1 and 1.0 nM/mL.

Results and Discussion: The results indicated that the BDE-209 changes different parameters in 24

hours of exposure, such as: lysosomal metabolism (↑~20%), gene expression of ABC transporters:

ABCB1 (↑~22%), ABCC1 (↑~10%), ABCC2 (↑~27%) and ABCC4 (↑~44%) and activity of the ABC

carrier proteins (↓~29%). With the exposure of 15 days, the following parameters were observed:

lysosomal metabolism (↓~13%), cell proliferation/adhesion (↑~10%), gene expression of ABCB1

(MDR1) (↓~3.5%); ABCB5 (MDR5) (↑~2.5%); ABCC1 (MRP1) (↑~3.5%); ABCC2 (↑~1.2%);

ABCC4 (~3.0%) and activity of the ABC carrier proteins (↑~30%). For the TCDD, the results also

showed alterations in the parameters in 24 hours of exposure as in the lysosomal metabolism

(↑~22%), cell proliferation/adhesion (↑ ~23%) and in the levels of ABC expression ABCB5 (↑~42%),

ABCC1 (↑~20%), ABCC2 (↑~15%) and ABCC4 (↑~33%) while for the 15 day exposure the changes

were in cell proliferation/adhesion (↓~11%) and ABCB1 gene expression (↓~0.2%); ABCB5 (MDR5)

(↓~1.3%); ABCC1 (MRP1) (↑1.2%); ABCC4 (MRP4) (↓~1.5%).

Conclusion: In general, the data presented suggest that the exposure to BDE-209 and TCDD may

potentiating the resistance of melanoma cells making them less sensitive to chemotherapeutic

treatment, mainly due to the activity and gene expression of ABC: PGP and MRP transporters. These

data reinforce the importance of understanding of the risk of human exposure to xenobiotics.


Acknowledgement: We apreciate Dra. Marianna Bóia and Dra. Sheila Maria Brochado Winnischofer

for their with contribution the project and Dra. Magda Clara Vieira da Costa Ribeiro and Dr. Silvio

Sanches Veiga by the collaboration. This research was supported by CNPq (Brazilian Agency for

Science and Technology).


QSOX1 extracellular interaction with plasma membrane proteins of aortic smooth

muscle cells

MARTÍNEZ, P. A. (1); NAKAO, L. S. (1)

1. REDOX Pathology Laboratory, Department of Basic Pathology, Biological Sciences Sector,

Federal University of Paraná, Curitiba - Brazil.


Introduction: Quiescin sulfhydryl oxidase 1 (QSOX1) is a predominantly secreted flavoprotein (b-

isoform) whose biological function is poorly understood. Because it is extracellular, it has been

demonstrated that QSOX1 contributes to the remodeling of the extracellular matrix and assembly of

extracellular proteins such as laminin and fibronectina, modulates adhesion and migration of tumor

cells and fibroblasts. In the cardiovascular system, QSOX1 was highly expressed in atherosclerotic

plaque and neointimal carotid tissue after balloon catheter injury, otherwise its deletion led to heart

failure. Overall, the role of QSOX1 in cardiovascular disease is still not fully understood.

Accordingly, our group have shown by in vitro assays that extracellular QSOX1 induces proliferation

and migration of aortic smooth muscle cells (VSMC) independently and dependently sulfhydryl

oxidase activity, respectively. Thus, our hypothesis is that QSOX1b binds to proteins on the cell

surface and activates the signaling that leads to these two processes.

Methods: We standardized experimental conditions to crosslink recombinant QSOX1b and VSMC

proteins using formaldehyde as a crosslinking agent. Recombinant cross-linked cell membrane

fractions were isolated, enriched in Ni-NTA resins and analyzed by SDS-PAGE, western blotting and

mass spectrometry to identify possible ligands. Indirect immunofluorescence assays were also

performed to demonstrate these ligations.

Results and discussion: Crosslinking with formaldehyde allowed stabilization and capture of

recombinant QSOX1b bound to VSMC membrane. The presence of the recombinant QSOX1b was

observed in the membrane fractions of the VSMC incubated with the recombinant, indicating the

binding of this to some plasma membrane component, both by silver nitrate-stained gels and by

western blotting. Still preliminarly, the results obtained in the mass spectrometer reveal a large range

of potentially candidate membrane proteins to interact with QSOX1b, with roles in the reorganization

of the actin cytoskeleton, cell-cell and cell-matrix adhesion, membrane repair and different signaling

pathways. QSOX1b binding to cell surface was also demonstrated by indirect immunofluorescence.

Adhered or suspended VSMC were incubated with QSOX1b and presented a positive immunostaining

with anti-QSOX1 antibody. The images acquired in the confocal microscope showed an intense and

distinctive staining of the surface but also intracellularly in cells incubated with 1μM of recombinant

wild type and mutated QSOX1b, indicating that the recombinant may be internalized by an yet

unclear mechanism. The immunostaining was absent when incubations were done at 4°C. Because

cell proliferation and migration appear to depend on different signaling pathways, understanding this

phenomenon of internalization will be important in differentiating the molecules involved in these

pathways.

Conclusions: Together, our data show that QSOX1b interacts with cell surface molecules that are

possibly responsible directly or indirectly for proliferation and migration, but the internalization of the

protein may also be involved in these processes.


Acknowledgment: to research funding agencies CAPES and CNPq and the Graduate Program in Cell

and Molecular Biology of the Federal University of Paraná.


Use of Alprazolam during pregnancy: impact on the central nervous system of the

offspring

MASCARENHAS, F.N.A.P. (1); SILVA, N. F. (1); REIS, L.T.M. (1); VIEIRA, L.G. (2); HIRANO,

L.Q.L (3); ZANON, R. G. (1)

1. Institute of Biomedical Sciences, Federal University of Uberlândia, MG, Brazil.

2. Institute of Biological Sciences, Federal University of Goiânia, GO, Brazil.

3. Faculty of Agronomy and Veterinary Medicine, University of Brasília, DF, Brazil.


Introduction: The use of benzodiazepines (BZDs) during pregnancy can lead to cellular changes in

the Central Nervous System (CNS). Despite this, these medicines, especially Alprazolam, are widely

used in the gestational period to treat anxiety and insomnia. Thus, the present project aimed to

investigate the effects of Alprazolam on the CNS of Wistar rat puppies exposed to the drug.

Methods: Thirty animals were used (CEUA protocol 014/17), from which eight females were stayed

with two males, which were mated and raised an offspring. The pregnant females were divided into

three groups (n = 8): control (CT, 0mg / animal), treatment 1 (T1, 1.25mg / animal) and treatment 2

(T2, 30mg / animal). Alprazolam was administered orally ten days before copulation and during the

entire gestation. After the birth of the offspring, the number of individual per litter, the percentage of

males and females per litter, the weight of the mothers and offspring, the external macroscopic

alterations of the encephalon, the morphology and the cellular density of the motor cortex and the

olfactory bulb, were evaluated.

Results and Discussion: The number of puppies per litter was similar between groups, but the

number of males per litter was smaller when compared to the amount of females, this occurred in all

groups, with no relation of sexing to the treatment. Body weight of mothers and offspring was similar

between groups. Regarding the encephalon, no external macroscopic changes were identified. The

motor cortex showed a tendency to increase the number of cells in the T1 group when compared to

the other groups (p = 0.09), but with the similar cellular area between the groups. In the olfactory

bulb, similar cell number and cell size were observed between groups. It can be explained the

tendency of a greater number of cells in the puppies of the T1 group by the presence of a gliosis or

neurogenesis in the motor region, in the attempt to repair some damage caused by the medicine.

Conclusion: Treatment with Alprazolam did not appear to affect the general morphology of the motor

cortex or the olfactory bulb of rats. However, complementary studies need to be performed in order to

try to clarify the trend of higher numbers of cells present in the CNS of these puppies. Weight and

number of live births were not affected by treatment.

Acknowledgement: This study was financed in part by the Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.


Cellular and molecular characterization of mesenchymal stem cells derived from

pluripotent cells and prospection of applications in regenerative medicine

MATOS, B. M. (1), STIMAMIGLIO, M. A. (1), CORREA, A. (1,2)

1. Program in Biosciences and Biotechnology, Carlos Chagas Institute (ICC/FIOCRUZ-PR).

2. Program in Cellular and Molecular Biology, Federal University of Paraná.


Introduction: Stem cells have been studied for their properties on tissue regeneration and functional

recovery of injured organs. Among those properties, intercellular communication through the release

of extracellular vesicles (EVs) have been pointed as one of the main mechanisms inducing tissue

healing. The EVs are able to actively modulate the cellular metabolism and promote the recovery of

lesions. Mesenchymal stem cells (MSCs) are prominent in this context as they promote the increase of

activity and viability of several cell types through distinct mechanisms, including communication via

EVs. However, biological samples of MSC bring great variability in behavior and viability while in

culture, making difficult the continuous and unswerving acquisition of EVs. Therefore, it is necessary

to establish a standardized and permanent protocol for the generation of stable cell cultures that allows

the continuous isolation of the vesicles. In this sense, the use of pluripotent stem cells in derivation

protocols for generation of MSCs is an alternative, since pluripotent cells maintain their

characteristics consistently under suitable culture conditions.

Methods: This project proposes the use of pluripotent stem cells to obtain MSCs based on exposing

the cells to specific culture conditions such as the presence of growth factors and other biological

molecules. The transcriptomic, morphological properties and surface proteins of generated cells will

be characterized by polysome profiling, electronic microscopy and flow cytometry, respectively.

Generated MSC populations will be expanded and their EVs isolated and characterized regarding their

molecular content by small RNA sequencing and mass spectrometry. The transcriptomic and

proteomic data will be compared to other cell types formerly characterized in the literature. This

project also proposes the evaluation of the therapeutic potential of both cells and EVs through in vitro

anti-inflammatory, anti-apoptotic and pro-angiogenic assays and in vivo skin wound healing models.

On the other hand, culture on 3D printed devices will be tested to observe the adaptation of the cells,

release of EVs and adsorption of the vesicles in the scaffolds.

Results and Discussion: Pluripotent stem cells (HES3) grown as monolayers or as embryonic bodies

were subjected to protocols with different combinations of type I collagen, dexamethasone and

ascorbic acid. Cells that look like MSC were obtained from one of the protocols tested. After 14 days

of derivation protocol, the cells were successfully expanded and passed two times until a preliminary

evaluation for MSC surface marker and others used as control. The derived cells were positive for

CD73 and negative for CD31, as expected. It was not possible to determine the positivity of a marker

for MSCs, CD105.

Conclusion: As up to now, we cannot state that we have derived MSCs from pluripotent stem cells.

However, our preliminary results are encouraging to further testing the same and new protocols.

Acknowledgement: We thank the Stem Cell Research Group, the Program in Biosciences and

Biotechnology and the Carlos Chagas Institute (ICC/FIOCRUZ-PR) for the opportunity project

development, and the funding agencies CAPES, Cnpq and Araucária Foundation for financial support.


Selective cytostatic effect on human melanoma cell line by a fucose-rich sulfated

polysaccharide from marine algae

MAXIMO, A. I. (1); REIS, M.B. (1); OLIVEIRA, C.C. (1); ROCHA, H.A.O. (2); TRINDADE, E.S.

(1)

1. Cell Biology Department, Laboratório de Células Inflamatórias e Neoplásicas e Laboratório

de Investigação de Polissacarídeos Sulfatados, Universidade Federal do Paraná (UFPR).

2. Biochemistry Department, Universidade Federal do Rio Grande do Norte(UFRN).


Introduction: Cancer comprises a heterogeneous group of diseases that share some characteristic and

unifying features, like the presence of abnormal cells that grow extensively, without stop, and beyond

their original boundaries. Melanoma is a challenging type of cancer. According to National Cancer

Institute latest Cancer Statistics Review, patients with stage IV metastatic melanoma have a 5-year

survival of approximately 18%. In terms of mutations patterns, cutaneous melanoma can be

categorized into four genetic subgroups, based on the most frequent mutation occurrence: BRAF

(~52%), RAS(~28), NF1 (~14%) and triple wild-type melanomas. Treatment scheme is frequently

based on prevalent mutation, with target therapy and immunotherapy being preferred. Despite all

scientific progress on treatment alternatives, non responsive phenotypes and resistance remains

significant obstacles Natural substances are promising candidates for antitumor therapies

development. Many studies, conducted in different experimental models, have found admirable

anticancer properties for some fucose-rich sulfated polysaccharides, components of marine algae,

such as antiproliferative (cytostatic effect) and proapoptotic (cytotoxic effect). Fucan A, the most

abundant among the three fucans type synthetized by brown seaweed Spatoglossum schröederi, is a

xylofucoglucoronan molecule of 21 kDa. So, our aim here was to investigate Fucan A cytostatic and

cytotoxic effects on human normal and metastatic melanoma cell lines.

Methods: A375 (BRAF mutant), SKMEL-147 (NRAS mutant), CHL-1 (triple-wild type) melanoma

cells; NGM (melanocyte) and THP-1 (monocyte-derived macrophages) normal cells were exposed to

a range of fucan A concentrations (1 – 100 µg/mL), for 24 to 72h. Cell viability was evaluated by

neutral red uptake assay and proliferation rate was assessed by crystal violet staining.

Results and Discussion: cell viability was not affect by polysaccharide presence, but 72h exposure to

fucan A significantly (p<0.01) decreased CHL-1 cells proliferation rate. No other melanoma or

normal cell lines were affected. Since BRAF mutations are the most common in melanoma, patients

with advanced stages disease are typically tested for this biomarker first, and the information is used

to guide treatment plans, with targeted therapy drugs such as BRAF inhibitors. Although NRAS and

NF1 mutated melanoma are responsive to immunotherapy, drugs that target these phenotypes are also

being studied and may have approved targeted therapies soon. But, due to the variety of other

mutations possibilities, triple-wild type patients have no defined good options targeted therapy yet.

Conclusions: considering that fucan A had no cytotoxic effect on any cell line tested, but reduced

proliferation rate of triple-wild type CHL-1, we can suggest that fucan A has no hazard effect on

normal cells and could be a good antitumor agent candidate to treat the most neglected case of triple-

wild type melanoma.

Acknowledgement: CAPES and CNPq.


Commercial pectin alters viability and cell cycle distribution of glioblastoma cells

MAZEPA, E. (1); AMARAL, S.C. (2); SPISILA, L.J. (2); SILVEIRA, J.L.M. (1); TRINDADE, E.S.

(1); WINNISCHOFER, S.M.B. (1,2)

1. Federal University of Paraná, Molecular and Cell Biology Department, 81.531-980, Curitiba-

PR, Brazil

2. Federal University of Paraná, Biochemistry and Molecular Biology Department, 81.531-980,

Curitiba-PR, Brazil


Introduction: Commercially pectin is usually produced from waste pulp during fruit juice production

(apple pomace or citrus peel). Recently, accumulated evidence has demonstrated that citrus pectin

(CP) exhibits inhibitory effects against cancer cells in breast, colon, prostate, melanoma, and multiple

myeloma cancers. However, no report has been found on the antitumor potential of CP against

glioblastoma cells. Glioblastomas are the most common type of primary brain tumors in adult. The

average survival rate for glioblastoma patients is about 15 months. In the present study, we evaluated

cytotoxic activity and cell cycle modifications to elucidate the effect of CP on glioma cells.

Methods: T98G and U87MG cells were exposed to 25, 100, and 400 µg mL-1 of a commercial citrus

pectin (CP, Sigma) for 48 h. Cell cytotoxicity was determined by crystal violet assay and results were

expressed as percentage of cells relative to control (100%) ± SD of 3 independent experiment each

one in sextuplicate (p<0.05). After treatment with CP (25, 100, and 400 µg mL-1) for 48 h, T98G and

U87MG cell cycle distribution was evaluated by flow cytometry through propidium iodide (PI)

staining. Samples were run in a FACS System and analyzed with BD Accuri C6 software. The results

were expressed as percentage of cells in each cell cycle stage relative to control (100%) ± SD of 2

independent experiment each one in duplicate (p<0.05). Cell viability of T98G and U87MG was

measured by crystal violet assay. Crystal violet dyes the DNA of adhered and fixed cells.

Results and Discussion: In this work, all concentrations of CP (25, 100 and 400 µg mL-1) decreased

significantly the percentage of adhered T98G cells at 21, 15 and 43%, respectively. On the other hand,

U87MG viability was decreased in 9.8% only with the higher concentration. In order to investigate

the cytotoxic action mechanism induced by CP, T98G cells were treated with CP (25, 100 and 400 µg

mL-1) and submitted to cell-cycle analysis. All concentrations tested arrested cells at S phase (4.2, 4.7

and 5.9%, respectively), with concomitant decrease of cell population in G2/M phase (5.0, 5.0, and

2.8%, respectively). In addition, cell cycle analyses showed a higher proportion of fragmented DNA

when compared to control, induced by 100 and 400 µg mL-1 concentrations (3.3 and 3.9%,

respectively). A similar effect was observed for U87MG after treatment with 100 and 400 µg mL-1:

2.4 and 3.3% of cells arrested at S phase, and 3.1 and 3.8% of decrease of cell population in G2/M

phase. The percentage of fragmented DNA in U87MG cells was detected only with the higher

concentration treatment (3.2%, CP 400 µg mL-1). Both cell lines presented fragmented DNA after

treatment with higher doses of CP, suggesting a cell death mechanism.

Conclusion: In this study, a commercial citrus pectin (CP) presented cytotoxic effect in T98G and

U87MG human glioblastoma cell lines. The treatment with CP promoted cells arrest at S phase and

decrease of cells percentage at G2/M phase, effect observed for both cell lines. Besides that, the

presence of fragmented DNA suggests apoptotic cell death mechanism, and to confirm this, further

studies are in progress.


Acknowledgement: The authors gratefully acknowledge the following Brazilian agencies for

financial support: National Council for Scientific and Technological Development-CNPq

(309225/2018-3; 141692/2018-9); Coordination of Superior Level Staff Improvement-CAPES

(88887.354741/2019-00) and Federal University of Parana-Brazil-UFPR.


Founder effect of cancer-associated genetic variants within the Mennonite population

reveals a new cell stress-variant of the ROS-activated ZBTB10 gene

MEISSNER, C.G. (1); WEIHERMANN, V. (1); STRUCK, G.T. (1); NABHEN, J.J. (1); OLIVEIRA,

L.C. (1); LOPES, F.L. (1); BOLDT, A.B.W. (1)

1. Federal University Of Paraná; Human Molecular Genetics Laboratory, Genetics Department,

Federal University Of Paraná.


Introduction: The Mennonites are a Christian European Anabaptist group with almost 500 years of

demographic isolation and at least three bottleneck events, with a possible founder effect for cancer

susceptibility. Objective: To evaluate cancer-associated coding mutations in the Brazilian Mennonite

population.

Methods: 433 Mennonites (M) and 65 non-Mennonites (NM) from Curitiba (PR), Witmarsum

(Palmeira-PR) and Colônia Nova (Aceguá-RS), were interviewed between October/2016 and

December/2018. Whole-exome sequencing data was available for 136 Mennonites (10 with a history

of cancer and 94, without). We selected the variants with Polyphen-2 score≥0.80 and CADD≥20,

passing Hardy–Weinberg equilibrium (p<10-6) and call rates >99%. A total of 27304 missense and 50

start lost variants were evaluated for cancer association with multivariate logistic regression (PLINK

v1.9).

Results and Discussion: 81.2% of the participants had familiar cancer history, while 11.7% have

been diagnosed with cancer, of which 27 were women (23 M, 4 NM) and 32, men (M). 19

Mennonites (5.23%) had personal basal cell carcinoma (BCC) cancer history (in Europe, the

estimated incidence per year is 0.098%). Of those affected, 68.4% reported sun exposure for more

than 30 minutes daily, but only 36.8% reported to presently use sunscreen. There was no evidence for

a founder effect of common, previously described BCC-associated polymorphisms, identified in the

exomes: rs11170164 (p.Gly138Glu) in the KRT5 and rs34135067 (pAla340Ser) in the SUFU genes.

Instead, 25 rare missense mutations were enriched among cancer-affected participants (OR>10,

P<0.01). Of these, 18 (72%) presented higher frequency (P<0.05) or were not reported before (8 or

28%) in non-Finnish Europeans and Brazilians. Seventeen (68%) of the mutations belong to genes

previously implicated in cancer, none currently screened in classical cancer gene panels. In particular,

a new variant in the ZBTB10 gene was found in two unrelated, relatively young male individuals with

recurrent BCC episodes. ZBTB10 (zinc finger and BTB domain containing 10) is a transcriptional

repressor that competitively bind GC-rich cis-elements and displace Sp (specificity regulatory

proteins) resulting in decreased Sp-regulated prooncogenic gene expression. Its translation is also up-

regulated by reactive oxygen species, through Myc repression and decreased expression of cMyc-

regulated microRNA miR-27a.

Conclusion: BCC was over five times more frequent among Mennonites than Europeans, possibly

due to of a founder effect increasing frequencies of rare, high-impact variants, revealing new targets

for cancer prevention and therapy.


Evaluation of food behavior, serum and biometric mother’s parameters of obese rats

puppies to metabolic imprinting by small litter

MIYASATO, J. P. (1); COMOTTI OLIVEIRA, B. (2); RUGINSK, B. (1); FISCHER, S. V. (1);

COUTINHO, D. S. S. (1); MARTINS, C.G. (1); IAGHER, F. (1); APPEL, M. H. (3); FERNANDES,

L. C. (1); NALIWAIKO, K. (2)

1. Universidade Federal do Paraná, Departamento de Fisiologia (Paraná, Brazil).

2. Universidade Federal do Paraná, Departamento de Biologia Celular e Molecular (Paraná,

Brazil).

3. Universidade Estadual de Ponta Grossa, Departamento de Biologia Estrutural, Molecular e

Genética (Paraná, Brazil).


Introduction: Interferences during critical periods of development, such as during gestation and

lactation, can cause metabolic changes such as obesity in the litter. Studies have shown that maternal

nutrition can affect animal’s life, such as malnutrition during gestation and lactation can result in

obesity of the puppy in adult life. Due to the importance of the gestational and lactational phase, it is

relevant to evaluate the feeding behavior as well as the body mass of the mothers during these phases.

The metabolic imprinting by small litter induces obesity to pups during lactation through

overnutrition.

Methods: Thirty females and ten males (Wistar 90 days old) were used for mating in the proportion

of 3:1/box. After confirming the pregnancies the females were separated by box until the end of the

lactation. The body mass of the mothers was evaluated twice weekly during gestation and lactation,

and feed consumption 3 times per week during lactation. The correlation of the body mass of the

mothers with the number of pups born was performed. On the first postnatal day, the litters were

adjusted to 10 pups per mother; on the third day the experimental groups were reduced to 3 pups,

giving the groups: (MNL, mother normal litter) with 10 pups (PNL, pups normal litter) and (MSL,

mother small litter) with 3 pups (PSL, pups small litter). The body mass of the pups was evaluated on

the first postnatal day and at 21 days of age. After the lactation, the mothers were separated from the

pups and fasted for 12 hours to perform the biochemical parameters of the plasma as total cholesterol,

glucose and triglycerides, which were dosed by commercial colorimetric kits.

Results and Discussion: During gestation the body mass of the MNL and MSL mother were not

different. The MSL mother showed 68.9% lower feed intake than the MNL mother during lactation,

this is probably due to the lower number of pups. As a consequence, this directly influenced the body

mass, which from the third lactational week the MSL mother had a lower body mass of 10.26% versus

MNL mother. These data suggest that the size of the litter interferes with the dietary intake of the

mother, probably related to the lower energetic demand to feed a smaller number of pups. The body

mass of the mothers compared to the number of pups born, the data didn’t show the relation between

the largest body mass and the largest number of pups. The evaluation of the biochemical parameters,

the mothers didn’t differ, showing that all the mothers received the same conditions. The evaluation of

the body mass of the pups on the first postnatal day, there was no difference. However, at 21 days of

age, PSL pups showed a 27.52% increase in body mass versus PNL pups. This shows before litter

reduction, all pups were kept under the same conditions and therefore had the same body mass. But

after the litter reduction PSL pups had a greater body mass gain, evidencing that metabolic imprinting


by litter size reduction in lactation induced the overweight in the PSL pups, probably causing an

imprinting and inducing a metabolic disorder generating an energy imbalance and greater body mass

gain. This reinforces the importance of the gestational and lactational period as important

developmental stages in the life of the puppies.

Conclusion: Reduction of litter size in the lactational period can affect the behavior of the mother

affecting the alimentary interest and reflect in a lower body mass gain. Interferences in critical periods

of development such as litter size reduction during lactation were able to induce overweight in pups.

Acknowledge ment: CAPES e CNPq.


The resistance of neuroblastoma cells submitted to different conditions of stress in vitro

MONDINI, G. (1); COGO, S. (2); ELIFIO-ESPOSITO, S. (1,2)

1. Bacharelado em Ciências Biológicas, Pontifícia Universidade Católica do Paraná.

2. Programa de Pós-graduação em Ciências da Saúde, Pontifícia Universidade Católica do

Paraná.


Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor in children with

highly clinical heterogeneity. To high-risk patients, chemotherapy is obligatory, although treatment

may not be effective due to resistance mechanisms developed by the tumor cells. Survivin (SVV) is

an antiapoptotic protein, related to cellular resistance to programmed cell death (PCD) and studied as

a potential target to the development of new treatments. A characteristic of the most tumors is the low

level of oxygen, called hypoxia. The Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that

functions as a master regulator of oxygen homeostasis. The objective of this work was to evaluate the

resistance of two neuroblastoma cells lines to different conditions of stress.

Methods: SK-N-SH and SH-SY5Y cell lines were submitted to different conditions of stress in vitro:

growth factor deprivation (incubation with DMEM supplemented with FBS 0.5, 1, 1.5 and 2%),

hypoxia (1% and 5% O2), and by the incubation with the topoisomerase inhibitor topotecan, in

different times of incubation. Viability was assessed by the methylene blue assay. Apoptosis was

determined by flow cytometry with the staining with Annexin-V7AAD kit. 7AAD was used for cell

cycle evaluation. Immunofluorescence was used to a morphological evaluation of the expression of

SVV with no stress condition.

Results and Discussion: The viability assay was used as a screening to analyze the susceptibility of

every cell line to the stress conditions. SH-SY5Y was the most resistant, as it was able to grow even at

the lowest FBS concentrations after 72 hours. Cell cycle analysis demonstrated no significant change

in hypoxia and FBS deprivation, and the apoptosis assay showed the same. Treatment with topotecan

induced a modest apoptosis response with 16% of cells positive for annexin-V. At the same

conditions, SK-N-SH cells were more susceptible over time in all of the stress conditions assayed,

with a reduction of 50% of viable cells in 24h in hypoxia 1%, compared to normoxia (p<0.05), or FBS

deprivation for 72h, compared to FBS 10% (p<0.001). Treatment with topotecan presented 81% of

annexin-V positive cells and a cell cycle arrest at the S-phase. The SK-N-SH cells have a non

differentiated phenotype, showing a bigger proliferative and metastatic potential, activating the cell

cycle constantly and being more sensible to the stresses conditions, differently of SH-SY5Y.

Immunofluorescence was used to a morphological evaluation demonstrating the SVV with no stress

condition in both cell lines.

Conclusion: The different stress condition simulate the tumor microenvironment and can help us to

understand better and assist the science to develop new treatments.

Acknowledgement: The authors thank CAPES/COFECUB, CNPq and Fundação Araucária-PR for

financial support.


Effects of gradual temperature increase on energetic metabolism of Antarctic fish

Notothenia rossii

NEUNDORF, A. K. A. (1); MARINS, E. A. (1); DE SOUSA, M. R. D. P. (1); KANDALSKI, P. K.

(1); GUILLEN, A. C. (1); PRESTES, J. G. (1): MOURA, M. O. (2); DONATTI, L. (1)

(1) Department of Cell Biology, Federal University of Paraná, Curitiba, Paraná, Brazil.

(2) Department of Zoology, Federal University of Paraná, Curitiba, Paraná, Brazil.


Introduction: Global analyses have reported accelerated warming in the Antarctic Peninsula, which

has presented, in recent years, average temperatures higher than double compared to previous

analyses. Due to stability of Antarctic environment, which maintains temperatures close to 0 °C,

organisms living in the region present adaptations to low temperatures. However, successive

environmental changes may damage their cellular metabolism, affecting, in long-term, their survival.

Thus, the present study evaluated the influence of gradual temperature increase in the energetic

metabolism of carbohydrates and lipids in muscle and heart of the Antartic fish Notothenia rossii

(Notothenidae).

Methods: For this pourpose, fishes (n = 56) were exposed to a gradual increasing of temperature

(0.5°C every 24 hours) from 0°C to 2°C, 4°C, 6°C or 8°C, remaining for 96h at these temperatures.

For each experimental group there was a negative control group maintained at a temperature of 0°C.

Sampling was n=7 individuals/per bioassay. The energetic metabolism enzymes analyzed were

hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH),

citrate synthase (CS), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH), from

carbohydrate metabolism; carnitine acyltransferase (CPT) and β-hydroacyl CoA dehydrogenase

(HOAD), from lipd metabolism; and intermediary metabolites such as glycogen, lactate, pyruvate and

total proteins.

Results and Discussion: Animals exposed to 4 and 8°C, showed a decrease concentration of muscle

glycogen, which may be related to the increase of PFK activity and pyruvate concentration at 8°C,

which can also explain the increase of lactate concentration. The increase in muscle lactate levels may

indicate an alternative use of pyruvate oxidation pathway as an organism response to hypoxia, caused

by the oxygen level reduction in water during its warming. The muscular HOAD activity decreases in

N. rossii at 6 and 8 °C in relation to other temperatures. In the heart, the decrease at 4°C and an

increase at 6°C of the HK activity explains, respectively, the increase and the decrease of the

glycogen concentration. The increase in cardiac CS activity at 8°C can be corroborated by the

increase in pyruvate concentration, however, PFK had decreased activity at 8°C (as well as at 6°C),

which may indicate that pyruvate used is originated in other paths than that of carbohydrates.

Conclusion: The observed results suggest thermal plasticity of N. rossi. Nevertheless, gradual

increase of temperature, even in conditions of acclimatization, may affect energetic metabolism of

carbohydrates and lipids in heart and white muscle.

Acknowledgement: We thank the following groups for their support: the Brazilian Ministry of

Environment (MMA), the Ministry of Science, Technology and Innovation (MCTI), the National

Council for the Development of Scientific and Technological Research (CNPq), the Brazilian Federal

Agency for Support and Evaluation of Graduate Education (CAPES) and the Secretariat of the Inter-


ministerial Commission for the Resources of the Sea (SeCIRM). The authors would like to thank Dr.

Edith Susana Elisabeth Fanta (in memoriam) and Dr.Yocie Yoneshigue Valentin, coordinator of the

National Institute of Antarctic Science and Technology of Environmental Research (INCT-APA), for

all their help and encouragement during the performance of the present work.


Detection of cross-reactivity of anti-IgG from Trypanosomatid-infected rats and cattle

by immunofluorescence

NEVES, G.B. (1,3); NASCIMENTO, L.F.N. (1); RAMOS, C.J.R. (1); MARQUES, J. (1); LUZ, S.P.

(2); MILETTI, L.C. (1)

1. Universidade do Estado de Santa Catarina, UDESC, Lages - SC, Brazil.

2. Centro Universitário UNIFACVEST, Lages - SC, Brazil.

3. Universidade Aberta do Brasil, UAB/UFSC, Pato Branco -PR.


Introduction: Trypanosomes found in mammals, including humans, belong to the genus

Trypanosoma, to the Trypanosomatidae family and to the order Cinetoplastida (CARNES et al.,

2015). Trypanosoma evansi and Trypanosoma vivax belong to the salivary section (HOARE, 1972)

and to the subgenus Trypanozoon and Dutonella, respectively. Transmission occurs the inoculative

way of the trypomastigote form, present in the salivary glands of hematophagous insects, such as

tabanids and Stomoxys spp. (SILVA et al., 2002). These are protozoa responsible for diseases with a

negative economic impact on livestock (OSÓRIO et al., 2008; JITTAPALAPONG et al., 2009). They

are distributed throughout the national territory and a large variety of mammals are hosts of the

parasites, especially the equines and cattle (STOCO et al., 2017; DESQUESNES et al., 2013).

Furthermore, Trypanosomes are diagnosed by parasitological, immunological and molecular methods

(DAGNACHEW and BEZIE, 2015). The indirect immunofluorescence (RIFI) and enzyme-linked

immunosorbent assay (ELISA) are the most commonly used serological techniques for detecting anti-

IgG antibodies in trypanosomatids (MADRUGA et al., 2006). Moreover, RIFI is one of the most

applied techniques, however it is a time-consuming and laborious technique, besides the possibility of

cross-reactions with other trypanosomes (UILENBERG, 1998). In this context, the work aims to

detect anti-IgG antibodies T. evansi and T. vivax by indirect immunofluorescence, using sera from

infected rats and cattle and altering the antigen for both, allowing cross-reaction evaluation.

Methods: Preparation of the RIFI slides consisted of blood collection (1x106 trypomastigotes/mL) in

EDTA tube and centrifugation for six minutes (Celm®). The leukocyte (buffy-coat) was transferred to

a falcon tube and made two consecutive washes with physiological solution. The thick blood smear

was made using 30μL of blood, fixed with ice-cold acetone for 5 minutes and stored at -20°C. The

blood was collected in tube without anti-coagulant to obtain the rat serum (Rattus norvegicus Wistar)

and bovine (Bos taurus), submitted to centrifugation (Celm®) for 10 minutes, at 2000g at room

temperature and stored at -20ºC. The fluorescence test for the detection of anti-IgG antibodies was

performed according to the methodology described by Camargo (1966) with modifications. Slides

with fixed antigen were delimited with enamel. Besides, positive and negative control serum were

diluted in PBS at the 1:40, 1:80, 1:160 dilutions and distributed on slides, containing the antigen of

each trypanosome. The FITC-labeled (Sigma®) corresponding species conjugate was used at 1:500

dilutions. Lastly, the slides were read on a Nikon® epifluorescent microscope.

Results and Discussion: Fluorescence reactions were considered positive with ≥ 1:80 titers for both

and cross-reactivity was observed with low fluorescence intensity. According to Uilenberg (1998) the

test is not specific enough to distinguish between different species of pathogenic trypanosomes. This

is due to the great genetic similarity between the different species of trypanosomes, since many


members of the Trypanosomatidae family may share surface antigens, which would justify the

appearance of cross reactions (OBISHAKIN et al., 2014).

Conclusion: There was a cross reaction with low fluorescence intensity; the RIFI test in question

proved to be insufficient in specificity to distinguish the different species of pathogenic trypanosomes.

Acknowledgement: FAPESC, CAPES, CNPq, UDESC.


Rare endoplasmic reticulum stress variants associated with gastrointestinal disorders in

the brazilian mennonite population

OLIVERA, L.C. (1); DORNELLES, A.C. (1); MEISSNER, C.G. (1); VIEIRA, W.A. (1);

NISIHARA, R.M. (2); LOPES, F.L. (3); BOLDT, A.B.W. (1)

1. Laboratório de Genética Molecular Humana, Departamento de Genética, Universidade

Federal do Paraná, Brasil.

2. Laboratory of Molecular Immunopathology, Department of Clinical Pathology, Hospital de

Clínicas, Federal University of Paraná, Curitiba, Brazil.

3. Human Genetics Branch, National Institute of Mental Health, Intramural Research Program,

National Institutes of Health, US Department of Health and Human Services, Bethesda,

Maryland.


Introduction: Cellular stress of gut epithelial cells is one of the main reasons generating

gastrointestinal symptoms (GIS). We investigated familial aggregation and searched for rare GIS-

associated variants in exomes of the South Brazilian Mennonites, a Christian European Anabaptist

group isolated for approximately 20 generations, which passed through three populational bottlenecks.

Methods: Using a modified version of the questionnaire of the National Health Research of 2013, we

interviewed 598 Mennonites from Colônia Nova (RS), Witmarsum (PR) and Curitiba (PR). Exomes

of 144 Mennonites were sequenced to >30× coverage, using CGI’s combinatorial probe–anchor

ligation. We also performed serological screening, Anti-DGP IgA in 230 individuals and genetic

screening of HLA-DQ2, HLA-DQ8 in up to 351 individuals.

Results and discussion: Among 358 Mennonites, 34 (9.49%) reported family history of celiac

disease (CD), 16 being (4.47%) first-degree relatives. CD prevalence was 4,72% (compared to

maximal 1% worldwide). Eight individuals were identified by serological screening, representing

1.34% underdiagnosed cases. We found familial aggregation of GIS and diseases, with affected

individuals presenting a higher chance to develop thyroid dysfunction (OR=2.48, p=0.038), and a

trend to develop musculoskeletal symptoms (OR=2.11, p=0.061). Among 77 Mennonite cases and 59

sequenced controls, the intronic rs13306513*A of the LDLR (low density lipoprotein receptor) gene

was associated with an additive effect for increased GIS susceptibility, independent of sex, age,

diagnostics of celiac disease, lactose intolerance or irritable bowel syndrome (OR=10.44, P=0.029).

The rs202192345*G and rs116256283*T variants of the MUC2 and MUC6 mucin genes,

respectively, presented a trend towards an additive protective effect against gastrointestinal disorders

(OR=0.33, p=0.08, and OR=0.15, p=0.09, respectively). Minor allele frequencies of rs202192345

were higher among Mennonites, than among non-Finnish Europeans and Brazilians (P=0.000059).

Furthermore, the mucin family plays a number of important roles in intestinal homeostasis. Muc2 is a

major component of mucus and contributes to intestinal barrier integrity. The LDLR and MUC genes

further regulate endoplasmic reticulum stress and their deregulated expression may inhibit intestinal

wound healing.

Conclusion: Epidemiological results favor a founder effect to explain CD prevalence among

Mennonites. The LDLR gene may play a role modulating inflammation in the gastrointestinal tract, as

well as the identified mucin variants.


Evaluation of internalization kinetics and stability of the disintegrin and

metalloprotease ADAM23

OLIVEIRA, N. H. (1); MELO DE SOUZA, I. L. (1); ZANATA, S. M. (1,2)

1. Department of Cell Biology, Universidade Federal do Paraná, Curitiba, PR, Brazil.

2. Department of Basic Pathology, Universidade Federal do Paraná, Curitiba, PR, Brazil.


Introduction: ADAM23 is a type I transmembrane glycoprotein expressed in the brain during

embryonic and adult development, being expressed mainly as a cell surface protein with probable

location in intercellular contact areas and acting in processes of cellular adhesion, neurite growth and

dendritic arborization. Recent data obtained by the group (BORGONOVO et al., 2018) point to

ADAM23 as a partially resident protein in rafts, susceptible to leave these microdomains and follow

the endocytic pathway. Objective: Evaluate the internalization kinetics and stability of protein

ADAM23 in N2A cells.

Methods: Biotinylation: Biotinylation assays were carried out followed by internalization assay of

ADAM23. For these assays, 4x10 5 cells were used per condition. N2A cells were transfected with

pCMV6-ADAM23V2-FLAG (Origene) with human full-length ADAM23, were incubated with biotin

(sulfo-NHS-SS-biotin) for 30 min at 4oC to label the cell surface proteins, followed of free-biotin

blocking with 0,1 M glycin. Then, cells were incubated at 37oC for 15, 30, 45 and 60 min. Control

cells were incubated for 1 hour at 4oC. After internalization, cells were submitted to biotin stripping

with reducing agent GSH (glutathione) in order to remove non-internalized biotin from the cell

surface (GABRIEL et al., 2009; STAUTZ et al., 2012). Internalized biotinylated ADAM23 proteins

were then purified through affinity chromatography to streptavidin-agarose (pull-down) and analyzed

through western blot with anti-ADAM23. Cycloheximide Chase Assay: In order to evaluate protein

stability, 4x 10 5 N2A cells, per condition, were transfected with pCMV6-ADAM23V2-FLAG, full

length ADAM23. After 48h of expression cells were treated with cycloheximide and chased for 16h

(t= 0, 0,5, 1, 3, 6, 12, 24 h). After chase cells where lysed, quantified and subjected western blot

analyze.

Results & Discussion: Considering western blotting results, ADAM23 suffers maximum

internalization at 30 and 60 minutes, recycling partly back to plasma membrane at 45 minutes,

indicating that a recycling cycle occurs for this protein in N2A model. This finding suggests that

internalization kinetics differs significantly between members of ADAM family, considering that

ADAM12 maximum internalization seems to is reached after 1h (STAUTZ et al., 2012).

Cycloheximide Chase Assay results do not show a significant decrease of ADAM23 after 16h of

treatment, suggesting that this protein has a long half-life in N2A cells.

Conclusions: ADAM23 appears to be a long-life protein constitutively internalized in cycles of 30

minutes.


Importance of the familiarization process in the application of the maximum loaded

carrying test in rats

OTTO, E.L. (1,3); LIMA, M.C.A.M. (1,3); HESPANHOL, L.A. (1,3); SELSKI, S.B. (1,3);

RAMALHO, L.G. (1,3); CORDEIRO, G.C. (1,3); MARTINS, L.F. (1,3); FERNANDES, L.C. (2,3);

NALIWAIKO, K. (1,3)

1. Departamento de Biologia Celular e Molecular - UFPR.

2. Departamento de Fisiologia - UFPR.

3. Laboratório de Metabolismo Celular - UFPR.


Introduction: There are different ways of determining the loaded load in the first session of

progressive resistance training, through body mass or a previous test. The maximum loaded carrying

test (MLCT) has been used to determine this initial training load. Besides that, the pre-intervention

and post-intervention MLCT results can be compared to verify the improvement on the animal’s

performance. The animals perform the MLCT voluntarily, thus, the maximum value carried by the

animal does not necessarily represent its maximum capacity of resistance strength. The process of test

familiarization is intended to reduce possible errors in the measurement of the maximum load. The

importance of familiarization in humans is known and studies about this process are needed in

rodents. The objective of this study is to analyze if the familiarization with the test causes the animal

to improve its performance in the pre-intervention MLCT.

Methods: The research included 205 male Wistar rats. The apparatus for the test’s application

consisted in a 110 cm of height staircase, with an 80º inclination and a 2 cm spacing between the

steps, in the top there was a 20x20x20 cm wooden box, used as shelter. The adaptation process to the

apparatus was performed for 5 consecutive days. The animals were kept for 2 minutes in the shelter,

then 4 climbs were made (15, 30, 55 and 110 cm away from the shelter, respectively) with a 1-minute

interval between each ascent. The Maximum Loaded Carrying Test (MLCT) was done with a

weightless initial climb with a minute rest. The next climb was performed with an overload attached

to the base of the tail with tape, corresponding to 50% of the animal’s body mass. After this climb, an

overload corresponding to 10% of the animal's body mass was added at each attempt. The animals had

2 minutes of rest between each climb. The maximum loaded load was defined as the last overload the

animal was able to load. The MLCT 1 (familiarization) was performed 48 hours after the end of the

adaptation period and 72 hours later the MLCT 2 (pre-intervention) was performed. The normality of

the data was verified through the Shapiro-Wilk test. The data weren’t normal, so Wilcoxon's test was

used to compare the animals' MLCTs. The level of significance was set at p <0.05.

Results and Discussion: The performance of animals in the MLCT 2 was better than the performance

in the familiarization MLCT (p<0,0001) The results obtained in the familiarization MLCT may not

correspond to the ability of the animal due to the lack of custom with the process, and the increase of

the loaded load by the animals after familiarization may be due to the learning of the proposed test.

Conclusion: The results indicate that the process of familiarization with

the Maximum Loaded Carrying Test improved the performance of the animals in the pre-intervention

test, thus, its valid application to discover a more adequate load for the initial prescription of the

resistance training load.


Histone Deacetylase directing sign identification to Toxoplasma gondii apicoplast.

PAULA, D. C. de (1); FRAGOSO, M. S. I. (1); NARDELLI, S. C. (1)

1. Carlos Chagas Institute – FIOCRUZ/PR


Introduction: Toxoplasma gondii is an intracellular parasite, that belongs to the Apicomplexa

phylum. It is the causative agent of toxoplasmosis, being very harmful to immunocompromised

patients or in case of congenital toxoplasmosis. This parasite has an intriguing organelle acquired

from a second endosymbiosis, the apicoplast. Although this organelle is essential to the parasite’s

metabolism, and to evolution, most of its proteins became translated into the parasite’s body and

needed assistance to get into the apicoplast. Therefore, a directing system was achieved through the

combination of a signing peptide and a transit peptide. The control of the genetic expression of the

parasite, associated with epigenetic mechanisms, may be mediated by the functioning of histone

deacetylases (HDACs). This research has an interest in TgHDAC4, a histone deacetylase located at

apicoplast of Toxoplasma, and we focused on characterize the signals responsible for protein

targeting.

Methods: The directing signs were predicted using in silico tools SignalP 4.1 Server and ChloroP 1.1

Server and primers were designed to amplificate the sequences from the parasite’s cDNA, adding the

BglII (New England Biolabs) restriction site for classic cloning. The PCR (Polymerase chain reaction)

was performed using a high-fidelity enzyme. To characterize the directing signs, an YFP tag was

added to its C-terminal region by cloning in the superexpression plasmid ptub-TUB-YFP/sagCAT

(Gubbels et al.,2003). The correct position of the insert into the plasmid was confirmed by digestion,

sequencing and PCR. 24h pos transfection, the parasites were fixed, and an immunofluorescent assay

was realized.

Results and Discussion: The tools for signs prediction showed as result a sign peptide of 27 amino

acids and a transit peptide of 77 amino acids with 95% and 51% of probability. The insert was cloned

in the vector and to verify if it was on the correct position, a confirmation PCR was performed, using

two pairs of primers to amplificate regions of specific length. It was also performed a digestion using

PmlI and SphI (New England Biolabs) and both tests confirmed that the insert orientation was correct.

The plasmid was transfected and after 24 hours, the parasites were fixed and the immunofluorescent

assay was performed using DAPI for coloring the cell’s nucleus, parasite’s nucleus and apicoplast,

and anti-GFP to increase the YFP fluorescence. As result, even though it was not possible to

distinguish the apicoplast, it was possible to visualize a strong green fluorescence in the supposed

location of the organelle, in between the parasite’s nucleus and apical complex.

Conclusion: The immunofluorescent assay results resembled to an apicoplast marking, but it wasn’t

possible to guarantee its location since it was not possible to observe the DAPI marking. Thereby the

next step is to realize new assays using an apicoplast protein marker to colocalize the directing sings

in the organelle.

Acknowledgment: CAPES, CNPq, Fundação Araucária, FIOCRUZ/PR


Nanotoxicity of silver nanoparticles from biological syntesis

PASCHOAL, A.C.C. (1); REIS, G.F (2); PANAGIO, L. (2); NAKAZATO, G. (2); KULIGOVSKI, C.

(1); ABUD, A.P.R. (1); AGUIAR, A.M. (1)

1. Instituto Carlos Chagas – FIOCRUZ/PR.

2. Universidade Estadual de Londrina.



Introduction: Silver nanoparticles (AgNPs) are nanomaterials (NMs) of great medical and economic

interest because of their germicidal activity. Conventional physicochemical methods of synthesis are

costly and have the potential to generate toxic byproducts. Alternatively the biological synthesis of

AgNPs from fungal derivatives has been studied as an economically and environmentally favorable

method. However, it is necessary that parallel to the synthesis process, the study of its interaction with

biological systems is performed. In this perspective, the objective of the present project is to evaluate

the interaction of AgNPs synthesized by Fusarium oxysporum, here called AgNPs_BIO, with

mammalian cells, evaluating cytotoxic and genotoxic effects in vitro.

Methods:The AgNPs were biosynthesized by reducing ionic silver present in a silver nitrate solution

in AgNPs, through the conditioned medium of F. oxysporum, which contains compounds responsible

for the reduction and stabilization of NMs. Next, the nanoparticles were characterized by ultraviolet -

visible spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS),

energy-dispersive x-ray spectroscopy (EDX) and zeta potential. Biological assays for evaluation of

the AgNPs_BIO were performed with the Balb / c 3T3 cells of clone A31, which consists of a murine

fibroblast line used as a reference for in vitro cytotoxicity testing. First, IC20, IC50, IC80 and a

concentration which did not interfere in cell viability (sub-toxic) were determined by the neutral red

and MTT relative cell viability dyes. These concentrations were used as a basis for evaluating DNA

damage through the comet assay, for the ultrastructural analysis of the interaction between the

AgNPs_BIO with the cells by MET and for evaluation of the possible mechanisms of cell death, being

apoptosis evaluated through the kit of TUNEL and by determining the potential of the mitochondrial

membrane with JC10 dye. Necrosis was evaluated by differential staining with ethidium bromide and

acridine orange. Finally, the antimicrobial effect of AgNPs_BIO was tested with Staphylococcus

aureus, Listeria monocytogenes, Escherichia coli and Candida albicans by determining the minimum

inhibitory concentration (MIC).

Results and Discussion: The results confirm the presence of AgNPs in the colloidal solution, which

shows brown coloration and a plasmid peak of 450nm, with spherical morphology and heterogeneous

size (52.25 ± 19.95nm), with a zeta potential of -36 ± 9.64mV . In the analysis of the cytotoxic profile

an average value of IC50 of 1.91 ± 0.55μg / mL was determined and the other concentrations

mentioned were evaluated. Ultrastructural analyzes demonstrated that the AgNPs_BIO were not

identified within the cells, however images suggestive of autophagy and necrotic cell death were

observed, which were subsequently confirmed and quantified. Damage to cellular DNA was

demonstrated at the concentrations tested. For the microorganisms, AgNPs_BIO presented a

germicidal effect, with MIC values ranging from 3.12 to 12.5μg/mL and selectivity index less than 1.

With this, we concluded that it was possible to produce AgNPs_BIO through fungus F. oxysporum,

which present antimicrobial effect, but with low selectivity index.


Conclusion: Thus, it is observed the need to indicate alternative or biotechnological strategies that

guarantee a greater effectiveness in the function of AgNPs_BIO.

Acknowledgement: UTFPR, through professor Arandi Ginane Bezerra Jr and his student Thiago

Neves Machado for the characterization of the material. FIOCRUZ / PR microscopy platform,

through Bruna Marcon technologist and Tabata Klimeck technique. Platform of biotechnological

bioassays of FIOCRUZ / AM, through the technique Ivanildes Santos Bastos. Funding source:

CAPES, Araucária Foundation, CNPq, Fiocruz and UEL.


Exploring the role of a long non-coding RNA during the cardiomyogenic differentiation

of hESC

PEREIRA, I. T. (1); CHAN, S. (2); KYBA, M. (2); DALLAGIOVANNA, B. (1)

1. Laboratório de Biologia Básica de Células Tronco - Instituto Carlos Chagas, Fiocruz Paraná,


2. Kyba Lab - Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, USA.


Introduction: Low rate cardiomyocyte proliferation contributes to the limited heart regenerative

capacity. Considering that differentiation of pluripotent stem cells is a highly coordinated process

involving trigger, maintenance and coordination of gene expression patterns, advances in knowledge

about stem cell differentiation can support its potential use in regenerative therapy. Long non-coding

RNAs (lncRNAs), arbitrarily characterized as longer than 200 bp, play crucial roles in gene

expression regulation at different levels, including chromatin organization, transcriptional regulation

and post-transcriptional control. There is evidence that lncRNAs are involved in many biological

processes, including the regulation of cell differentiation. RNA-seq data of human embryonic stem

cell (hESC) cardiomyogenesis was preliminarily generated by our group and can be used to

investigate about differential expression of lncRNAs. This data was produced using polysome-bound

RNA and can also reveal lncRNA association with polysomes, which might indicate the coding

potential of these transcripts or could suggest their role in translational regulation. The lncRNA

LINC-X was found expressed in the cardiac progenitor stage and was chosen for the investigation of

its potential role during cardiomyogenesis. Therefore, the aims of this study are to characterize the

lncRNAs expression patterns along hESC cardiomyogenesis and their differential association to

polysomes, and also functionally analyze the lncRNA LINC-X.

Methods: Five time-points during cardiomyogenesis were used to generate our preliminary

polysome-bound RNA-seq data which was analyzed to search for differentially expressed lncRNAs

(log Fold Change≥±2, FDR≤0.05). Human ESCs (H1 cell line) were used to derivate a new cell line

carrying an inducible overexpression system containing the encoding sequence of LINC-X (H1

iLINCX). Also, CRISPR/Cas9 technology was used to knock-out LINC-X (H1 LINCX-KO). For

validation experiments, total and polysomal RNA were isolated, cDNA was synthetized by in vitro

reverse transcription and transcript levels were quantified by qPCR. Cardiomyogenic differentiation

was performed using small molecules that modulate WNT signaling and cardiomyocyte functional

analysis were performed, including differentiation efficiency, cardiomyogenic markers expression and

electrophysiological measures.

Results and Discussion: Cardiomyogenesis stages represented by pluripotency, embryoid body (EB)

aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte showed 6693 lncRNAs

expressed. We also identified differentially expressed lncRNAs associated with polysomes that

showed a clear temporal expression pattern. LINC-X was shown as highly expressed in cardiac

progenitor stage, including associated to polysomes. For its functional characterization, the new cell

line H1 iLINCX was derived and showed the maintenance of pluripotency after induction of LINC-X

expression by doxycycline, suggesting that this gene is not involved with pluripotency scape. On the

other hand, cells in which LINC-X was expressed during day 5 to day 10 of cardiogenic


differentiation showed improvement in maturation and more atrial physiological properties. In

addition, when H1 LINCX-KO cells were submitted to cardiomyogenic differentiation, they were able

to differentiate to mesoderm stage, but not to cardiomyocytes, suggesting an essential role of this

lncRNA in cardiac development.

Conclusion: Polysomal RNA-seq data showed dramatic differential expression of lncRNAs during

cardiogenesis, illustrating the molecular complexity of cardiac differentiation and suggesting not only

that lncRNAs could differentially associate with polysomes but also that they could play a crucial role

in the differentiation process. Between these genes, LINC-X showed a specific cardiac progenitor

expression and functional analysis suggests that it could have a crucial role in maturation and

determination of cardiomyocytes.

Acknowledgement: FIOCRUZ; Capes; CNPq; Fundação Araucária.


Protective response against toxic effects of Loxosceles spider venoms using recombinant

mutated phospholipases-D as antigens

POLLI, N. L. C. (1); ANTUNES, B. C. (1,2); DA SILVA, T. P. (1); VUITIKA, L. (1); JUSTA, H, C.

(1); MINOZZO, J. C. (2); VEIGA, S. S. (1); GREMSKI, L. H. (1)

1. UNIVERSIDADE FEDERAL DO PARANÁ (UFPR), CURITIBA - PR - BRASIL.

2. CENTRO DE PRODUÇÃO E PESQUISA DE IMUNOBIOLÓGICOS (CPPI),

PIRAQUARA - PR - BRASIL.


Introduction: Loxoscelism is the group of clinical symptoms triggered after brown spider bites. The

species most commonly involved in accidents in Brazil are L. intermedia, L. gaucho and L. laeta.

Phospholipases D (PLDs), or dermonecrotic toxins, can be considered the most important toxins in

their venom, since alone they can reproduce experimentally the main symptoms of loxoscelism. In

view of the limitations of serum therapy, a preventive strategy could be applied in endemic areas.

Moreover, the production of a second-generation serum with enhanced neutralization capacity even

when used several hours after the bite, with lower cost and less animal suffering is proposed here. In

this context, this work proposes the use of recombinant mutated PLDs as antigens to prevent and

reduce the onset of the major symptoms of loxoscelism.

Methods: Recombinant mutated PLDs (LirecDT1Y228A, LlrecDT1H12A/H47A and

LgrecDT1E32A/D34A) expressed in BL21(DE3)pLysS cells were tested for immunogenic potential

through Western Blot assay. Sphingomyelin cleavage was tested using Amplex Red® (Invitrogen).

Biological activity was evaluated by dermonecrosis assay. To evaluate the protective response elicit

by them, 2 groups of rabbits received 3 doses (30 days interval) with increasing amounts of PLDs and

Aluminum Hydroxide. Group 1 received 50, 100 and 300 µg of PLDs and group 2 100, 150 and 400

µg. Control animals received PBS and adjuvant in the same immunization schedule. 15 days after last

dose serum was collected and analyzed through ELISA and the protection of the vaccine was

evaluated. For this, 10 μg of Loxosceles venom was injected intradermally into a shaven area of rabbit

dorsum skin. Area of lesion was evaluated analyzing edema, erythema and bruise. Recombinant

mutated PLDs alone or enriched with venom were used as antigens to produce a second-generation

serum in rabbits, whose efficiency will be tested by in vitro and in vivo tests. In vivo assays were

approved by CEUA UFPR nº 1205 and 1238.

Results and Discussion: PLDs chosen as antigens showed a high immunogenic potential, did not

hydrolyze sphingomyelin in vitro and develop dermonecrosis in vivo, even with high masses (50μg).

Reductions of up to 93,41% in edema and a significant decrease in dermonecrose, erythema, bruise

were observed in immunized animals. Furthermore, the serum of these animals was analyzed by elisa,

showing a higher production of antibodies in animals of group 2.

Conclusion: PLDs without activity were successfully produced and showed a high immunogenic

potential. Animals that received the PLDs in the vaccine showed visual reductions in the lesions

caused by Loxosceles whole venom. However, this assay showed a better protection in animals that

received higher masses of PLDs, reinforcing the need to use larger masses of proteins in the vaccine

formulation to obtain a better immune response. Besides that, aiming a production of a antiloxoscelic

serum, rabbits were immunized with PLDs or venom enriched with PLDs. These sera will be

compared with a serum produced in rabbits with the whole venom of Loxosceles. Therefore, is


proposed in this work the production of a antiserum with low cost, less animal suffering and enhanced

neutralization capacity even when used several hours after the envenoming.

Acknowledgements: CAPES, CNPq, Fundação Araucária, UFPR, CPPI.


Anterior tibialis muscle morphometric changes of obese Wistar rats submitted to whole

body vibration

RETAMEIRO, A.C.B (1), KIRSCH, C.B. (1); BOARO, C.D.T. (1); THEODORO, J.L. (1);

ZAZULA, M.F. (1); ANDRADE, B.Z. (1); BERTOLINI, G.R.F. (1); RIBEIRO, L.C. (2)


– UNIOESTE.




Introduction: Obesity is a chronic multifactorial disease, which is characterized mainly by the body

fat increase. A lot of experimental models have been used to study and treat the alterations and

associated comorbidities caused by this disease; one of these treatments is physical exercise. Based on

this, the aim of this study was use the chemical obesity model induced by monosodium glutamate to

understand the effects of the Whole Body Vibration (WBV) under the tibialis anterior muscle

morphological characteristics and its main components.

Methods: For this, 32 male Wistar rats were used and divided into 4 groups: Control Group (CG),

Control treated with WBV (CWBV), Obese Group (OG) and obese treated with WBV

(OWBV).Obesity was induced in the OG and OWBV groups in the neonatal phase using intradermal

monosodium glutamate injection, which causes lesions in hypothalamic nuclei, which ones are

directly related to body energy balance. The WBV treatment happened through vibratory platform,

which started at 80th day of animals’ life, 3 times a week for 8 weeks with a 60Hz frequency. After

euthanasia, anterior tibialis muscle samples were fixed and processed in ascending series of alcohol,

diaphanized in xylol and sectioned in 7 μm to obtain histological slides, which were stained in

hematoxylin and eosin for general morphological analysis. Subsequently, the slides were

photomicrographed and analyzed morphometrically by Image Pro Plus 6.0 program.

Results and Discussion: Regarding the muscle fibers cross-sectional area means (S), there was no

interaction between the factors (p=0, 88742; F=3,1096), but analyzing the simple effects it was

possible to notice a decrease in the OG and OWBV means in relation to CG and CWBV groups

(p=0,018024; F= 6,2414; CV= 10,12%). The same was found in largest diameter means (D), without

interaction between the factors (p=0,98128; F= 0,0076), also in the effects simple analysis were found

means lower in OG and OWBV groups in relation to CG and CWBV groups (p=0,03918, F=4,6812,

CV=6,53%). Concerning the smallest diameter values (d), a significant interaction between the factors

(p= 0,028127; F= 5,3618) was found, where the CWBV group obtained the highest means when

compared to the other groups, showing that exercise was able to positively alter this parameter

(p=0,073943; F=3,4464; CV= 4,17%). On the other hand, the obese animals obtained de smaller

means (p=0,073943; F= 3,4464; CV=4,71%). In relation to the nuclei variables, both total nuclei (N)

and nuclei by fiber relation (N/F) showed significant interactions of the factors (p=0,78941; F=0,073)

and CG animals had higher N and N/F means when compared to OG (p=0,00020; F=18,240;

CV=6,09%), while the trained animals of both CWBV and OWBV had higher N and N/F means when

compared to untrained animals (p=0,00000; F=65,296; CV=6,09%). The internalized nuclei (NC%)

percentage mean, showed no interaction between the factors (p=0,84467; F=0,0391), in the simple


analysis of factors only the trained animals had lower mean values when compared to control

(p=0,00197; F=11,6528; CV=27,57%).

Conclusion: As expected, the hypothalamic injury obesity model yielded animals smaller than

control, but training was not effective in recovering the S, D and d values. In the nuclear parameters

the training proved being effective, what suggests a better nuclear activity capacity in non-sedentary

animals.

Acknowledgment: To Fundação Araucária, for the financial assistance through the call 016/2016 –

UNIOESTE/ basic and applied research.


Correlation between reck isoforms and genes expression in glioblastoma: a

bioinformatic tool

RIBAS, H. T.(1); RIBAS, G. T.(2); WINNISCHOFER, S. M. B.(1,3,4)

1. Postgraduate Program in Biochemistry Sciences, Sector of Biological Sciences, Federal

University of Paraná, Curitiba, Paraná, Brazil.

2. Bioinformatics and Systems Biology Lab , Federal University of Parana, Polytechnic Center,

Curitiba, Paraná, Brazil.

3. Department of Biochemistry and Molecular Biology, University of Paraná, Paraná, Brazil.

4. Postgraduate Program in Cell and Molecular Biology, University of Paraná, Paraná, Brazil.


Introduction: Glioblastoma (GBM) is the most aggressive type of central system nervous tumors

(HANIF, F. et. al., 2017). This aggressiveness is explained by its high cellular density, necrosis and

endothelial proliferation, which leads to poor prognosis with a ratio survival of 14 months

(WESTPHAL e LAMSZUS, 2011). There have been advances in the glioma biology understanding,

however, the current therapies are still inefficient in stop GBM to invade and proliferate. Therefore, a

seeking for better understanding of pathways involved in invasion and migrations are needed. Matrix

metalloproteinases (MMPs) are enzymes responsible for degrading extracellular matrix, playing an

important role in cancer invasion and growth (TURUNENA, S. P.; TATTI-BUGAEVAB, O. AND

LEHTIA, K., 2017). The protein RECK, an inhibitor of MMPs, was elucidated and it is

downregulated in different cancers (TAKAHASHI et al., 1998). Alternative splicing variants of the

gene encode for the protein RECK was also described (TROMBETTA-LIMA, et. al., 2015). Those

splicing variants were shown to be correlated with glioblastoma patient prognosis; a higher ratio

expression between RECK and its splice variant 3 leads to an increasing in survival rate. Therefore, to

better understand the mechanisms involved in the expression of RECK and its splicing variants the

aim of this work was to find gene candidates that would be correlated to splicing variants expression,

by using TCGA analyses.

Methods: TCGA analyses were performed by using a software developed for this propose. The

programing was developed using the software library Pandas, which is written in Python language.

Three RECK isoforms were analyzed : the canonical variant and the splice variant 3 and 5. RNAseq

data (n=160) of gene and isoform expression, already normalized, were collected from the Firebrowse

platform, which is sponsored by Broad Institute. The correlation between each isoform expression and

all genes expression was performed by calculating slope, intercept, r value and p value. Only those

correlation with a r>0.6 and with p<0.05 were considered to be plot in the heatmap graph.

Results and Discussion: The expression of the gene coding for canonical RECK was best correlated

to gene named ATP8B2 (r=0.71) which codes a translocase membrane protein responsible for

transport phosphatidylserine and phosphatidylethanolamine in membranes (HARRIS M. J. AND

MARIAS I., 2003). This protein was described to be important for lipid rafts formation, vesicles

biogenesis, and it was linked to different diseases (Folmer, D. E. R., Elferink P.J. O. and. Paulusma,

C. C., 2009). However, the relationship between this protein and cancer; nonetheless, how ATP8B2

and RECK is correlated, have still being unknow. The splice variant 5, on the other hand, has been

positively correlated to discoidina neuropilin-like membrane protein (DCBLD2) (r=0.66). DCBLD2


was first identified in the vascular injury process (Kobuke K., et. al., 2001). Furthermore, DCBLD2

was described to play an important role in tumorigenesis of GBM by activating the cariogenic

EGFR/TRAF6/AKT signaling (FENG, H., et. al., 2014). A bioinformatic tool to find correlation

between splicing variant isoforms and genes expressions using TCGA data has been created.

Conclusion: Now, further investigation has to be elaborated in order to elucidate how those correlated

genes and RECK isoforms are linked in glioblastoma.

Acknowledgement: This work was sponsored by CAPES/CNPQ.


Effects of bismuth nanoparticles produced by lasis on mesenchymal stem cells

RIBEIRO, A. L. (1); MACHADO, T. N. (2); BEZERRA-JR, A. G. (2); REUS, T. L. (1);

PASCHOAL. A. C. C. (1); HORINOUCHI, C. D. S. (1); DALLAGIOVANNA, B. (1), AGUIAR, A.

M (1);

1. Laboratório de Biologia Básica de Células-Tronco, Instituto Carlos Chagas, FIOCRUZ

Paraná, Rua Prof. Algacyr Munhoz Mader, 3775 CIC, 81350-010 Curitiba, PR.

2. Laboratório FOTONANOBIO, Universidade Tecnológica Federal do Paraná, Avenida 7 de

Setembro 3165, 80230-901 Curitiba, PR, Brazil.


Introduction: Due to their physical-chemical properties, such as high surface contact area and high

penetration capacity, nanoparticles (NPs) have been widely used in industry, technology and medical

fields. Among all types of NPs, bismuth nanoparticles (BiNPs) have been used for bactericidal,

antifungal and antibiolfim purposes. Despite having many applications, little is known about the

toxicity related to BiNPs. Mesenchymal stem cells (MSCs) can be isolated from different sources,

such as adipose tissue, and represent one of the cell types that can be used for the in vitro toxicity

assessment of NPs. In this study, we aimed to assess the cytotoxicity of BiNPs using MSCs as an in

vitro model for toxicity evaluation.

Methods: Two batches of BiNPs (BiNPs_01 and BiNPs_02) were synthesized by laser ablation

synthesis in solution (LASiS) and characterized by UV/Vis spectroscopy, dynamic light scattering

(DLS) and transmission electron microscopy (TEM). MSCs derived from adipose tissue were

cultivated in DMEM medium supplemented with 10% fetal bovine serum and 4mM L-glutamine.

These cells were maintained at 37 °C in 5% CO2 atmosphere. The cytotoxicity of BiNPs was

evaluated at concentrations ranging from 550 to 144.88 μg/ml (dilution log factor: 1.21) through the

neutral red uptake (NRU) and MTT assay.

Results and Discussion: The solution BiNPs_01 were produced at concentration of 1.1 mg/mL and

had an average size of 60 nm. For the BiNPs_02 solution, the concentration were at 1.7 mg/mL and

had an average size of 100 nm. Both solutions presented a plasmon peak around 250 nm. The

BiNPs_01 solution was evaluated in a NRU assay and the inhibitory concentrations (IC) values

obtained were as follows: IC20 = 381.80 µg/ml, IC50 = 408.55 µg/ml and IC80 = 448.10 µg/ml.

Additionally, the predicted LD50 was 989 mg/kg, suggesting that the BiNPs could belong to the GHS

class 4 for acute toxicity. According to this scale, class 4 compounds are considered harmful but not

fatal or toxic. A concentration-response curve of BiNPs could not be evaluated by the MTT assay. We

visualized the formation of a precipitate of BiNPs on the plate wells which might interfered on the

absorbance measurement.

Conclusion: So far, bismuth nanoparticles have shown a low toxicity on MSCs according to the NRU

assay. It is important to point out that BiNPs production by LASiS is still being standardized in order

to obtain accurate concentrations and average sized nanoparticles among distinct batches. Also, the

protocol for the MTT assay needs few adjustments to be effective in the prediction of the nanoparticle

cytotoxicity. For future studies, differentiation of stem cells will be addressed in order to evaluate the

specific role of BiNPs on human cells.


Acknowledgement: CAPES, CNPq, Instituto Carlos Chagas, Universidade Federal do Paraná.


Kappa carrageenan and its chemically oxidized derivatives: synthesis and cytotoxicity

evaluation on melanoma (B16-F10) and fibroblasts (BALB/3T3) cells

RIBEIRO, C. L. G. (1); SANTOS, B. P. D. (2); EIFLER, N. T. (1); DUCATTI, D. R. B. (1); SIMAS,

F. F. (1)

1. Department of Cell Biology of UFPR.

2. Department of Biochemistry of UFPR.

E-mail: [email protected]; [email protected].

Introduction: Cancer is a set of more than 100 diseases that has the characteristic of disordered

growth of cells. Skin cancer is a type of cancer and can be subdivided into: melanoma and non-

melanoma. The first, although less recurrent in the world population, is very metastatic and

aggressive. Current treatment for this type of cancer includes surgical removal, chemotherapy and

radiation therapy. Non-cytotoxic polysaccharides has been used for melanoma’s treatment. In this

work, extraction and cytotoxicity of kappa carrageenan, a polysaccharide extracted from red seaweed

Kappaphycus alvarezii, and its chemically oxidized derivatives on murine melanoma cells (B16-F10)

and fibroblasts (Balb/3T3) were evaluated.

Methods: Native kappa-carrageenan (KEQ-D) was extracted with hot water (70°C) with 41.6% yield.

KEQ-D was submitted to a selective oxidation with TEMPO reagent, 2.5 or 0.5 TCCA (co-oxidizing

trichloroisocyanuric acid) equivalents, to give fractions PO-KC (partially oxidized kappa carrageenan)

and TO-KC (totally oxidez kappa-carrageenan), respectively. At the end of reactions NaBH4 was

added to eliminate the carbonyl intermediates. The degree of oxidation in the samples were

determined by colorimetric assays (dosage of uronic acid), and 1H nuclear magnetic resonance

(NMR). HPSEC analysis were carried out to confirm absence of degradation and molar mass of the

samples. Each sample was solubilized in PBS LPS free and diluted in DMEM culture medium

without fetal bovine serum (FBS) at different concentrations to perform a final well concentrations

(0,0002; 0,002; 0,02; 0,2; 2; 20; 200; 2000 µg/mL) of each polysaccharide. The cell viability and cell

proliferation were assayed by neutral red and crystal violet methods, respectively (ICCVAM, 2006;

VEGA-AVILA et al, 2011). Cells were plated in DMEM medium supplemented with FBS and treated

24 hours later. After 72 h, neutral red was added and the cells were incubated (2h). An extraction

solution was added and the absorbance was read at 540 nm. The wells were washed and then stained

with violet crystal. A solution of elution was added and the obtained absorbance was read at 570 nm.

Results and discussion: The partially and almost totally oxidized derivatives were successfully

produced from kappa carrageenan. The selective oxidation was confirmed analyzing changes in 1H

NMR spectra of all fractions. The signal at 3.79 ppm, assigned to H6 of the β-D-Galp-4-sulfate units,

was reduced at PO-KC and TO-KC, confirming the changed of these units in its galacturonic acid

derivative. The degree of oxidation, calculated by the area integration of the signal at 3.79 ppm,

resulted in 80% and 21% for the TO-KC and PO-KC, respectively. All three polysaccharide fractions,

in all tested concentrations, did not show toxicity on non-tumor cell. Experiments with the tumor cells

are being performed to analyze the potential antitumor activity of the polysaccharides.

Conclusion: It was produced partially and totally oxidized kappa carrageenan derivatives using

TEMPO oxidation. Biological assays showed that all three studied sulfated polysaccharides were no

cytotoxic to normal cell line under a large range of concentrations (0.0002 to 2000 μg/ml). The

potential antitumor activities of these fractions are under analysis.


Acknowledgement: Authors would like to thank to UFPR/TN and CNPq for the scholarships grants

and CAPES (CIMAR 1985/2014, PROCAD 2965/2014 and Finance code 001) for the financial

supports.


Extracellular vesicles modulate aggressiveness phenotype in human glioblastoma cells

RIBEIRO, C. S. P. (1); RAMIREZ, M. I. (2,3); WINNISCHOFER, S. M. B. (2,4)

1. Postgraduate Program in Biochemistry Sciences, Federal University of Paraná, Paraná, Brazil.

2. Postgraduate Program in Cell and Molecular Biology, Federal University of Paraná, Paraná,

Brazil.

3. Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.

4. Department of Biochemistry and Molecular Biology, Federal University of Paraná, Paraná,

Brazil.


Introduction: Gliomas have low incidence with high mortality rates and represents 80% of the

malignant tumors from CNS with high aggressiveness and largely resistant to treatment. Astrocytoma

are the most common and can be classified according aggressiveness, from I to IV (IV is also known

as glioblastoma - GB). Although there is progress in therapy, glioblastomas have, high resistance

mechanisms (SAYEGH et al., 2014). GB secrete extracellular vesicles (EVs) and their content may be

associated with the aggressiveness profile (NAKANO et al., 2015). The aim of this work was to

evaluate the secretory profile of EVs derived from GB cells and the modulation of the aggressiveness

phenotype mediated by EVs.

Methods: Human GB cells A172 and T98G were cultured in DMEM-AG 10% FBS. For the isolation

and purification of EVs the protocol were cells maintained in DMEM-AG with EVs-depleted FBS for

48 hours (EVs from FBS removed by ultracentrifugation). Then, conditioned medium was collected,

and serial centrifugations were performed (2x 30min 4000g and 90min 100,000g). Protein content of

EVs for each cell (A172 or T98G) was quantified by Bradford, with a mean of 2.73 µg/µL. EVs

isolated were analyzed by Nanoparticle Tracking Analysis (NTA) to determine vesicle concentration

and size distribution of particles. The mean particle size obtained for both cell lines was 200 nm,

which is characteristic of microvesicles. To evaluate the uptake of vesicles from T98G and A172 in

A172 cells, was performed a confocal microscopy analysis. A172 cells were seeded at a density of

1.105 cells/well in 24-well chamber slides. After 24 h, cells were incubated with PKH26-labeled EVs

for 1 hour. Subsequently, cells were washed with PBS and fixated with 4% paraformaldehyde in PBS

at room temperature for 20 min. After fixation, slides were washed with PBS and mounted using

glycerol. Fluorescence imaging was done on a Nikon scanning confocal microscope. Images were

processed using Fiji. Considering that A172 cells show lower proliferative capacity in comparison to

U87MG cells, it was evaluated the effects of EVs derived from T98G cells on the phenotype of A172

cells. For this, we performed a cellular-growth curve under different conditions: (1) A172 and T98G

cells maintained in culture medium containing 10%FBS, (2) A172 cells maintained in conditioning

medium derived from T98G or A172 cells and (3) A172 cells maintained in culture medium 10%FBS

with EVs derived from T98G or A172 cells. The culture medium was replaced every 2 days, and the

quantity of vesicles added to the culture medium was proportional to number of vesicles

physiologically released during this period. After 196 hours, a change in the growth profile of A172

cells was observed, suggesting that T98G-derived EVs increase the proliferative capacity of A172

cells.


Results and Discussion: Our results indicated that EVs derived from T98G and A172 cells have

mean sizes of 278 and 242 nm, respectively, which are characteristic of microvesicles, with a mean

protein concentration of 2.73 µg/µL. EV internalization was observed from both cells. Finally, a

change in the growth profile of A172 cells was observed, suggesting that EVs derived from T98G

were able to modulate the proliferative phenotype of A172 cells.

Conclusion: These results show that EVs confer pro-survival signals and alter the proliferative

phenotype of glioma cells.

Acknowledgement: Capes, CNPq


Analysis of the Keap1 / Nrf2 pathway in cells during uremic toxicity

RODRIGUES, S.D. (1); SANTOS, S.S. (1); MEIRELES, T. (1); ROMERO, N. (2); GLORIEUX, G.

(3); PECOITS-FILHO, R. (4); ZHANG, D.D. (5); NAKAO, L.S. (1)

1. Department of Basic Pathology, Universidade Federal do Paraná, Curitiba, Brazil

2. Cell Analysis Division, Agilent Technologies, Lexington, MA, USA.

3. Department of Nephrology, Ghent University Hospital, Ghent, Belgium.

4. Center for Health and Biological Sciences, Pontific Catholic University of Paraná, Curitiba,

Brazil.

5. Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA.

Introduction: Uremic toxins are substances that accumulate in the blood and tissues of patients with

chronic kidney disease (CKD), and in high concentrations become toxic. Some of them bind to

proteins and are not easily removed by dialysis. These include indoxyl sulfate (IxS), p-cresyl sulfate

(pCS) and indole acetic acid (IAA), which can cause deleterious effects in human cells and are

associated with increased incidence of cardiovascular events and mortality in patients with CKD. The

Keap1/Nrf2 pathway has been recognized as an important adaptive mechanism of antioxidant

response. Thus, in this study, we investigated the effect of uremic toxins on the Keap1/Nrf2 pathway.

Methods: HeLa cells were exposed for 4 h and 16 h to increased concentrations of IxS, pCS and IAA

or a mixture of them. The expression of Nrf2, as well as some Nrf2 target genes, was assessed by

Western blotting. Nrf2 nuclear translocation analysis was performed in endothelial cells by

immunofluorescence, captured with microscope confocal. We also analyzed the viability of Nrf2 null

MEF cells by MTT.

Results and Discussion: Hela cells exposed to uremic toxins showed an increase in Nrf2 protein

expression and its target genes. Nrf2 activation was also demonstrated in endothelial cells, by its

nuclear translocation after 4h of treatment. After longer exposure times, such as 48 h, Nrf2

immunostaining was not present at the nucleus. Also, Nrf2-null MEF showed a significant decreased

cell viability after 48 h of incubation with uremic toxins when compared with WT MEF, suggesting

the important role of the Nrf2 pathway in uremia.

Conclusion: Our data showed that uremic toxins lead to transient activation of Nrf2, but followed by

an inactivation of the Nrf2 pathway. These data may contribute to the understanding of uremic

toxicity mechanisms.

Acknowledgement: We gratefully acknowledge the fluorescence microscopy core facility CTAF-

Centro de Tecnologias Avançadas em Fluorescência from UFPR (CAPES/FINEP). scholarship was

supported by CNPq (207359/2015-6) and CapesFundação Araucária


Aging and resistance training affect the MMP-2 activity in femurs of rats

RUIVO, A. L. (1); NETO, I. V. S. (2); FARIAS JUNIOR, G. C. F. (2); MARQUETI, R. C. (3)

1. Graduation of Physiotherapy, University of Brasilia, Brasília, Brazil.

2. Graduate Program of Sciences and Technology of Health, University of Brasilia, Brasília,

Brazil.

3. Graduate Program of Sciences and Technology of Health, University of Brasilia, Brasília,

Brazil.


Introduction: Matrix metalloproteinases (MMPs) are calcium and zinc dependent endopeptidases.

They are capable of modeling the extracellular matrix. During aging process, the bone quality is

impaired and became the bone fragile then susceptible to fracture. The aim of the present study was to

verify the effect of ageing and strength training on MMP-2 activity in rat femurs.

Methods: 20 Wistar Novergicus Albinos rats aged 3 and 20 months were divided into four groups:

young sedentary (YS); young trained (YT), old sedentary (OS), and old trained (OT). Animals it

belongs to this group of trained animals performed a Resistance Training (RT) protocol for a period of

12 weeks, at a frequency of once every two days. After RT, the femurs were extracted from the

animals and the MMP-2 activity was measured by zymograph.

Results and Discussion: The aging process shows a negative factor on MMP-2 activity. The

proposed RT was not able to attenuate the deleterious effects of aging, but increased the activity of

MMP-2 in the young group.

Conclusion: The aging process is shown to be a negative regulator for MMP-2 activity in rat femurs.

However, RT it is a useful tool to increase the activity of this endopeptidase in young rats.


Understanding the diabetic wound healing by extracellular vesicles perspective

SANTOS, M. L. F. dos (1); KERKHOVEN, N. C. (1); BEBBER, B. A. (1); BRITO, L. G. C. (1);

RAZENTE, I. C. (1); SANTOS, H. G. dos (1); MARANGONI, C. G. P. S. (1); FOTI, L.(1)

1. Carlos Chagas Institute (ICC) – FIOCRUZ/PR


Introduction: There are approximately 13 million Brazilians with Diabetes Mellitus (DM),

generating an estimated cost of R$ 5,200 patient/year to SUS. In these individuals, unhealed skin

lesions associated with poor circulation are responsible for most limb amputations. Cicatrization is a

dynamic and complex process which is composed of complementary phases: hemostasis,

inflammation, proliferation and remodeling. In diabetics, healing remains arrested in inflammatory

phase, and full cicatrization may never occur. Extracellular vesicles (EV) - exosomes (EXO),

microvesicles (MV) and apoptotic bodies (AB) - have a role in intercellular communication during the

wound healing process. Analyzing the differences between number, size and content of EV comparing

healthy and diabetic animals may provide important information for understanding of EV function in

healing process. Therefore, the objective of this work is to establish EV’s role on murine epithelial

tissue in non-DM and DM model.

Methods: Diabetic murine model was obtained by injecting 200 mg/kg intraperitoneal streptozotocin

(STZ) in Balb/c mice, whereas the control group received SHAM. After STZ induction, animals were

confirmed diabetic by glycemic index (GI) measurement. Then, a 8 mm circular wound was made on

the animals’ back and, on the day 3, 7, 10, 14, 18 and 21 after surgery, they were weighed, GI

measured and euthanized. Tissue was collected from a 0.5 cm radius within the wound border then

stored at -80 °C. For macroscopic analysis, the wound was photographed and wound’s area was

calculated by pixel counting with support of scale ruler. For EV’s extraction, tissue was thawed and

subjected to a differential centrifugation/filtration combined methodology. Obtained EV were

confirmed by Transmission Electron Microscopy (TEM) and Flow Cytometry (FC).

Results and Discussion: In order to obtain a GI cutoff value between non-DM and DM animals, area

under the curve (AUC) analysis was made. We applied a confidence interval of 10%, resulting in a

cutoff = 275 mg/dL to consider the animal as diabetic. There was a significant (P = 0.0021) weight

difference between the non-DM 25.45 ± 2.65 g and DM group 23.52 ± 3.18 g in the euthanize day.

Both GI and weight corroborate the pathology status. When cicatrization area average from Non-DM

15.21 ± 5.96 mm2 and DM 27.28 ± 24.95 mm2 was compared, a great deviation on the wound size

healing could be observed. EVs with sizes corresponding to EXO (50 nm) and MV (> 150 nm) could

be observed by TEM. When the EV fraction tagged with Annexin V was analyzed by CM, results

showed a positive population, consistent with EV characteristics.

Conclusion: Weight and GI measurement were able to confirm DM animals. By comparing wound

sizes between both groups, we showed that DM animals are arrested in the inflammation phase. We

were able to obtain intercelular MVs from tissue. As next work steps, EV’s proteomics will be

performed by mass spectrometry. Further tissue microscopic analysis to understand the changes

undergoing in tissue will be also done. It is expected that this project may contribute existing

knowledge about the differences in healing between healthy and diabetic patients, in special the MVs

role in the wound process.


Acknowledgement: PPSUS, Araucaria Foundation, Physiology department (UFPR).


Characterization of a potential linker histone in Toxoplasma gondii

SEVERO, V. R. (1); FRAGOSO, M. S. I. (1); DE SIQUEIRA, C. M. (1); KLIMECK, T. D. F. (1);

AVILA, A. R. (1); NARDELLI, S. C. (1)

1. Carlos Chagas Institute – FIOCRUZ/PR


Introduction: The chromatin is a natural barrier to all DNA-dependent processes such as

transcription. The chromatin compaction levels are regulated mainly by histones and their post-

translational modifications (PTMs) that may act facilitating or preventing access to DNA. Toxoplasma

gondii has the four canonical histones (H2A, H2B, H3 and H4), but so far, the fifth histone (H1 or

linker histone), has not been identified. In other eukaryotes, H1 acts linking nucleosomes and its

absence could interfere to the chromatin condensation. The aim of this work was to characterize a

small and basic protein identified in Toxoplasma gondii, similar to H1-like of bacteria, which we

named TgH1-like.

Methods: Initially, the epitope tagging of the endogenous gene was performed using a 3xHA tag. By

immunofluorescence assay, it was possible to analyze the location of this protein. To verify the

association of TgH1-like with chromatin, a histone extraction protocol by cell fractionation was

performed. In search of functional data, a classical knockout of tgh1-like was performed, where the

gene of interest was replaced by a selection gene hxgprt through homologous recombination. Using

the knockout parasites, a plaque assay was performed to verify if the parasites would show some

alterations in the replication and invasion process. In addition, a replication and invasion assay was

performed carried out after 1 hour of infection by 105 parasites, intracellular parasites were fixed and

an immunofluorescence assay was performed using the IMC antibody that helps to visualize the

parasites. The number of parasites per vacuole in the replication assay, and the number of vacuole per

field in the invasion assay were analyzed. The nuclei architecture of knockout parasites was

investigated by transmission electron microscopy using ethanolic phosphotungstic acid staining,

which allows determining the location of basic proteins such as histones and therefore chromatin.

Results and Discussion: Using the endogenous TgH1-like tagged, we found the protein located

exclusively in the nucleus of tachyzoites, which is expected from a histone that aids chromatin

compaction. Performing standards histone extraction protocols, we observed TgH1-like in the same

fraction of histone H4, which was confirmed by co-immunoprecipitation assays. This data gives us a

clue of the histone potential of this protein, strengthening our hypothesis that this protein is a histone.

Plaque assay experiments using TgH1-like knockout parasites showed a discrete but significant

increase in the plaque number, which may be related to an alteration in parasite replication.

Replication assay showed an increase in the number of parasites per vacuole. Whereas in the invasion

assay, it was observed an increase in the number of vacuoles per field. When we analyzed the

knockout parasites by immunofluorescence assay, it was possible to observe the presence of some

vacuoles in an asynchronous replication, demonstrating a probable alteration in the parasite

replication. Next, we investigated the nuclei architecture by transmission electron microscopy, Dtgh1-

like showed a different chromatin distribution compared to the control, suggesting TgH1-like has a

role in the chromatin organization.

Conclusion: To our knowledge this would be the first linker histone identified in Apicomplexa

parasites and will provide new insights about the chromatin dynamics in Toxoplasma gondii.


Acknowledgement: CAPES, CNPq, Fundação Araucária, FIOCRUZ/PR.


Functional characterization of Bar-III, a metalloproteinase from Bothrops barnetti

venom

SCHLUGA, P.H.C. (1); MELO, A.M. (1); OLIVEIRA, D.P.L. (2); VEIGA, S.S. (1); SANCHEZ,

E.O.F. (2); GREMSKI, L.H. (1);

1. UNIVERSIDADE FEDERAL DO PARANÁ, UFPR, CURITIBA - PR - BRAZIL.

2. FUNDAÇÃO EZEQUIEL DIAS, FUNED, BELO HORIZONTE - MG - BRAZIL.


Introduction: The World Health Organization recently included accidents with snakes to the list of

neglected tropical diseases. Among the venomous snakes are those of Bothrops genus. They are

responsible for most of the snakebite accidents in South America. Bothrops barnetti snake belongs to

the northern coast of Peru. Proteomic analysis of B. barnetti venom gland revealed the predominance

of proteins characterized as metalloproteinases (SVMP-Snake Venom Metalloproteinases) totalizing

51.2% of B. barnetti venom proteins. These SVMPs are commonly divided into classes P-I, P-II and

P-III. Typically, Class III SVMPs are more hemorrhagic. Most of the B. barnetti venom proteins are

P-III class SVMPs, but none was characterized. This class of toxins has been the object of studies due

to its action in the venoms, in order to understand its role in envenoming, and for its potential for

biotechnological and therapeutic application, particularly due to its disintegrin domain, which shows

to be relevant in the interaction with surface receptors of different cell types. Recently, a P-III class

metalloproteinase, named barnettlysin-III (Bar-III), was purified from B. barnetti. We aim to evaluate

and characterize the main activities of this enzyme. By investigating its activity in endothelial cells in

culture, we will also investigate if Bar-III is able to interfere with the stability of extracellular matrix

(ECM).

Methods: The cell viability assay was performed using rabbit endothelial cells (RAEC) in culture, by

trypan blue method, comparing Bar-III and B. barnetti venom at different amounts. We also

investigated if Bar-III was able to digest some ECM components by performing in vitro digestion of

some ECM proteins followed by SDS-PAGE analysis.

Results and Discussion: Cell viability results showed that B. barnetti venom reduced cell viability,

whereas Bar-III alone had no effect on endothelial cell viability. Results of effects Bar-III over ECM

components evidenced that Bar-III degrades gelatin and fibronectin, and partially digested collagen I

and collagen IV. Digestion studies of Bar-III on laminin, that is a major component of the basement

membranes, showed no proteolytic effect.

Conclusion: Bar-III did not affect endothelial cell viability, unlike most SVMP-III described in

literature. However, Bar-III showed proteolytic activity upon important ECM components, such as

fibronectin and collagen IV, pointing to an activity pattern of interaction with both basal membrane

and interstitial stroma ECM components. We intend to further characterize Bar-III to understand its

role in envenoming and to seek for potential applications of this molecule in different areas.

Acknowledgement: UFPR, CAPES, CNPq, Fundação Araucária e FAPEMIG


Proteomic prospection to identify plasma biomarkers associated with type 1 diabetes

mellitus

SILVA, L.P. (1); REGO, F.G.M. (1); PICHETH, G. (1); MÜLLER-SANTOS, M. (2); ALBERTON,

D. (1)

1. Postgraduate Program in Pharmaceutical Sciences, Federal University of Paraná, Curitiba,

PR, Brazil.

2. Postgraduate Program in Sciences-Biochemistry, Federal University of Paraná, Curitiba, PR,

Brazil.

E-mail: [email protected]/[email protected]

Introduction: Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by

autoimmune β-cell pancreatic destruction, usually leading to absolute insulin deficiency. T1DM

affects more than 500 million people, and its incidence rate increases 3% each year. Diabetes can be

diagnosed based on glucose concentration measured under different conditions, either as the fasting

plasma glucose or the 2-h plasma glucose during a 75-g oral glucose tolerance test or through

glycated hemoglobin (HbA1C) criteria. Therefore, the continued search for new biomarkers is

extremely important, because only these two biomarkers (glucose and HbA1C) are available to

diagnose and monitor diabetes. Given this, this study aimed to determine differential expression of

plasma proteins, through comparative proteomic analysis, of T1DM patients with adequate

(HbA1c<7) or inadequate (HbA1c≥7) glycemic control and healthy patients (Control).

Methods: The Ethics Committee on Human Research of the Federal University of Parana approved

this study (CAAE: 01038112.0.0000.0102). Blood samples were collected from the T1DM and

healthy patients and processed to obtain the plasma. The plasma samples were pooled according to the

study groups, T1DM HbA1c<7 (n = 10), T1DM HbA1c≥7 (n = 10) and Control (n = 10). Proteome

Purify™ 2 commercial kit was used to remove the most abundant proteins from plasma. After this

treatment, the plasmatic proteins from each group were separated on a 10% sodium dodecyl sulfate–

polyacrylamide gel electrophoresis (SDS-PAGE), which was stained with PhastGel® Blue R. The

relative volumes (%Vol.) of protein bands were determined by densitometric analysis, and protein

identification was carried out by MALDI-TOF mass spectrometry of tryptic peptides. Tukey's test was

applied for the comparative analysis between the %Vol. of the protein bands in the 3 groups studied.

Data analysis was performed using Statistical for Windows 10.0 software (StatSoft Inc., Tulsa, OK,

USA), and a P<0.05 was considered significant for all analyses.

Results and Discussion: Four protein bands identified as α-2-macroglobulin (α-2-M), ceruloplasmin

(Cp), haptoglobin (Hp) and apolipoprotein AI (ApoA-I) presented higher and statistically significant

relative volumes in T1DM with adequate and inadequate glycemic control than the control group.

T1D has as main characteristic the chronic hyperglycemia that stimulates the production of

inflammatory cytokines through the activation of pro-inflammatory transcription factors, resulting in

elevated plasma acute protein concentration, such as α-2M, Cp, and Hp. Increased expression of

ApoA-1 in the DM1 group was also verified in other published studies, and one of the hypotheses

would be a compensatory mechanism due to a reduced HDL function.

Conclusion: T1DM patients with adequate glycemic control (HbA1c<7) and inadequate glycemic

control (HbA1c≥7) present an inflammatory clinical condition due to increased expression of acute

phase proteins.


Acknowledgment: This project was supported by CNPq (National Council for Scientific and

Technological Development) and the Araucaria Foundation (PPSUS).


Modulatory effect of vitamins A, D and E on the redox and inflammatory profile in

blood cells of patients with breast cancer

SILVA, J.C. (1); SCANDOLARA, T.B. (1,2); RECH, D. (1,3); PANIS, C. (1,4)

1. Laboratory of Tumor Biology, State University of West Paraná, Unioeste, Francisco Beltrão,

Paraná, Brazil.

2. Rio de Janeiro Federal University, Rio de Janeiro, Brazil.

3. Francisco Beltrão Cancer Hospital, Francisco Beltrão, Paraná, Brazil.

4. Post Graduate Program of Applied Health Sciences, State University of West Paraná,

Unioeste, Francisco Beltrão, Paraná, Brazil.


Introduction: Reactive species (RS) are widely associated with carcinogenesis, through several

mechanisms, as lipoperoxidation of cell membranes and DNA damage. RS are produced as a

consequence of inflammation in conjunction with other mediators, as Cytokines. Both mediators act

together and can be regulated simultaneously, improving inflammation in a deregulated way. Several

studies have investigated the role of antioxidants as putative regulators of this process in cancer.

Considering the potential anti-inflammatory and antioxidant role of vitamins, our purpose was to

investigate if the treatment of blood cells from breast cancer patients with vitamins A, D and E could

affect the levels of interleukin 1 β (IL1β) and cause changes in their hydroperoxides profiling.

Methods: This research was approved by the Institutional Ethics Committee (CAAE

35524814.4.0000.0107). After signing the informed consent form, heparinized whole blood was

collected from breast cancer patients by vein puncture and incubated with pre-established

physiological concentrations of vitamins D, A and E (4,96e-8; 4,75e-4; 0,0125 mg/mL) for 1 hour at

370C. The material was then centrifuged at 4000 rpm for 5 minutes and the plasma stored at -20 ° C

for further analysis. Each analysis battery containing a positive and a negative sample for treatment.

To quantify the secreted IL1β we used the enzyme-linked Immunosorbent Assay (ELISA) method (e-

Biosciences, USA). The determination of hydroperoxides was determinedby high sensitivity

chemiluminescence, and the results were expressed as the area under the curve in the Origin 9.0

program and by the paired t test in the statistical software GraphPad Prism 7.0, used for IL- 1ß as

well. It was considered significant when p <0.05.

Results and Discussion: Vitamin E treated samples showed a significant increase in the secreted IL-

1β levels, ranging from 120.4pg/mL to 159.2pg/mL (p=0.0313). For vitamin A, no significant

difference was observed, while for vitamin D treatment a significant reduction of IL1- β was

observed, varying from 132.3pg/mL before to 122.5pg/mL after exposition (p=0.0233). These

findings indicate that acute exposition of whole blood cells to vitamin E promotes the immediate

secretion of IL-1 β, a cytokine that is crucial for destroying breast cancer cells. On the other hand,

reduction of this cytokine by vitamin D can alter immune functioning, since this vitamin is a positive

modulator of this response. Regarding the levels of hydroperoxides, a marker of lipid peroxidation,

vitamin A treatment showed a significant decrease, from 1.259006 to 1.064006 URL (p=0.0387).

Vitamin E demonstrated a downward trend from 1.600006 to 1.262006 URL, but not statistically

significant (p=0.1194). Furthermore, vitamin D increased hydroperoxides levels from 1.164006 to

1.336006 URL (p=0.9038). As expected, vitamin A exerted an antioxidant effect on the whole blood,

while vitamin D showed the contrary. It suggests that vitamin D have triggered some pro-oxidant


effect on breast cancer patients blood. Although their antioxidant roles are widely discussed, such

results show the complexity related to these micronutrients, dependent on multiple factors, such as the

types of free radicals formed, where and how they are generated, in addition to optimal doses and

synergy of nutrients to obtain protection, acting on the modulation of the immune response and

inflammation.

Conclusion: These results shown that even at physiological levels, vitamins may exert antagonistic

effects on the blood of patients with breast cancer. More studies are being conducted in order to

understand which mechanisms permeate these phenomena.


DNA vacines for cancer treatment

SILVA, M.C. (1); VARGAS, B.J. (1); GUIMARÃES, T.D. (1); BENTO, J.F. (1)

1. Centro Universitário Campos de Andrade

E-mai: [email protected]

Introduction: Immuno-oncology is a rapidly growing field of cancer research dedicated to

developing novel cancer therapies by understanding and harnessing immune pathways and has

become an effective way to combat cancer, which can be combined with surgery, chemotherapy and

radiation.therapy. Therapeutic cancer vaccines are one of the most developed part of immuno-

oncology. Unlike prophylactic vaccines which are used for the prevention against oncogenic agents,

such as human papilloma virus, therapeutic cancer vaccines utilize tumor antigens to stimulate the

immune system of cancer patients to fight against cancer. Cancer treatment vaccines work by

activating B cells and killer T cells and helping them to recognize and act against cancer cells which

involves isolating autologus tumor antigens and creating a vaccine that present the antigen back to the

patient, thereby stimulating the immune system to attack the cancer. DNA vaccines represent one of

cancer immunotherapy. DNA vaccines are bacterial plasmids containing the coding nucleic acid

sequence of a target antigen under the control of a eukaryotic promoter. Immunization with DNA

vacines has been shown to elicit both humoral and T cell–mediated immune responses against a

variety of tumor antigens in multiple preclinical models and in early human clinical trials, altough

they fail to generate sufficient CD4+ T cell responses.

Methods: It consists of review of the use of DNA vaccines at cancer treatment. The mains focus is to

describe the advanages of DNA vacines over other types of immunization and to analyze data from

preclinical studies to verify the potencial of DNA vaccines against several tumors.

Results and Discussion: DNA vacines have advantages relative to other antigen-specific approaches:

plasmid DNA is easy and inexpensive to manufacture and the ability to manipulate the backbone of

plasmid DNA offers another advantage. Besides that, plasmid DNA is more temperature stable than

peptides and proteins, making DNA vaccines easier to transport and store with a longer shelf life. An

additional advantage of DNA vaccines is the adjuvant property of the bacterial plasmid DNA itself.

The bacterial backbone of DNA vaccine has been shown to elicit innate immunostimulatory

properties through the recognition of unmethlyated CpG-rich regions present in noneukaryotic DNA.

Thus, administration of bacterial DNA can engage immune cells and inflammatory cytokines at the

vaccination site, effectively acting as a vaccine adjuvant. The method of direct injection of tumors, its

use becomes attractive not only due to the decrease of side effects and because it is less invasive but

also as a benefit the increase of survival, effectiveness in potentiating the systemic therapy. The head

and neck carcinoma makes use of the ONCORINE therapy and has as product the oncolytic virus in

the treatment of the disease; T-VEC is directed to treatment in melanomas and makes use of herpes

simplex virus type 1 (HSV-1); SIPULEUCEL-T to treat prostate cancer this vaccine in turn uses

double-stranded tumor cells from the individual's own that stimulates proliferation and maturation of

several types of defense cells, these are examples of vaccine already in administration for treatment

and released by the FDA organization.

Conclusion: DNA vaccines have significant potency in stimulating innate and adaptive immunity

against a wide variety of tumor antigens, although this stimulation remains to be achieved in patients.

Further studies must focus on testing vaccines and adjuvant strategies in humans. Finally, combining


DNA vaccination with other immunotherapeutic approaches, such as checkpoint inhibitors, may be

the most effective way to develop a comprehensive immune-based treatment for cancer.


Caracterization of the histone deacetilase 2 of Toxoplasma gondii

SIQUEIRA, C.M. (1); FRAGOSO, M.S.I. (1); SEVERO, V.R. (1); LIMA, L.P.O. (2); SILVA, M.S.

(2); AVILA, A.R. (1); SABBAGA, M.C.Q.B.E. (2); SOUZA, T.A.C.B. (1); NARDELLI, S.C. (1)

1. Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brazil.

2. Instituto Butantan, São Paulo, SP, Brazil.


Introduction: Histone deacetylation is a form of epigenetic regulation that is associated with gene

silencing, since the removal of acetyl groups by histone deacetylases (HDACs) results in a more

condensed chromatin. In Toxoplasma gondii little is known about these enzymes, but it is believed

they are an essential part of gene expression regulation. T. gondii has 7 HDACs, including HDAC2

(TgHDAC2), the aim of our study. Although it is considered a classic HDAC of class I very similar to

other eukaryotes, the TgHDAC2 has 2 amino acid insertions inside the HDAC domain, which is

unique to Toxoplasma, and whose function remains unknown. In addition, RNA-seq data from T.

gondii database showed an increase of tghdac2 expression during S phase of the cell cycle. Due to its

particularity, our goal is to characterize HDAC2 of T. gondii (TgHDAC2).

Methods: To this end, we have obtained the knockout of tghdac2, which was replaced by

homologous recombination, by hxgprt gene.

Results and Discussion: So far, we observed a lower infectivity and proliferation rate of Dtghdac2

parasites, suggesting a role during cell cycle progression, possibly during the S phase. Proliferation

assays with EdU incorporation indicated that the lack of tghdac2 leads to a delay during the S phase

of the cell cycle, which may indicate that these parasites have fewer replication forks or the

replication is slower. In addition, knockout parasites also showed a lower rate of adhesion and host

cell invasion, indicating that TgHDAC2 affects the virulence of these parasites. Besides the knockout

we also obtained the overexpression of the protein, which was located in the nucleus of the parasite,

again indicating a nuclear function. Furthermore, circular dichroism analyzes had shown that

recombinant HDAC2 is a very stable and enveloped protein, and in this way we are trying to obtain its

crystal to analyze its three-dimensional structure by X-ray crystallography.

Conclusions: To date, it has been possible to partially characterize the histone deacetylase 2 of

Toxoplasma gondii, indicating that this protein may be involved in the invasion and replication

processes, but further studies are still needed on how it participates in these processes.

Acknowledgement: Instituto Carlos Chagas/Fiocruz-PR; Fiotec; CAPES; CNPq


Multigenerational exposure to manganese alters the antioxidant homeostasis and

functional conditions of the male reproductive system of mice

SOUZA, T. L. (1); BATSCHAUER, A. R. (1); MANUITT, P. (1); MARTINO-ANDRADE, A. J(2);

ORTOLANI-MACHADO, C. F. (1)

1. Laboratory of Embryotoxicology. Department of Cell Biology. Federal University of Paraná,

Curitiba – PR, Brazil.

2. Laboratory of Endocrine Physiology and Animal Reproduction. Department of Physiology.

Federal University of Paraná, Curitiba – PR, Brazil.


Introduction: Manganese (Mn) is an essential element widely distributed throughout the planet and is

used for the several metabolic functions. However, many studies have shown neurotoxic aspects of

this element under certain exposure conditions. Nevertheless, little is known about its effect on the

male reproductive system. Thus, the present study aimed to analyze the effect of exposure to Mn at

realistic doses, over a generation, on the physiology of the male reproductive system of Swiss mice.

Methods: Forty females and 20 males (F0 generation) were treated for 60 days with MnCl2 via

gavage at doses of: 0, 0.013, 0.13 and 1.3 mg/kg/day. Then, copulation was performed to obtain the

F1 generation and treatment with MnCl2 was continuous during gestation and lactation. After weaning

the offspring were divided into two categories, respecting the same doses used in the F0 generation:

animals that received Mn only via parental (PE), during gestation and lactation; and those who were

exposed via parental and direct (PDE), in the same condition as the parents. After 60 days the animals

were euthanized and analyze of sperm parameters, such as motility and concentration were performed.

In addition, the following antioxidant components were evaluated in the testis and seminal vesicles:

catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), non-protein thiols

(NPT) and lipid peroxidation (LPO).

Results and Discussion: There was a drastic reduction in the number of motile spermatozoa as well

as sperm concentration in animals 0.13 and 1.3 mg/kg/day in the PE and PDE conditions. Significant

reductions in the activity of the enzymes of the antioxidant defense system, CAT and GST, were

observed in the testis of the EP animals. In addition, for the seminal vesicle, there was also a reduction

in the activity of CAT and SOD, as well as an increase in LPO at the dose of 1.3 mg/kg/day.

Conclusion: It was found that Mn, even at low concentrations, is capable of promoting the alteration

of sperm quality and oxidative parameters of the male reproductive system in a multigenerational

exposure.

Acknowledgement: We would to thank CNPq (Conselho Nacional de Desenvolvimento Científico e

Tecnológico) for the financial support.

Determination of the effects of the mesoionic compound 4-phenyl-5- [4-nitrocinamoyl] -

1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) over tumoral cells (HT29) employing

chorioallantoic membrane (CAM) as a in vivo model


STOLTZ, I.R. (1); TABORDA ROCHA, L. (1); VICTOR RODRIGUES, A.L. (1); GONÇALVES,

P.G. (1); STUELP-CAMPELO, P.M. (1); ECHEVARRIA, A. (1); PEREIRA, L.F. (1)

1. Curso de Bacharelado em Ciências Biológicas, Laboratório de Biologia Experimental, Escola

de Ciências da Vida, PUCPR, Curitiba - PR.


Introduction: Mesoionic compounds have been studied in the search for antineoplastic drugs, since

its structure confers a biological and biochemical activity. The MI-D, mesoionic compost 4-phenyl-5-

[4-nitrocinamoyl] -1,3,4-thiadiazolyl-2-phenylamine chloride reduces the phosphorylation efficiency

of mitochondria. The chorioallantoic chicken embryo membrane, is a fusion of membranes chorion

and allantoic formed between five and six days of embryo development. CAM is a low cost and

simple handling experimental model. In consequence of high vascularization, it is used for studies of

angiogenic, let tumors grow fast. The membrane also can be used as model for pharmacological tests,

as the MI-D. Objective: The goal of this study was to analyze the effects of MI-D on HT29 cells

(colon carcinoma) in angiogenic and antiangiogenic CAM’s properties. Macroscopically and with

histological procedures.

Methods: All animal procedures were approved by the institutional ethical committee, numbers

850/2016 (first version), 850/2016, 850A/2016 and 850G/2016. For the tests, were used 60 fertilized

eggs of Gallus gallus (Chácara Amazônia - metropolitan area of Curitiba). The eggs were cleaned,

incubated and then opened after 9 days of incubation. The test (N=60) was distributed in six groups:

control, negative control (dexamethasone 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL), positive control (sodium

hydroxide 0.008 g/mL, 0.04 g/mL, 0.08 g/mL) collagen control, collagen + HT29 cells, collagen +

MI-D (10 eggs per group). Prior to CAM test, HT29 cells were cultured in RPMI 1640 growth

medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 0.1 mg/mL

streptomycin. Cells were added to collagen on a concentration of 1x107 and polymerized in a

concentration of 200 µL using sterile PBS 1 x. Controls test were performed containing only collagen,

collagen + MI-D (5 µM, 25 µM, 50µM) and collagen + HT29 cells. After the shell opening and

inoculation, the eggs were closed with tape and incubated to three days. After twelve days, the eggs

were reopened, analyzed macroscopically and photographed with a SONY DSCW - 610. 14.1

megapixels coupled to a stereoscopic microscope in a 30x magnification. To analyzes was used a

software IMAGE PRO PLUS, to determine the blood vessel density. For the histological analyzes

(H&E, Sirius Red) part of CAM with the tumor was removed, fixed during 10 minutes in Formalin

10%. The embryos were euthanized by injection of 0.2 mL of Ketamine:Xylazine, after that were

clean with distillated water, photographed and preserved in Formalin 10% to morphological analysis.

For the statistical analysis, grids with 9 quadrants totalizing 1 mm2 were positioned between two

caliber vessels for a random quantization of the average number of vessels per mm2. The statistical

study was based in the sample of the 10 independent experiments, performing M ± SD, ANOVA and

Tukey’s test.

Results and Discussion: The results were significantly, changing the number of blood vessels per

mm2 against the collagen control 12,7 ±0,32 vessels / mm2. The tests containing MI-D and tumor

cells shown significant decreasing on all concentrations. Of 15,74% in 5 µM (10,70 ± 0,84 v/ mm2),

of 26,29% with 25 µM (9.49 ± 0.25 v/mm2), and 36,61% with 50 µM (8,05 ± 0,44 v / mm2). The

thickness and depth of CAM shown significantly decrease among the experiments with MI-D, 60%

with 5 µM, 30% with 25µM, and 28% with 50µM.


Conclusions: The MI-D changed CAM’s vascularization, decreasing the blood vessels density

(aprox. 37% with 50 µM, 27% with 25 µM and 15% with 5 µM) and triggered embryo malformation.

This effect corroborated to the MI-D anti-inflammatory effect described on the literature.

Acknowledgment: Pontifical Catholic University of Parana (PUCPR).


Evaluation of decavanadate salts (([V10O28]6-); {(NH4)2[Cu(H2O)6]2[V10O28]}2H2O) in

the development process of HET-CAM vessels

TABORDA-ROCHA, L. (1); STOLTZ, I.R. (1); FARIA, E.M.M. (1); LOPES, R.H.L. (1); PIRES,

D.C. (1); RODRIGUES, A.L.V. (1); LEME, L.B.P. (2); POSTAL, K. (2); SÁ, E.L. (2); NUNES, G.G.

(2); PEREIRA, L.F. (1)

1. Curso de Bacharelado em Ciências Biológicas, Laboratório de Biologia Experimental, Escola

de Ciências da Vida, PUCPR, Curitiba/Paraná.

2. Laboratório de Química Inorgânica, Dep. de Química, UFPR.


Introduction: Vanadium compounds have been extensively studied due to their interaction with

proteins and their potential application in medicinal chemistry. Among them, decavanadate, [V10O28]6-

, has demonstrated a wide medical application. The chicken embryo chorioallantoic membrane

(CAM) is a tissue that forms a wide network of blood vessels being very useful to visualization and

determination of biological phenomena of angiogenesis. Therefore, CAM is of relatively easy

manipulation and is a good model to evaluate the effect of chemicals in the angiogenesis. The copper

element plays an essential role in the biochemistry of aerobic organisms, being essential to catalyze a

wide range of enzymes. Thus the presence of copper inserted in a decavanadate structure should

improve his biological activity. Objectives: Evaluation of decavanadate effects combined with a first

transition metal element in the formation of blood vessels using chick embryo chorioallantoic

membrane as a model.

Methods: Heterometallic decavanadate were prepared by the addition of MCl2 (M = CuII) to freshly

prepared (NH4)6[V10O28] aqueous solution in the proportion of 2:1. Products were characterized by

spectroscopic and diffractometric methods. All animal procedures were pre-approved by the

institutional ethical committee nº 850/2013 (1st version) and 850/2016. The fertilized eggs of chicken

(Leghorn variety) were incubated for 7 days. A window was opened in the shell and CAM injected

with different concentrations (1.0, 2.5, 5.0, 7.0, 10, 15, 20, 35, 70, 100, 500, 1000 and 3000 µg·mL-1)

of the products and sealed with adhesive tape. Seven days later CAM was photographed with a SONY

DSCW-610 14.1 MP coupled to a stereoscopic microscope (30 x magnification). CAM was removed

and fixed with formalin 10% and analyzed to count the blood vessels. The embryos were euthanized

with 0.2 mL of ketamine-xylazine and fixed to further morphological studies. The pictures were

analyzed with Image-Pro Plus program. Statistical analyses were performed with meanSD, ANOVA

and Tukey's test.

Results and Discussion: Assays with CAM revealed that solutions of copper decavanadate in

concentrations above 15 µg·mL-1 were lethal to the embryos, causing desmoplasia (opacity,

degradation) and disintegration of CAM. The doses up to 15 µg·mL-1 were not embryotoxic, but

triggered an increasing of CAM’s blood vessel density in a dose-dependent manner, as compared with

a control without the addition of products. On the other hand, only the decavanadate was lethal at all

concentrations evaluated.

Conclusion: The studies showed that the general response of the system was dose-dependent,

resulting in an angiogenic effect. All concentrations above 15 µg·mL-1 decreased the morphological

development of the embryos, in this sense is possible to infer a toxic effect of decavanadate and


copper decavanadate on the CAM. More assays should be conducted to verify of decavanadate effects

on CAM and on the embryo.

Acknowledgment: Pontifícia Universidade Católica do Paraná, Universidade Federal do Paraná

(UFPR).


Antioxidant Kraft lignins induced DNA oxidation and increased intracellular reactive

oxygen species in biological system (HepG2 cells)

THÁ, E.L. (1); SOUZA, I.R. (1); MAGALHAES, W. L. E. (2); LEME, D.M. (1)

1. Department of Genetics, Federal University of Paraná (UFPR), Curitiba-PR/Brazil;

2. Embrapa Forestry – Brazilian Agricultural Research Agency.


Introduction: Lignin, a major component of biomass, is a polymer that promotes impermeability and

structural stability to plant cell walls. Every year, tonnes of lignin are produced worldwide as by-

products of the paper industry, and thus, studies have been proposed to identify biological properties

that can make lignin value-added products. Antioxidant capacity, a health benefit property, has been

demonstrated to lignin, making this natural phenolic compound a good alternative to synthetic phenol-

based antioxidants. However, the potential pharmacological use of lignin requires safety assessments.

For that, we evaluated the genotoxic potential of two fractions of lignin derived from Kraft process by

the in vitro Comet assay.

Methods: The two fractions of kraft lignin were obtained from a sequential acid precipitation of kraft

black liquor donated by a Paper industry, and they were named as fraction 3 (F3, pH 3) and fraction 5

(F5, pH 5). The genotoxicity test Comet assay (alkaline and oxidative versions) with an in vitro liver

cell model (human hepatocarcinoma cell line - HepG2) was used to determine the potential of lignin

fractions in inducing DNA damages. The intracellular reactive oxygen species were also quantified on

HepG2 cells by H2DCFDA probe (flow cytometry).

Results and Discussion: The tested lignin fractions are considered antioxidants by the DPPH assay;

however, the results of the oxidative Comet assay (hOGG1) showed that these fractions were able to

oxidize DNA bases. In addition, the lignin fractions also increased the levels of intracellular reactive

oxygen species (ROS). The discrepant results between DPPH assay and the tests with HepG2 cells

may relate to the fact that DPPH assay is based on chemical reactions to determine free radical

scavenging activity, and, consequently, the biological relevance of this assay might be uncertain in

some cases. However, as other phenolic compounds, lignin may present pro- and anti-oxidant

activities depending on the testing conditions.

Conclusion: The Kraft lignin fractions, pH3 and pH5, may pose risks to human health by stimulating

the production of ROS and inducing DNA oxidation; however additional studies are needed to better

determine the hazard of Kraft lignins. Comparing our findings with the literature results from DPPH

assay of those fractions, we can suggest that in chemico tests should be carry out with other biological

system-based tests to increase the predictivity of determining free radical scavenging activity of

compounds.


Evaluation of in vitro influence of Nyssomyia neivai saliva on infective potential of

Leishmania braziliensis stains from the state of Paraná.

TIRADO, T.C. (1,2); FIGUEIREDO, F.B. (1); RIBEIRO, M.C.V.C. (2); DE ANDRADE, A.J. (2)

1. Instituto Carlos Chagas.



Introduction: American Cutaneous Leishmaniasis (ACL) is a serious disease caused by protist

parasites of the genus Leishmania, distributed worldwide and growing every year. It is a pathology

that is linked to population where the poverty index is high and there are few treatment alternatives,

characteristics that make it a neglected one. ACL is transmitted to the host through the blood meal of

the female phlebotomine, belonging to the subfamily Phlebotominae. Saliva from sand flies has been

much studied in recent years. The salivary components are endowed with high pharmacological

properties, such as those very important for parasite-host interactions. Patients who are pre-

immunized with the saliva proteins of the vectors become more protected from infection against

Leishmania. Nyssomyia neivai is one of the main vectors of Leishmania braziliensis in the state of

Paraná, so it is in the process of disseminating most of the cases of ACL in the state. The lack of

studies was an issue of interest for the development of the present study.

Methods: In total, 130 female specimens of Nyssomyia neivai were identified and analyzed. In vitro

infections were made in THP-1 cells with 4 strains of Leishmania braziliensis using 4 different

dilutions of the salivary gland, aiming at the differences between strains and between the

concentrations. The numbers of cells and amastigotes were counted automatically by an equipment

called Operetta, and the data provided by the reading allowed the calculation of the Infection Index,

which was the comparison factor between the groups and strains.

Results and Discussion: Regarding the Control without saliva, only in the P5 strain was observed

statistical difference. The concentration of 1 gland per well infected more, implying that this is an

ideal dilution and favors the entry of the parasite into the cell. Interestingly, the female sand fly uses

only the contents of one of the two glands at the time of the blood meal.

Conclusion: In this context, the use of this dilution could be a starting point for immunization tests,

considering that producing vaccination to this patology is a great public interest.

Acknowledgment: We thank Instituto Carlos Chagas, Universidade Federal do Paraná, CNPq and

Fundação Araucária.


Aerobic physical training associated to omega-3 reduce inflammatory cytokines in the

prostate of Wistar rats submitted to high fat diet

VERAS, A.S.C. (1); GOMES, R.L. (2); TAVARES, M.E.A. (3); CORREIA, R.R. (3); LENQUISTE,

S.A. (2); VANDERLEI, L.C.M. (4); TEIXEIRA, G.R. (1,3).

1. Postgraduate Program in Movement Sciences, Department of Physical Education, School of

Technology and Sciences, UNESP campus of Presidente Prudente-São Paulo.

2. Faculty of Health Sciences - Universidade do Oeste Paulista (UNOESTE), Presidente

Prudente-São Paulo.

3. Department of Physical Education, School of Technology and Sciences, UNESP campus of

Presidente Prudente-São Paulo.

4. Department of Physiotherapy, São Paulo State University (UNESP), School of Technology

and Sciences, Presidente Prudente-São Paulo.


Introduction: The poor lifestyle such as excessive consumption of high fat diets, insufficient physical

activity and sedentary lifestyle are important factors for the onset and progression of diseases. The

high fat diet (HF) promotes an inflammatory/obesogenic environment triggering metabolic changes

responsible for the onset and development of diseases such as cardiovascular diseases, obesity and

lesions on prostate morphology. Inflammation is part of the host's normal response to infection or

injury. However, excessive inflammation contributes to a number of acute and chronic human

diseases and is characterized by the production of inflammatory cytokines. At sufficiently high

intakes, long-chain omega-3 (n-3) polyunsaturated fatty acids (PUFAs), as found in fish oils, decrease

the production of inflammatory eicosanoids, cytokines, and reactive oxygen species. The physical

exercise use these lipids as fuel during the training session, protecting against prostate disorders and

acting in metabolism. The ingestion of n-3 associated with physical exercise practice has its

potentiated and recent indicative benefits related more to the performance of aerobic activities due to

the vasodilating properties of n-3, improving the oxygen flow and nutrients to the muscle tissues

during the exercise. The aim of study was to evaluate the effects of aerobic physical training

associated with omega-3 in inflammatory markers in prostate of Wistar rats supplemented with a high

fat diet.

Methods: 35 male Wistar rats (±200g) were underwent in 7 groups (n=5): CT - control animals that

were fed standard feed and water ad libitum; HF - animals supplemented with high fat diet; HF+n³ -

animals supplemented with high fat diet and received omega-3; HF+Ex - animals supplemented with

high fat diet and performed physical exercise; HF+n³+Ex - animals supplemented with high fat diet,

received omega-3 and performed physical exercise; HF+Chia - animals supplemented with high fat

diet and received chia oil; HF+Chia+Ex - animals supplemented with high fat diet, received chia oil

and performed physical exercise. The high-fat diet was composed of roasted peanuts, milk chocolate

and cookies (3:2:2), omega-3 and chia were applied by gavage, as well as water in the other groups to

induce the same type of stress. The swimming training protocol was performed 3 times a week for 30

minutes and the study was approved by CEUA (protocol number 3962). The One-way ANOVA with

a Bonferroni post-hoc test was used to statistically analyze, the intensity of cytokines was examined

using Image-J software.


Results and Discussion: The immunoreactivity of NF-KB presented no significant difference

between the groups. The HF+n³ animals increased the TNF-α labeling (13,46±1,09) compared to CT

group (10,89±0,73), when associated with exercise, the HF+n³+Ex and HF+Chia+Ex groups reduced

the TNF-α expression (8,82±1,79 and 8,54±0,59 respectively), compared to HF (12,69±1,02) group.

Comparing with HF groups yet, the HF+n³+Ex group decreased this expression too. On the other

hand, the IL-6 expression were most pronounced in HF (11,55±1,15), HF+Chia (11,93±0,64) and

HF+Chia+Ex groups (12,18±0,85) compared to CT group (8,98±0,95). The HF+n³ group decreased

IL-6 labeling (9,08±1,41) compared to HF (11,55±1,15) and HF+Chia (11,93±0,64). The animals of

HF+Ex (9,54±0,32) and HF+n³+Ex (9,83±0,54) minimized this expression compared to HF+Chia+Ex

(12,18±0,85).

Conclusion: Nevertheless, the aerobic physical training associated to omega-3 intake were able to

decreases partially the inflammatory cytokines and prevents prostate injuries.

Acknowledgement: This study was financed in part by the Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior - Brasil (CAPES)-Finance Code 001.


Identification and characterization of carbazoles derivatives as new inhibitors of

ABCG2 transporter

ZANZARINI, I. (1); ZATTONI, I. (1); KITA, D.H. (1); REGO, F.G.M. (1); PICHETH, G. (1); LE

BORGNE, M. (1); MOURE V, R. (1); VALDAMERI, G. (1)

1. University Federal of Paraná, Clinical Analysis Department.


Introduction: Cancer is one of the diseases with the highest mortality rates worldwide and the

emergence of neoplasms presenting resistance to chemotherapy, also known as multidrug resistance

(MDR), makes this conjuncture even worse. The overexpression of transmembrane proteins named

ABC transporters is considered the main cause of this clinical condition. The human genome encodes

48 ABC transporters and three of them are highlighted for their involvements in the MDR

phenomenon: P-gp, MRP1 and ABCG2. These transporters can recognize and promote the efflux of a

broad spectrum of antineoplastic agents; thus, many studies have been carried out to develop

compounds and evaluate their ability to inhibit this activity. Despite its pronounced relation with

MDR, there are still no promising inhibitors of ABCG2 to be forwarded to clinical steps of drug

development, which endorses the urgency to identify and characterize new selective inhibitors of this

protein. The ascending development of new drugs from heterocyclic scaffolds relates to the role of

this type of molecules in biological processes. Carbazol, an aromatic compound with many

pharmacological activities, was reported as an inhibitor of CK2 protein, which is highly

comprehended in numerous pathologies, including cancer. The circumstances presented above reveal

the relevance of testing carbazoles derivatives (CDs) as ABCG2 inhibitors, aim of this study.

Methods: HEK293 cells stably transfected to overexpress ABCG2 were mostly used. For the

inhibition assays, cells were treated with mitoxantrone/CDs and intracelular fluorescence was

measured by flow cytometry. Cell viability was performed according to MTT method. The ATPase

assay was conducted using a commercial kit. Confocal microscopy was executed with cells treated

with Hoescht 33342/CDs. CDs cytotoxicity was conducted with a range of 0.09-100 μM and the

reversion assay was performed using 0.49 and 10 μM of CDs in co-treatment with SN-38 in a range of

0.1 nm-20 μM. Antibody 5D3 binding assay was performed by flow cytometry.

Results and Discussion: Five carbazoles derivatives were tested to inhibit ABCG2 activity. All

compounds showed positive results (IC50 values: 0.49 to 3.39 μM) and, positively, were also highly

selective for ABCG2 when tested as P-gp/MRP1 inhibitors. Moreover, the compounds showed a very

low cytotoxic (IG50 values higher than 100 μM). The most auspicious compound, with a therapeutic

ratio [TR (cytotoxicity/inhibition)] notably higher than 200, had its inhibition mechanism

characterized and were established as a noncompetitive and substrate independent inhibitor with no

allosteric effects neither on the recognition of 5D3 conformational antibody nor on the ATPase

activity of ABCG2. Additionally, the capacity to reverse the resistance phenotype mediated by

ABCG2 was verified after long-term assays, which are closer to clinical reality. Finally, molecular

docking analysis demonstrated that the bonds between ABCG2 amino acid residues and the fused

rings of the compound are mainly responsible for their interaction, emphasizing the importance of its

hydrophobic core.

Conclusion: We identified the first carbazoles derivatives capable of inhibiting the substrate transport

mediated by ABCG2 in a potential, selective and minimal cytotoxic behavior. Properties as substrate


independence, inhibition kinetic and absence of allosteric effects corroborates with the literature and

the ability to reverse the resistance mediated by ABCG2 enable future preclinical trials. Also, docking

studies listed the prior chemical attributes that must be considered in future design of new compounds

for this purpose. Further assays using cancer cells overexpressing ABCG2 will be conducted.


Characterization of the effects of porphyirinic derivatives in ABCG2 transporter

ZATTONI, F, I. (1); KITA, D.H. (1); ZIASCH, M. (1); GUANAES, L.D. (2); MORAES; F.G DE M.

(1); PICHETH, G. (1); GOLÇALVES, A.G. (2); VALDAMERI, G. (1)

1. University Federal of Paraná, Clinical Analysis Department.

2. University Federal of Paraná, Pharmacy Department.


Introduction: Multi-drug resistance (MDR) is the capacity to avoid cell death in cancer cells. The

expression of ABC transporters can cause major implications on chemoterapy failure. Forty-eight

genes are known to code ABC transporters but three of them are the most implicated on cancer

resistance: P-gp, MRP1 and ABCG2. ABCG2 is the second most prominent transporter in neoplastic

cells, but there is no selective inhibitor testable on clinical trials due the cytotoxicity presented by the

inhibitors. Porphyrins are widely used in Photodynamic Therapy (PDT) and are classic substrates for

ABCG2. The efflux of porphyrins represent a limitation on PDT since their efflux can limit the

therapy results, conversely, these compounds can be used as probes by flow cytometry due their

fluorescence. A common approach to develop new inhibitors is to take advantage of a substrate

scaffold and perform chemical modifications to turn the molecule into an inhibitor, however, there is

no porphyrin described as ABCG2 inhibitor. The aim of this work was to study the effects of 25

porphyrins on the ABCG2 transporter as either substrates or inhibitors.

Methods: For the inhibition assays HEK 293 cells stably transfected to overexpress ABCG2 were

treated with mitoxantrone/porphyrin and intracelular fluorescence was measured by flow cytometry.

Confocal microscopy was performed using the same cell lineage treated with Hoescht

33342/porphyrin, the excitation/emission wavelength was 405/425-475 nm, respectively. Cell

viability was performed by MTT assay. Porphyrin citotoxycity was conducted with a range of 0,09-

100uM and the reversion assay was performed using 10 uM of porphyrin in co-treatment with SN-38

in a range of 0,1nm-20uM. The ATPase assay was performed using a commercial kit. Antibody 5D3

binding was performed by flow cytometry.

Results and discussion: Twenty-five porphyrins were tested as substrates and inhibitors. None of the

porphyrins were identified as substrate, which is very interesting since non transported porphyrins can

represent a promising class for PDT in some types of cancer. The same structures were tested as

inhibitors and one porphyrin was able to inhibit the mitoxantrone transport mediated by ABCG2 in

84% at 10uM, concentration at which the porphyrin shows no toxicity, and an IC50 of 1,3uM. The

same pattern was observed by confocal microscopy, using Hoescht 33342 as substrate, showing that

the inhibition is substrate-independent. Until now, the porphyrin did not inhibit P-gp and the tests with

MRP1 are in progress. Long-term cell viability assays showed that the porphyrin in co-treatment with

chemoterapic SN-38 was able to reverse in 7 times the resistance phenotype when compared with the

sensitive parental cell lineage. ATPase activity measurement and conformational 5D3 antibody

binding were performed to evaluate the allosteric modifications on the protein, but alterations were

not observed.


Conclusion: We identified one porphyrin capable of inhibiting the ABCG2 transporter and also able

to reverse the resistance in transfected cells lineage in co-treatment with SN-38 with no allosteric

modifications detectable by ATPase activity or 5D3 conformational antibody. This result represents

the first porphyrin described as inhibitor and non-substrate on the literature. Further experiments will

check the resistance reversion on cancer cells overexpressing ABCG2 transporter, the effect on the

expression with long-term treatment, the inhibition mechanism and tests in silico to verify the

porphyrin’s binding pocket on the protein structure.

Aknowledgments: CNPq (400953/2016-1), Fundação Araucária (Code 006-09/2016), CAPES

(Finance Code 001), PPGCF and LCDR.


Morphological comparison of the skeletal striated muscle of different experimental

designs after application of a histopathological analysis protocol

ZAZULA, M.F. (1); BOARO, C.D.T. (1); KIRSCH, C.B. (1); LUDWIG, A.F. (1); RÖHL, R.C.T. (1);

COSTA, R.M. (1); GUIMARÃES, A.T.B. (1); MACEDO, A.B. (2); RIBEIRO, L.C. (2)


– UNIOESTE.




Introduction: The use of histopathology is one of the simplest and most accessible ways of

evaluating changes in the tissue studied, however, there are few standardized tools that can transform

the descriptive data into values that can be compared quantitatively.

Methods: For the application of the protocol, we used 06 experimental groups, where 48 histological

slides of the anterior tibial muscle were stained in hematoxylin and eosin, being: GC: adult males that

did not submitted to any intervention; GE: elderly females; GI: adult males immobilized for 15 days;

GL: adult males with sciatic nerve compression; GO: adult males obese by chemical induction

(MSG); GP: oophorectomized adult females. The slides were blinded by specific adaptations to the

skeletal muscle of the histopathological protocol proposed by Bernet et al, 1999. The values obtained

were submitted to the analysis of variance test. The categories applied were tissue damage index (Itec)

and percentage damage index (Itec%), related to total changes, circulatory disorders index (IprI),

index of regressive alterations (IprII), index of progressive alterations IprIII) and inflammation index

(IprIV). After the application of the histopathological protocol, the histological description of the

experimental groups was carried out to compare the obtained results.

Results and Discussion: A strong categorization was achieved for Itec in all classes of variables, and

the most representative damages were always from the GE, GI and GL groups, followed by the GO

and GP groups when compared to the CG (F = 135.44; p <0.0001; CV = 10.12%). The same

categorization pattern was observed for IprII (F = 99.667, p <0.0001, CV = 12.07%), IprIII (F =

16.45, p <0.0001, CV = 26.4% and IprIV (F = 38,752, p <0.0001, CV = 34.16%). The IprI values

were less categorized, however, the mean values were those of the GC and the highest of the GE,

while the GI and GL were like both the GE and the GO and GP, which were like the CG (F = 6,

37778, p = 0.0001, CV = 34.16%). The CG animals presented normal histological characteristics,

without marked alterations, while the GE, GI and GL animals had a high presence of atrophic fibers,

degeneration processes, presence of inflammatory infiltrate and alteration in the associated tissues.

The GO and GP animals presented more subtle alterations when compared to the other groups. Both

histological and quantitative evaluation were similar in the presentation of the categorization of the

experimental models.

Conclusion: The most invasive nature of the aging, immobilization and nerve injury models were

responsible for the high values of the reaction patterns, as well as the presence of important

morphological changes.

Acknowledgement: To Fundação Araucária, for the financial aid through the public notice 016/2016-

UNIOESTE/ basic and applied research.


Selection of a more resistant and infective population in Trypanosoma cruzi by high

exposition to normal human serum

ROSSI, I (1); NUNES, M. A. F. (1); RAMIREZ, M.I. (2,3).

1- Programa de pós-graduação em Biologia Celular e Molecular, Universidade Federal do

Paraná.Curitiba/PR - Brasil

2 – Departamento de Bioquímica e Biologia Molecular, Universidade Federal do

Paraná.Curitiba/PR - Brasil

3 - Instituto Oswaldo Cruz. Rio de Janeiro/RJ - Brasil


Introduction: The protozoan Trypanosoma cruzi is the etiologic agent of Chagas' disease, initially

restricted to the Americas, but has spread throughout the world, reaching millions of individuals. T.

cruzi has a complex biological cycle where it needs to evade the immune system and invade host cells

to complete the infection. One of the most effective mechanisms in innate immune defense against

pathogens is the complement system, which consists of a set of proteins that are activated in cascade

and which culminate in the formation of a pore in the membrane of the microorganism, causing its

lysis. T. cruzi have developed several escape mechanisms to the complement system, expressing

different molecules and releasing extracellular vesicles. Extracellular vehicles (EVs) are small

vesicles composed of a lipid bilayer which comprises microvesicles and exosomes, according to their

size and biogenesis. Our group has shown the release of EVs during the interaction between the

parasite and host cells promotes complement system inhibition and increases the invasion of

metacyclic forms of T. cruzi to host cells.

Methods: We selected a population of epimastigotes from Trypanosoma cruzi G strain through 30%

normal human serum exposition (2R population). The 2R parasites were recovered and evaluated their

ability to infect eukaryotic cells and resist the complement system.

Results and Discussion: The population of selected parasites presented greater resistance to lysis by

the complement system and also greater infectivity to eukaryotic cells when compared to the wild

type parasite. Metacyclic trypomastigotes from 2R were at least three times more infective to

eukaryotic cells even in the presence of normal human serum. The production of EVs was similar

among populations, but 2R parasites presented greater resistance in the presence of EVs. It was

evaluated that the resistance phenotype became unstable and was lost over time, but the parasites

remained more infective and seems to express more gp82 on its surface.

Conclusion: It is possible to select a population of parasites more resistant to SNH. The resistance to

the complement system is transient and becomes unstable over time. The selected parasites are more

infective, even in the presence of normal human serum, possibly due to increased exposure of gp82,

the main antigen related to metacyclic invasion.

Acknowledgement: We would like to thank Dr. Wanderson Da Rocha for

sharing his laboratory at the Universidade Federal do Parana. Finally, this

study has received support from FIOCRUZ, CNPq, CAPES and Programa de Pós-graduação em

Microbiologia, Parasitologia e Patologia, Curitiba, Brazil. M.R is currently fellow from CNPq-Brazil.

2° simpósio araucária em biologia celular e molecular · 2° simpósio araucária em biologia...

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