biomolecules – identification and...

1

13th

International Seminar:

"Biomolecules – Identification

and Functions"

Krakow, 19-20.10.2018

www.biomolecules2018.pl

2

Scientific organizers

Department of Biochemistry and Neurobiology

Faculty of Materials Science and Ceramics

AGH University of Science and Technology

Mickiewicza 30, Krakow, Poland

Piotr Suder, Ph.D.

e-mail: [email protected]

Anna Drabik, Ph.D.

e-mail: [email protected]

ISBN 978-83-66016-21-7

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The event has been kindly sponsored by

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Scientific Program

19th

October (Friday)

AGH Building B8, Room 010

9:00-10:00 Registration / Coffee Break

Chair person: Marek Smoluch

10:00-10:45 Gaetano Malgieri, Universita degli Studi Della Campania, Caserta, Italy

„Protein structure, folding, and amyloid formation: an NMR view”

10:45-11:30 Marcin Chmielewski, Future Synthesis „Functional chemical modifications in

the nucleic acids”

11:30-11:50 Jerzy Jankowski, Bio-Rad “Automated protein purification with NGC

chromatography system”

11:50-12:20 Coffee Break

12:20-13:30 Poster Session

Chair person: Anna Antolak

13:30-14:15 Frederic Duconge, CEA, Molecular Imaging Research Center, Paris, France

„Preclinical evaluation of aptamers with imaging”

14:15-15:00 Dorota Kwiatkowska, Head of Department of Anti-Doping Research,

WADA Accredited Laboratory, Institute of Sport - National Research Institute,

Warsaw, Poland „Antidoping analysis - interpretation of results, search for new solutions”

15:00-15:20 Wojciech Bieniek, Perlan Technologies „Real-time measurement of functional

metabolism in living cells by means of Agilent SeahorseXF technology”

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Scientific Program

20th

October (Saturday)

AGH Building H-B3/B4, Room 131

Seminar Room 1

10:00-10:45 Bio-Rad, NGC chromatography system

10:45-11:30 Perlan, Agilent SeahorseXF technology


12:30-13:15 Bio-Rad, NGC chromatography system

13:15-14:00 Perlan, Agilent SeahorseXF technology

Seminar Room 2

10:00-10:45 Polygen, Multi-Angle Light Scattering system

10:45-11:30 Biogenet, Multifunctional ELISA Reader


12:30-13:15 Polygen, Multi-Angle Light Scattering system

13:15-14:00 Biogenet, Multifunctional ELISA Reader

6

Oral communications

7

L01

Protein structure, folding and amyloid formation: an NMR view

G. Malgieri1

1Universita degli Studi Della Campania, Caserta, Italy

Abstract

Amyloid fibrils and plaques are the distinctive feature of those known as “protein deposition

diseases”1-3

, that include Alzheimer’s, Parkinson’s, Prion diseases and numerous forms of

amyloidosis. In the cellular environment, amyloid formation has been reported to be favored

by protein mutations, chemical modifications and metal ions.

This places an increasing importance on the development of approaches to characterize the

influences of these factors on the structural, folding and aggregation properties of proteins and

to clarify the links between these properties and the associated biological functions.

Challenges associated with such characterization have thus resulted in the increasing

development of an array of biophysical methods. NMR spectroscopy is perhaps the most

readily suited technique for providing high-resolution structural information on protein states

in solution.

Over the past years, our group has been involved in the application of the most advanced

solution NMR techniques (in an integrated approach with other biophysical techniques) to the

study of proteins involved in neurodegenerative diseases. The recent advances in the

application of these methods and our contribution to the field will be discussed.

1 M. Stefani and C. M. Dobson, J. Mol. Med., 2003, 81, 678–699.

2 F. Chiti and C. M. Dobson, Annu. Rev. Biochem., 2006, 75, 333–366.

3 F. Chiti and C. M. Dobson, Nat. Chem. Biol., 2009, 5, 15–22.

8

L02

Functional chemical modifications in the nucleic acids

M. K. Chmielewski1,2

1FutureSynthesis sp. z o.o. ul. Rubież 46H 61-612 Poznań, Poland

2Institute of Bioorganic Chemistry Polish Academy of Sciences, ul. Noskowskiego 12/14

61-704 Poznań, Poland

Abstract

Nucleic acids carry a wide range of different chemical modifications and employ these

chemical marks to exert essential or critical influences in a variety of cellular processes but

also in biomolecular application.

A wide variety of modifications can be incorporated directly during the synthesis or after

synthesis. Certain modifications (notably Digoxigenin and some fluorescent dyes) are not

available to be incorporated during synthesis and must be attached to the oligo after synthesis

using NHS ester chemistry. Furthermore, all modified oligonucleotides require purification

before using. Desalting, HPLC or PAGE purification is offered for synthetic nucleic acids.

Key Words: chemical synthesis on solid phase, modified oligonucleotides, methylation, thermolabile protecting

groups

Acknowledgements: Research co-financed from the funds of the European Union. Grants no. POIR.01.01.01-

00-0487/17 and POIR.01.01.01-00-0027/17.

Correspondence: [email protected]

9

L03

Automated protein purification with NGC chromatography

system

D. Pianka1, J. Jankowski

1

1Bio-Rad Laboratories | Central Eastern Europe

Abstract

Bio-Rad's NGC Preparative Chromatography Systems are designed to rapidly

automate the purification of biomolecules. The flexible, modular, and economical design

makes the NGC Systems the instruments of choice for method development and scale-up.

NGC Systems are available in pre-plumbed, factory-tested configurations with two different

flow rates. Each preconfigured system can be further customized and upgraded by adding

valves, detectors, or pumps to meet specific application needs, such as tandem and 2D

chromatography.

Multi-D allows users to convert an optimized purification scheme requiring multiple

columns into a single combined method using ChromLab Software method templates. Simply

set up the method and system, push a button, and then walk away to do other work, all while

increasing productivity and reproducibility between purification runs.

Any system can be configured for operation at either high or low flow rate by simply

changing the pump head on the system pump module. With the NGC System, a single

hardware platform can be modified as needed to suit changing applications and scales.

10

L04

Preclinical evaluation of aptamers with imaging

F. Ducongé1

1CEA, DRF, Institute François Jacob, MIRCen, Unité de Recherche Associée CEA-CNRS

UMR 9199, Université Paris Saclay; 18 route du panorama, BP n°6, F-92265, Fontenay-aux-

Roses, France

Abstract

Aptamers are short oligonucleotides (< 100 bases) selected from large combinatorial

pools of sequences (from 1012

to 1015

) for their capacity to bind a target (amino acids,

antibiotic, proteins…). Such ligands are isolated by a method of directed molecular evolution

usually named SELEX (Systematic Evolution of Ligands by Exponential enrichment). Since

2005, our group and others have been adapting the SELEX against whole living cells. Using

this cell-SELEX strategy, we selected several nuclease resistant 2'-Fluoro-pyrimidines (2'-F-

Py) RNA aptamers against cell surface biomarkers that can represent surrogate markers of

cancer cells. Although these aptamers represent promising tools for in vitro experiments

(cytometry, microscopy, biochips...), their translation for in vivo uses is still difficult to

predict.

To solve this problem, we have developed fluorescence imaging techniques to

investigate their biodistribution and tumour targeting capacities in small animal models.

Particularly, we demonstrated that fluorescence diffuse optical tomography (fDOT) can

measure nanomolar concentration of oligonucleotides, even in a deep-seated organ. Hence,

six nuclease resistant 2'-fluoro-pyrimidine (2'-F-Py) RNA aptamers identified from cell-

SELEX were screened in nude mice bearing subcutaneous tumour xenografts. Despite high

affinity against cells in vitro, only one aptamer (named ACE4) exhibited significant tumor

uptake compared to a scramble sequence. This aptamer was then used to purify and identify

its target, annexin A2 (AII), a known cancer biomarker involved in metastasis and

angiogenesis.

In conclusion, due to the high discrepancy between in vitro models and what occurs in

vivo, we postulate that in vivo imaging based screening represents an efficient method to

discriminate aptamers that can bind a target of therapeutic interest and, most importantly, that

have the highest chance of being rapidly translated for in vivo use.


mailto:[email protected]

11

L05

Antidoping analysis - interpretation of the results, search for the

new solutions

D. Kwiatkowska1, P. Kaliszewski

1

1Department of Anti-Doping Research, WADA Accredited Laboratory, Institute of Sport -

National Research Institute, Warsaw, Poland

Abstract

Anti-doping laboratories are the key element in the fight against doping. Only laboratories

accredited by the World Anti-Doping Agency (WADA) can analyze samples collected from the

athletes. Currently, there are only 32 such institutions worldwide and one of them is located in

Warsaw. All anti-doping laboratories must perform in accordance with WADA documents such as the

Prohibited List, Technical Documents, Guidelines, and Technical Notes. The documents contain strict

rules that each anti-doping laboratory has to follow while analyzing and interpreting the results. The

crucial features of the Prohibited List are that it is updated annually, e.g. by introducing new groups

of substances and methods, and its open character. The latter results from the fact that in addition to

the examples of specific substances, the list includes the phrase: "(...) and other substances with a

similar chemical structure or similar biological effect".

Moreover, the Prohibited List also includes a group of unapproved substances (i.e. "any

pharmacological substance that is not included in the following sections of the list, and for which no

government healthcare unit has issued a marketing authorization as a medicinal product for human

use, e.g. substances undergoing preclinical or clinical trials, as well as medicines that have been de-

registered, modified drugs, or veterinary medicines”). The introduction of the next WADA documents

impose continuous method development on the laboratories, forcing them to look for the new

solutions.

One of the main challenges to the techniques and methods used in doping control analysis,

apart from the ability to detect and determinate numerous compounds during a single analysis, is to

distinguish compounds with endogenous and exogenous origin. Additionally, it is also necessary to

develop models of interpretation of the results, search for new metabolites of known compounds, and

to identify compounds that appear, e.g. on the basis of a modification of the known ones. Therefore,

the laboratories have to constantly modify their methods working in one network.

During the lecture, examples of identification of various organic compounds forbidden in

sports will be discussed. Furthermore, the importance of the reliable approach in the identification of

the substances in view of evidentiary proceedings adjudicating on the athletes guilt (i.e. violation of

the anti-doping rules) will be proven.

Key Words: Anti-doping, mass spectrometry


12

L06

Real-time measurement of functional metabolism in living cells by

means of Agilent Seahorse XF technology

W. Bieniek1

1Perlan Technologies Polska

Abstract

Mitochondrial function and glycolysis play critical roles in a variety of vital cellular

processes, including cellular activation, proliferation, differentiation, cell death and disease

progression. Therefore, functional metabolic data is required to tell a complete story of

cellular processes and pathologies. Agilent Seahorse XF technology was developed to provide

the capability of examining the intact and functional cellular system, enabling comprehensive

investigations into metabolic pathways, substrate preference and utilization, catabolic and

anabolic processes, and metabolic phenotypes. Agilent Seahorse XF technology allows

simultaneous measurement of mitochondrial respiration (oxidative phosphorylation;

OXPHOS) via the oxygen consumption rate (OCR), and glycolysis via the extracellular

acidification rate (ECAR) using live cells, in real time, in a microplate.

So far, a variety of cell lines has been analyzed by means of Agilent Seahorse XF technology,

including primary cells, adherent and suspension cell lines, isolated mitochondria, islets, 3D

spheroids, Zebrafish embryos, C. elegans, D. melanogaster, yeast, and other biological

materials. Due to the fact that Agilent Seahorse XF technology employs a label-free, non-

invasive methodology, cells can be used post-measurement for other applications or chronic

(long-term) experiments. Agilent Seahorse XF technology has been applied to multiple

research areas, including cancer, obesity, diabetes, metabolic disorders, immunology,

cardiovascular function, neurodegeneration, virology, and aging. In disease application,

Seahorse XF technology can be used to assess energy expenditure, substrate utilization,

mitochondrial function, and general cellular homeostasis.

Key Words: glycolysis, mitochondrial function, metabolism, Seahorse XF


Perlan Technologies Polska Sp. z o.o.

ul. Puławska 303, 02-785 Warszawa

Tel: (+48)-225491400 Fax: (+48)-225491401


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Poster communications

14

P01

Adipogenic differentiation of human umbilical cord mesenchymal

stem cells and human dental pulp stem cells

N. Bryniarska1,2,4

, A. Kubiak3,4

, A. Łabędź-Masłowska4, E. Zuba-Surma

4

1Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish

Academy of Sciences, Cracow, 12 Smetna St, 31-343 Cracow, Poland 2Department of Neurology, Jagiellonian University Medical College, 12 Santa Anny St, 31-

008 Cracow, Poland 3Department of Biophysical Microstructures (NZ55), Institute of Nuclear Physics Polish

Academy of Sciences, PL-31342 Krakow, Poland 4Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology,

Jagiellonian University, 7 Gronostajowa St, 30-387 Krakow, Poland

Abstract

Purpose: The aim of study was to investigate potential of human umbilical cord

mesenchymal stem cells (hUC-MSC) and human dental pulp stem cells (hDPSC) for

adipogenic differentiation.

Experimental: For adipogenic differentiation hUC-MSC and hDPSC were seeded at cell

culture plates coated with 0,1% solution of gelatine. Cells were cultured in StemPro®

Adipogenesis Differentiation Kit (Gibco) for 21 days. At days: 7, 14 and 21 staining with Oil

Red O and qRT-PCR (mRNA for: PPARγ and CEBPα) were performed to assess progress of

adipogenic differentiation.

Results: During hUC-MSC differentiation gradual increase in expression of mRNAs for both

PPARγ and CEBPα was observed. For hDPSC expression those mRNAs also tend to increase

over time. Nevertheless bigger variations in trend of its expression were observed. For both

investigated cell lines cytochemistry staining reveal lipid droplets formation. For hUC-MSC it

occurred after 7 days, while for hDPSC it tends to occur generally after 14 days of

differentiation process.

Conclusions: hUC-MSC as well as hDPSC undergoes adipogenic differentiation what

confirm their mesenchymal character. In case of hDPSC molecular signs of differentiation are

detectable far earlier than changes in morphology.

Key Words: stem cells, differentiation, dental pulp, adipose tissue, qRT-PCR Acknowledgements: FBB of Jagiellonian University is a partner of the Leading National Research Center

(KNOW) supported by the Ministry of Science and Higher Education,

The work was carried out as part of the project financed in the SYMFONIA program of NCN (2015-2019) - No.

2015/16 / W / NZ4 / 00071

Correspondence: [email protected], [email protected]


15

P02

New UV-absorbing mycosporine with strong photoprotective and

antioxidant activity from chlorolichen Cladonia arbuscula

E. Chrapusta1, K. Duchnik

1, B. Bober

1, A. Kaminski

1, J. Białczyk

1

1Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and

Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland

Abstract

Purpose: Previous high-performance liquid chromatography and mass spectrometry analysis

revealed the biosynthesis of a novel mycosporine-like amino acid (MAA) in Cladonia

arbuscula thalli. Due to the fact that the identification of new compounds requires further

analysis aimed to determine their function and ecological role, in the current study, we tested

its photoprotective and antioxidant activity.

Experimental: The antioxidant property of pure isolated compound was evaluated using

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, while its UV protection

ability was assessed spectrophotometrically by determination of UV transmittance through

aqueous solutions at various concentrations.

Results: Performed experiments showed that mycosporine exhibits antioxidant activity,

particularly in a concentration range from 66 to 166 μg mL-1

, providing strong protection

against damage caused by reactive oxygen species generated during excessive exposure to

UV. In addition, it is characterized by high UV-protective properties expressed by efficient

inhibition of UV-B and UV-A transmission. Its solutions with concentrations from 250 to

3000 μg mL-1

fully stop transmission of a wavelength of λ = 310 nm.

Conclusions: The obtained data clearly indicate that the extracted compound may play a vital

role in survival of C. arbuscula in changing environmental conditions by its antioxidant

function and ability to screen shorter wavelengths. Moreover, these results confirmed its

promising therapeutic activity and great significance for future applications.

Key Words: mycosporine-like amino acids, lichens, UV rays, UV protection, antioxidant activity

Acknowledgements: This work was carried out with the financial support of the National Science Centre

(NCN), Poland (Preludium grant 2015/17/N/NZ8/01575). Ewelina Chrapusta received financial resources for the

preparation of a doctoral dissertation from the NCN as part of the Etiuda scholarship funding based on the

decision no. UMO-2015/16/T/NZ8/00153. Faculty of Biochemistry, Biophysics and Biotechnology is a partner

of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.


16

P03

Salivary protein pattern in pregnant cows

Ł. Chrobak1, M. Franczyk

1, J. Wawrzykowski

1, M. Kankofer

1

1University of Life Sciences in Lublin, Faculty of Veterinary Medicine, Department of

Biochemistry, Akademicka 12, 20-033 Lublin

Abstract

Pregnancy in cows lasts around 276 days and is related to deep modifications in the

metabolism of mother which could be reflected in the content of body fluids. From clinical

point of view it is important to obtain valuable information about the health status of patient

based on samples collected in non invasive way. Such matrix containing possible biomarkers

of course of pregnancy could be saliva. Experiments in human medicine already confirmed

that saliva could be a good source of biomarkers, similar studies in animals, however, are

scarce. The present study focused on the description of protein pattern of saliva from pregnant

cows for searching for possible markers of pregnancy course.

Saliva was collected during routine veterinary inspection from healthy, pregnant (3-4 months)

Polish White Black cows aged between 4 and 8 years (n=8) from the same farm. Biological

material was analysed by use of 2D electrophoresis (first dimension – 17 cm IPG strips, pH

range, 4-7, second dimension 17x21 cm gels). Based on statistical calculations (Delta 2D

software) selected spots were cut from gels and identified by MS.

Statistical analysis allowed for the detection of 155 spots in saliva. Out of these numbers 37

proteins which expressed the highest abundance were identified in saliva.

Among identified spots there are proteins which could be related to pregnancy (eg.

Apolipoproteins I and II as well as to regular cellular processes (eg. pyruvate kinase, aspartate

aminotransferase or lactoperoxidase).

The development of sophisticated laboratory techniques, including proteomic approach,

allowed for deeper and specific analysis of bovine saliva. These results can be used for

comparative analysis of different physiological and pathological conditions.

Although further identification of spots as well as the comparison between other periods of

pregnancy are necessary it is already visible that bovine saliva can be considered as valuable

diagnostic matrix containing potential markers of physiological and pathological status.

Key Words: bovine saliva, protein pattern, pregnancy, apolipoproteins

Correspondence: M. Kankofer, University of Life Sciences in Lublin, Faculty of Veterinary Medicine,

Department of Biochemistry, Akademicka 12, 20-033 Lublin, [email protected]

17

P04

Glycoproteome changes as a result of aptamer interactions with

cancer cells

A. Drabik1, J. Ner-Kluza

1, J. Silberring

1,2

1AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department

of Biochemistry and Neurobiology, Mickiewicza 30, 30-059 Krakow, Poland

2Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Curie-Skłodowskiej 34,

41-819 Zabrze, Poland

Abstract

Purpose: Aptamers interacting directly with cancer cells as therapeutics can modulate the biological

pathways for the intervention of many types of diseases such as cancer, infectious diseases, and

cardiovascular diseases. The underlying mechanisms of aptamer-mediated anti-proliferation effect

have not been fully understood, but have been shown to involve the inhibition of cell proliferation via

multiple signaling pathways. In this study, we have identified the proteomic changes of breast cancer

cells as a result of their interactions with aptamers. We have also validated their potential importance

as a tool for biomedical applications in cancer treatment.

Experimental: MCF7 cells were selected as a most popular breast cancer model of human mammary

gland adenocarcinoma. A series of experiments were performed, where target cells were incubated

with two aptamers A26, A33, and a scrambled sequence A33sc inert to MCF7 cells. The capillary

liquid chromatography combined with tandem mass spectrometry approach was designed for proper

protein recognition.

Results: Enriched N-glycoproteins fractions were analyzed using nanocapillary liquid

chromatography combined with tandem mass spectrometry. It was possible to identify 522 proteins in

all types of samples A26, A33, A33sc, and BB. Fourteen proteins were prevalent in samples incubated

with both aptamers A26 and A33 that were not present in control samples A33sc and BB. Finally, it

has been recognized, for the first time, the presence and localization of the specific N-glycan in amino

acid sequence among identified proteins.

Conclusions: In summary, the strategy enables efficient discovery of N-glycosylation patterns and

their localization in cancerous cells as a result of aptamers interactions. The presented approach

represents an effective identification method of potential malignancy - related biomarkers, facilitates

the development of innovative diagnostic and therapeutic tools, and significantly improves the

understanding of cancer disease mechanisms.

Key Words: Breast cancer, MCF7, aptamers, cancerogenesis, theranostics

Acknowledgements: Grant “META” No. 5/EuroNanoMed/2012


18

P05

Upregulation of MUC5AC mucin versus colonization of gastric

epithelial cells by Helicobacter pylori

W. Gonciarz1, M. Walencka

1, K. Hinc

2, M. Obuchowski

2, M. Chmiela

1.

1Division of Gastroimmunology, Department of Immunology and Infectious Biology, Institute

of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental

Protection, University of Łódź, 90-237 Łódź, Poland;

2Laboratory of Molecular Bacteriology, Intercollegiate Faculty of Biotechnology UG-MUG,

Medical University of Gdańsk, 80-210 Gdańsk, Poland

Abstract

Helicobacter pylori colonize gastric epithelial cells of more than half of the human

population. These bacteria cause chronic inflammatory response, peptic ulcer disease and

cancer development. Colonization of gastric mucosa is mediated by surface adhesins of H.

pylori, which bind mucin components, including mucin 5 (MUC5AC). The aim of this study

was to evaluate the role of H. pylori and their components in enhancing the MUC5AC

production and bacterial adhesion using in vitro and in vivo Cavia porcellus (guinea pigs)

models. Primary gastric epithelial cells were isolated from gastric tissue and propagated for

24 h in the culture medium alone or with H. pylori antigens: glycine acid extract (GE), 10

μg/ml; cytotoxin-associated gene A (CagA) protein, 1 μl/ml; UreA urease subunits, 5 μg/ml;

H. pylori or Escherichia coli lipopolysaccharide (LPS) 25 ng/ml or with the live H. pylori

CCUG17874 reference strain (2h, 2×107 CFU/ml). Animals were treated per os with H. pylori

and 7/28 days after inoculation the infection and inflammation caused by H. pylori were

assessed by histopathological, molecular (PCR) and serological examination. Cells or gastric

tissue were stained with anti-MUC5AC antibodies and secondary FITC antibodies to assess

MUC5AC production or anti-H. pylori FITC antibodies to visualize H. pylori adhesion. After

stimulation with H. pylori components (GE, CagA, UreA, LPS) or live bacteria the production

of MUC5AC by primary gastric epithelial cell significantly increased and was related to

elevated adhesion of H. pylori. In gastric tissue MUC5AC was produced more intensively in

the acute (7 days) than chronic (28 days) phase of infection. Blocking of mucin by anti-

MUC5AC antibodies resulted significantly decreased H. pylori adhesion. Upregulation of

MUC5AC production in the milieu of H. pylori and soluble components of these bacteria

especially in the acute phase of infection, was correlated to enhanced colonization of guinea

pigs gastric mucosa by these bacteria. These is an important mechanism maintenance the

infection and induction of pathological disorders by H. pylori.

Key Words: MUC5AC, H. pylori, guinea pigs,


19

P06

Analysis of estrogens in samples using the molecularly imprinted

polymers (MIP) or magnetic molecularly imprinted polymers

(mag-MIP) with FAPA-MS

M. Guć1, M. Pawlaczyk

1, G. Schroeder

1

1Faculty of Chemistry, Adam Mickiewicz University in Poznan, Umultowska 89b, 61-614

Poznan, Poland

Abstract

Purpose: The main aim of study was to create a new analytical technique combining the

molecular imprinting with flowing atmospheric-pressure afterglow plasma ion source mass

spectrometry (FAPA-MS) for the selective determination of hormones in water samples.

Experimental: General method for synthesis of mag-MIP is shown the scheme. Non-

magnetic polymers were obtained in a similar manner, omitting

the stage of application of magnetic particles. Prepared polymers

were characterized with FTIR and TG analysis. SEM images

were also taken to imagine the structure of their surfaces.

Desorption studies under various pH: 5, 7 and 9.5 were also

conducted. Obtained polymers with selective cavities were used

for detection of particular hormones using innovative FAPA-MS

technique.

Results: Desorption temperatures used in FAPA-MS were

defined on the basis of TG analysis. Moreover, maximal

desorption of templates were observed at pH 5 in 30 mins,

afterwards degradation proceed. Limit of detection for

ESI-MS were determined at 10-7

M, while for MIP-

associated FAPA-MS at 10-9

M.

Conclusions: MIP and mag-MIP for selective solid extraction and pre-concentration of

estrogens have been successfully prepared with E1 and β-E2 as templates. Obtained polymers

were efficiently applied to analysis of hormones by ESI-MS and FAPA-MS. The use of the

molecular imprinting technique allowed for direct analysis of analytes from the solid form,

which also prevented subsequent transformation of hormones. Combining of molecular

imprinting with FAPA-MS, gives a promising method for determining hormones in

environmental applications.

Key Words: estrone, β-estradiol, MIP, FAPA-MS, ESI-MS

Acknowledgements: This work was supported by National Grant no. 2016/21/B/ST4/02082, provided by the

National Science Centre.

Correspondence: Faculty of Chemistry, Adam Mickiewicz University in Poznan, Umultowska 89b, 61-614

Poznan, Poland, [email protected]

20

P07

Does Pb toxicity increase the accumulation of plumbagin in

Plumbago zeylanica L.?

M. Hanula1, K. Tokarz

1, W. Makowski

1, B. Piwowarczyk

1, R. J. Jędrzejczyk

2

1University of Agriculture, Faculty of Biotechnology and Horticulture, Institute of Plant

Biology and Biotechnology, Unit of Botany and Plant Physiology, Al. 29 Listopada 54, 31-

425 Krakow, Poland 2Plant-Microorganism Interactions Group, Małopolska Centre of Biotechnology, Jagiellonian

University Kraków, Poland

Abstract

Purpose: Plumbago zeylanica L. (Plumbaginaceae) is a plant known for its wide application

in natural medicine. This is associated with high accumulation of pharmacologically active

compounds in Plumbago tissues, especially plumbagin (5-hydroxy-2-methyl-1,4-

naphthoquinone). In natural habitats, Plumbago zeylanica L. grows in soils contaminated with

lead and zinc. Lead is classified as one of the most toxic element causing many malfunctions,

both on the morphological and physiological levels in cells. The aim of presented study was

to investigate the effect of lead on the accumulation of secondary metabolites, especially

plumbagin as well as determination of Pb accumulation rate in tissues of Plumbago zeylanica

L.

Experimental: Shoot fragments of Plumbago zeylanica L. in vitro plants were transferred to

the media containing different concentration of Pb (0.0, 0.025, 0.05, 0.1 g Pb/dm3). After 4

weeks of culture, plumbagin content, phenols content and Pb content were evaluated in

collected plant material (shoot and roots).

Results: Phenol compounds accumulation increased in roots but decreased in shoots of

Plumbago zeylanica L. plants cultivated on media with higher Pb concentrations (0.05 and 0.1

g Pb/dm3). In turn, content of phenylpropanoids, flavonols and anthocyanins decreased (or did

not change) both in shoots and roots again on the medium with the highest Pb concentration.

Moreover, plumbagin content decreased in shoots on the same medium and in roots on the

media with Pb regardless of its concentration. Notwithstanding, Plumbago accumulated lead

both in shoot and root tissues (Pb concentration in root was 2500 mg/kg and in shoot 37

mg/kg after treatment with 0.1 Pb/dm3).

Conclusions: Lead accumulation in roots can affect the quality of Plumbago zeylanica L.

extracts used in medicine. The presence of lead in the medium causes an increase in the

content of phenolic compounds, however, it does not increase the synthesis and accumulation

of plumbagin.

Key Words: heavy metals, lead, naphthoquinones, phenols, plumbago,

Acknowledgements: This Research was financed by the Ministry of Science and Higher Education of the

Republic of Poland

Correspondence: University of Agriculture, Institute of Plant Biology and Biotechnology, Unit of Botany and

Plant Physiology, Al. 29 Listopada 54, 31-425 Krakow,

[email protected]; [email protected]

21

P08

Synthesis and characterization of Carbon Quantum Dots (CQDs)

prepared from poly(lysine) and poly(succinimide)

Ł. Janus1, J. Radwan-Pragłowska

1, M. Piątkowski

1, D. Bogdał

1

1Cracow University of Technology, Faculty of Chemical Engineering and Technology,

Department of Biotechnology and Physical Chemistry, Warszawska 24, 31-155 Krakow,

Poland

Abstract

Purpose: The main aim of the research was to obtain new type of advanced nanomaterials

with fluorescent properties applicable in theranostics.

Experimental: Poly(lysine) was obtained by polycondensation reaction of L-lysine

hydrogenchloride under microwave radiation in propylene carbonate as a high boiling solvent.

For the reaction 1 g of L-lysine hydrogenchloride and 5 ml of propylene carbonate were

mixed and exposed to microwave radiation (100 W, 35 min). Poly(succinimide) was

synthetized by polycondensation reaction of L-aspartic acid also in propylene carbonate as a

reaction medium. During synthesis 1 g of L-aspartic acid was placed into reaction vessel and

5 ml of propylene carbonate was added. The reaction was carried out for 30 min and 100 W

microwave radiation was used. In the next step Carbon Quantum Dots (CQDs) were obtained

by degradation of poly(lysine) and poly(succinimide) under elevated temperature (200 - 230 oC) in the presence of concentrated sulphuric acid. To 1 ml of polymers solutions in propylene

carbonate obtained from the 1st stage 0.05 – 0.1 ml of concentrated sulphuric acid was added.

After reaction the mixtures were neutralized by 20% NaOH solution and dialyzed for 3 days

using dialysis tubes with MWCO 500 – 1000 Da. Fluorescence spectra were recorded by

Ocean Optics spectrometer. Fluorescence quantum yield was determinated by comparison

method using quinine sulphate in 0.1 M H2SO4 as a standard.

Results: Obtained N-doted carbon nanomaterials had strong fluorescent properties. The

fluorescence stability was over 98% after 30 days. MTT assay showed that obtained

nanobiomaterials were non-cytotoxic.

Conclusions: Poly(lysine) and poly(succinimide) appeared to be an excellent carbon source

which enabled obtainment of carbon fluorescent nanomaterials. Rich in nitrogen atoms

polymers degraded under elevated temperature in propylene carbonate, while the presence of

sulphuric acid enabled their transformation into high fluorescent N-doted carbon

nanomaterials.

Key Words: carbon quantum dots, fluorescence, nanobiomaterials

Acknowledgements: This work was supported financially by National Science Centre, Poland, grant number:

2017/UMO-2017/25/N/ST8/02952

Correspondence: Warszawska 24 Street, 31-155 Cracow, [email protected]

22

P09

Titanium dioxide for rapid degradation of neurotoxic anatoxin-a

A. Kaminski1, C. Edwards

2, E. Chrapusta

1, L. Lawton

2

1Department of Plant Physiology and Development, Faculty of Biochemistry, Biophysics and

Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland 2School of Pharmacy and Life Sciences, Robert Gordon University, Garthdee Road, Aberdeen

AB10 7GJ, United Kingdom

Abstract

Purpose: Occurrence of harmful cyanotoxins in raw water is a worldwide problem.

Unfortunately, the efficiency of techniques used for their removal is inadequate (traditional water

treatment) or expensive (ozonation). Therefore new methods are still being developed. TiO2

photocatalysis under UV irradiation seems to be simple and can effectively degrade common

cyanotoxins such as the hepatotoxic microcystins and emerging pollutants in water. The aim of

this study was to evaluate the TiO2 photocatalysis for the decomposition of the neurotoxin

anatoxin-a (ANTX-a).

Experimental: In order to determine the kinetic of ANTX-a concentration changes and confirm

the formation of potential decomposition products, the samples were analyzed on Waters

Acquity UPLC with Xevo quadrupole time of flight mass spectrometer. Samples (100µL) were

collected in the T0, and after 2, 4, 6, 8, 10, 15, 20, 25, and 30 min. At first it was evaluated

degradation of ANTX-a in pure water. The toxin was purified from Anabaena flos-aquae strain

SAG 30.87[1]. Its control aqueous solution (10 µg·mL-1

) was irradiated with UV-A range at a

distance of 7.5 cm by 30 min in the photoreactor [2]. In turn, reaction samples containing

ANTX-a and TiO2 (10µg·mL-1

, w/v) were stored in darkness or UV-A illuminated under

conditions described above. In the second set of the experiments impact of TiO2 on living

cyanobacterial culture or its extract was studied.

Results: Chromatographic and mass spectrometric data confirmed that purified ANTX-a

subjected to UV-A irradiation, and stored in darkness with the addition of TiO2 was stable, while

treated simultaneously with UV-A and TiO2 was rapidly degraded within 30 min with, the

formation of two main decomposition products with m/z equal to 198.12 (retention time, RT =

2.04 min) and 168.15 (RT = 2.36 min). The studies on living cells showed that TiO2

photocatalysis is able to lyse cyanobacteria and degrade toxin accumulated in their cells within

30 min. The fastest rate of toxin degradation, up to 10 min, was determined in cells extracts.

Conclusions: TiO2 photocatalysis seems to be a very efficient method to lyse cyanobacterial

cells and degrade completely dissolved ANTX-a.

Key Words: Anatoxin-a, degradation, titanium dioxide, photocatalysis

Acknowledgments: This work was funded by Jagiellonian University, Faculty of Biochemistry, Biophysics and

Biotechnology (grant no. BMN19/2017). Ariel Kaminski in 2016/2017 was supported by the Foundation for Polish

Science (FNP). Faculty of Biochemistry, Biophysics, and Biotechnology of Jagiellonian University is a partner of the

Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.


23

P10

Factors affecting ESI-MS analysis of peptides: mobile phase

composition and peptide modifications

A. Kluczyk1, R. Bąchor

1, B. Setner

1, M. Cebrat

1, M. Kijewska

1, P. Stefanowicz

1,

Z. Szewczuk1

1University of Wroclaw, Faculty of Chemistry, F. Joliot-Curie 14, 50-383 Wrocław, Poland

Abstract

Purpose: The efficiency of peptide detection by electrospray mass spectrometry (ESI-MS) is

related to their ionizability, which depends, i.a. on peptide sequence, hydrophobicity and

stability, as well as solution pH and composition. To improve the detection limits, various

derivatization strategies are employed, involving the introduction of easily ionizable groups or

fragments bearing permanent charge.

Experimental: In our studies we designed and synthesized peptides conjugated with linear

and bicyclic trialkylammonium salts, pyridinium salts and 5-azoniaspiro[4.4]nonyl

derivatives, and analysed their influence on ESI-MS signal intensity and collision-induced

dissociation (CID). To compare the effects of specific groups on peptide detection, two series

of conjugates were developed, of general formula QAS-CH2CO-(Asp or Ala)-Val-Tyr-Thr-

NH2 (QAS represents quaternary ammonium salt motif) and compared with unmodified

peptides H-Gly-(Asp or Ala)-Val-Tyr-Thr-NH2 and tris(2,4,6-trimethoxyphenyl)

phosphonium analogues. The concentrations of analytes were adjusted according to NMR

spectra. The equimolar mixtures were subjected to ESI-MS experiments to investigate the

impact of permanent charge moiety, carboxyl group in peptide sequence, mobile phase

composition, pH and additives on relative signal abundance.

Results: The results show that the modification may change the lipophilicity of peptides, thus

shifting the retention time in gradient elution LC-MS analysis. The series of isocratic elution

experiments indicated that the amount of organic component in mobile phase affects the

ionizability of analytes despite the permanent charge introduced by modifications.

Conclusions: In quantitative proteomics the chromatographic conditions of LC-MS studies

may have a significant effect on the observed participation of selected analytes in peptide

mixture.

Key Words: mass spectrometry, LC-MS, electrospray ionization, ionization reagents, quantitative proteomics

Acknowledgements: This work was supported by grant UMO-2013/09/B/ST4/00277 from the National Science

Centre, Poland and Wroclaw Centre of Biotechnology, the Leading National Research Centre (KNOW) for years

2014-2018. The authors would like to thank Shim-pol and Perlan Technologies for providing access to LC-MS

instruments.

Correspondence: Alicja Kluczyk, Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383

Wrocław, Poland; [email protected]

24

P11

Identification of secondary metabolites of the epiphytic lichen

Hypogymnia physodes (L.) Nyl. accumulated in spruce bark

E. Latkowska

1, B. Bober

1, E. Chrapusta

1, J. Bialczyk

1

1Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department

of Plant Physiology and Development, Gronostajowa 7, 30-387 Krakow, Poland

Abstract

Purpose: Dense populations of epiphytic lichens colonising a large part of tree’s trunk and

branches may have harmful effects on their host, mainly via secondary metabolites, which

they produced. The aim of our research was to estimate the allocation of phenolic compounds

synthesized by lichen Hypogymnia physodes growing on spruce branches into tree’s bark.

Experimental: Methanol extracts of Hypogymnia physodes (L.) Nyl. thalli and Picea abies

(L.) H. Karst. bark were analysed using ultra-performance liquid chromatography-tandem

mass spectrometry (UPLC-MS/MS) method. The detected compounds were identified on the

basis of their UV, MS and MS/MS spectra.

Results: Ten lichen phenolics were determined in the extract of H. physodes thalli. Among

them were previously known compounds such as atranorin, chloroatranorin and acids:

protocetraric, physodalic, 3-hydroxyphysodic, physodic and 2’-O-methylphysodic.

Furthermore, three new compounds that have not been described until now in this species

were identified: conphysodalic, 4-O-methylphysodic and α-alectoronic acids. The major

phenolics produced by H. physodes, i.e. physodalic, 3-hydroxyphysodic and physodic acids as

well as atranorin, have been also detected in the bark of spruce branches abundantly covered

by this lichen.

Conclusions: The presence of H. physodes secondary metabolites in the bark of host tree

might be the result of the natural translocation of these compounds and/or their extraction

from the hyphae, penetrating the tree tissues. Our findings confirm the possibility of

participation of these substances in allelochemical lichen-plant interactions in nature.

Key Words: epiphytic lichen, Hypogymnia physodes, lichen secondary metabolites, spruce, translocation,

UPLC-MS/MS

Acknowledgements: Faculty of Biochemistry, Biophysics and Biotechnology is a partner of the Leading

National Research Center (KNOW) supported by the Ministry of Science and Higher Education.


25

P12

Elicytation of phenolic compounds in tissue cultures of plants

from Droseraceae family

W. Makowski1, K. Tokarz

1, B. Piwowarczyk

1, K. Witek

1

1University of Agriculture in Krakow, Faculty of Biotechnology and Horticulture, Institute of

Plant Biology and Biotechnology, Unit of Botany and Plant Physiology, 29 Listopada 54,

31-425, Krakow, Poland

Abstract

Purpose: Carnivorous plants from Droseraceae family are rich in biologically active

compounds, such as: phenolic acids, flawonols, fenylopropanoides and naphtoquinones. One

of the possible way to increase accumulation of valuable molecules in plant tissues is

elicitation, that uses biotic or abiotic stress factors to enhance plant secondary metabolism.

Therefore, the aim of presented study was to determine accumulation of phenolic compounds

in tissues of examined plants cultured under red + UV-A radiation (abiotic stress factor) or in

medium with addition of bacterial extract from Pseudomonas syringae (biotic stress factor).

Experimental: Dionaea muscipula and Drosera indica were cultivated in ½ MS medium

(3% sucrose, pH = 5.5, no growth regulators) solidified with agar (control) or in liquid

medium in controlled conditions (fluorescence light 80 [μmol×m-2

×s-1

], temperature 24ºC,

photoperiod 16 h light/8 h darkness). Additionally, one part of plant culture cultivated in

liquid medium were grown under red + UV-A LED radiation (80 [μmol×m-2

×s-1

]) and the

other one in medium supplemented with 2,5% of Pseudomonas syringae extract. After one

month of experiment total phenolic content in both carnivorous genera were estimated using

spectrophotometrically method.

Results: Phenolic compounds content increased significantly in plants cultured in liquid

medium in each tested options. However, the efficiency level of secondary metabolites

synthesis was dependent on plant genus. The highest phenols accumulation in D. muscipula

was obtained using bacterial extract, while in D. indica using red + UV-A LED.

Conclusions: Accumulation of phenolic compounds in tissue cultures of carnivorous plants

depends on the type of elicitor and plant genus, what is connected with different stress

response strategy of D. muscipula and D. indica. Nevertheless, both applied stress factors can

be use as a tool for enhancement of phenolic compounds production.

Key Words: plant secondary metabolites, carnivorous plants, elicitation, in vitro conditions

Acknowledgements: This research was financed by the Ministry of Science and Higher Education of the

Republic of Poland.



26

P13

Pharmaceutical availability of prolactin from selected ointment

bases

A. Ostróżka-Cieślik1, B. Dolińska

1,2, F. Ryszka

2

1Medical University of Silesia in Katowice, School of Pharmacy with the Division of

Laboratory Medicine in Sosnowiec, Department of Pharmaceutical Technology, Kasztanowa

3, 41-200 Sosnowiec, Poland

2“Biochefa” Pharmaceutical Research and Production Plant, Kasztanowa 3, 41-200

Sosnowiec, Poland

Abstract

Purpose: Transdermal drug delivery is an alternative route for the delivery of active

substances to the body. Prolactin, as a peptide hormone with multidirectional effects on the

human body, can be used in topical therapy in the form of a pharmaceutical ointment. The

aim of the study was to develop a technology for preparing an ointment with prolactin in

order to obtain a formulation with the desired quality parameters.

Experimental: We made model ointments containing prolactin in the amount of 1 mg/g to 7.5

mg/g, in accordance with the recommendations of the Polish Pharmacopoeia XI using the

Unguator mixer. In order to develop the optimal composition of the formulation, two types of

the ointment bases were analyzed: eucerin with absorption properties and Lekobaza with

amphiphilic properties as potential carriers of the PRL. We examined the in vitro diffusion of

the hormone from the formulations through the model membrane.

Results: Lekobaza is a suitable base for the release of prolactin. The release of prolactin is

non-linear, similar to pulsatile release in the human body. There was no release of PRL from

an ointment based on eucerin.

Conclusions: The concentration of prolactin does not affect its pharmaceutical availability

from the Lekobaza. The obtained release profiles are similar.

Key Words: prolactin, ointment, eucerin, Lekobaza, pharmaceutical availability

Acknowledgements: This work was supported by the Medical University of Silesia in Katowice (Statutory

Funds SUM 2018 “Research on the development of the drug form (solutions, ointments) with protein-peptide

substances, including hormones”).

Correspondence: Aneta Ostróżka-Cieślik, Department of Pharmaceutical Technology, Medical University of

Silesia in Katowice, Kasztanowa 3, 41-200 Sosnowiec, e-mail: e-mail: [email protected]

27

P14

Attempt use of electrospray ionization mass spectrometry on the

characterization of ring-chain isomers of 3-hydroxy-isoindol-1-

ones and 3-hydroxy-5-azaisodolin-1-ones

B. M. Pasternak1,2

, A. Jóźwiak1, Z. Malinowski

1

1University of Lodz, Faculty of Chemistry, Department of Chemistry, Tamka12, 91-403 Lodz,

Poland 2University of Lodz, Faculty of Chemistry, Department of Chemistry, Laboratory of

Molecular Spectroscopy, Tamka 12, 91-403 Lodz, Poland

Abstract

Purpose: The 3-substituted iso-1-ones have attracted our attention since they represent an

important structural unit found in biologically active compounds, natural products and

synthetic intermediates. According to literature these substances are sodium channel blocking

activity of voltage-gated. This is of great importance in the treatment of pain disorders. On the

other hand, for example pyrrolo[3,4-c]pyridine-1,3(2H)-diones are active agent substances for

Mycobacterium tuberculosis.

Experimental:

ESI-MS and ms/ms were recorded using a Varian 500-MS LC ion-trap mass spectrometer

(Palo Alto, CA, USA). All sample was introduced into the ESI-MS source by continuous

infusion by means of the instrument syringe pump at a rate of 10 μl min-1

. The ESI-source

was operated at 5.00 kV and the capillary heater was set to 350 ˚C. The experiments were

performed in positive ion-mode and negative ion mode.

Results: An analysis of the spectra of various derivatives 3-hydroxyisoindol-1-ones and 3-

hydroksy-5-azaisodolin-1-ones showed that the ratios of the intensities of the ion peaks

depend on substituent. Particularly interesting are the fragmentations using the ion trap. We

presented a series of subsequent tandem reactions.

Conclusions: In the study we identified new compounds and presented their pathways of

fragmentation of the open and ring isomeric form.

Key Words: mass spectrometry, ring-chain isomerism, isoindol-1-ones, ms/ms spectra


http://www.chemia.uni.lodz.pl/psm/

http://www.chemia.uni.lodz.pl/psm/

28

P15

Silica-based PAMAM dendrimer-grafted materials as adsorbents

for Cu (II) ions

M. Pawlaczyk1, M. Guć

1, G. Schroeder

1

1Adam Mickiewicz University in Poznan, Faculty of Chemistry, Umultowska 89b, 61-614

Poznan, Poland

Abstract

Purpose: The aim of the study was to synthesize a series of poly(amidoamine) dendrimers

(PAMAM), which exhibit strong chelating properties toward heavy metal ions and their

simultaneous utilization as functionalizing agents for silica surface grafting, obtaining novel

hybrid materials, which adsorptive properties were studied, using Cu (II) ions as a model.

Experimental: General synthetic route for obtaining of PAMAM dendrimers and their

forward grafting onto silica materials is depicted below. Dendrimers were obtained in two-

step synthesis: (1) Michael addition of methyl acrylate (MA) to amine core and (2)

simultaneous amidation using 4 various amines, e.g. ethylenediamine (EDA), which were

used as functionalizing agents for SiO2-based hybrid materials. Adsorptive properties toward

Cu (II) ions, including adsorption isotherms, kinetic and thermodynamic studies, were

conducted using spectrophotometric assays.

NH2

N

NH2 NH2

N

O

NHNH

O

N

N

O

NH

NH O

N

O

NH

NHO

NH2NH2

NH2

NH2NH2

NH2

N C O

NH

O

NH

N

O

NHNH

O

N

N

O

NH

NH O

N

O

NH

NHO

NH2NH2

NH2

NH2NH2

SiO2

1. MA, MeOH, 7 days

2. EDA, MeOH, 5 days

SiO2

MeOH, 24 h

Results: Synthesized dendrimers were characterized with ESI-MS and NMR analysis, while

hybrid materials with IR analysis. Adsorption studies of Cu (II) ions concluded that materials

reached maximal adsorption capacity in the range of 19.8 – 104.6 mg g-1

. All materials

followed pseudo-first-order kinetic model, and they exhibited endothermic, spontaneous

character.

Conclusions: A series of silica-based PAMAM dendrimer-grafted materials was synthesized.

According to chelating feature of bioinspired functionalizing agent, obtained materials

showed good adsorptive character toward Cu (II) ions, thus they are the successful analytical

tools for pre-concentration of trace metal ions. Satisfactory results might be a breakthrough in

designing of novel adsorbents, finding utilization in heavy metal ions and organic pollutants

removal, as well as in targeted delivery of bioactive molecules.

Key Words: dendrimer, silica, hybrid materials, Cu (II) adsorbent

Acknowledgements: This work was supported by Project no. POWR.03.02.00-00-1026/16

Correspondence: Umultowska 89b, room 4.14, 61-614 Poznan; [email protected]

29

P16

Microwave-assisted synthesis and characterization of N-acylated

chitosan

M. Piątkowski1, J. Radwan-Pragłowska

1, Ł. Janus

1, B. Ulmaniec

1, A. Sierakowska

1



Poland

Abstract

Purpose: The main aim of the research was to develop fast and efficient method for chitosan

N-acylation without significant deterioration of its biological properties such as lack of

cytotoxicity and biodegradability.

Experimental: To obtain N-acylated chitosan, biopolymer with 80, 85, 90 and 95% DD was

used (CS-80, CS-85, CS-90 and CS-95 sample respectively). Saturated fatty acid chloride was

used as acylation agent. All reactions were performed in aquatic environment under

microwave-assisted conditions. Microwave radiation enabled shortening of reaction time from

6 hours to one hour. Ready product was purified from fatty acid residues using methanol and

acetone. Chemical structure was evaluated by FT-IR analysis. N-acylated chitosan was tested

over its susceptibility to biodegradation by Sturm Test method according to OECD 301B

norm. Cytotoxicity was evaluated by MTT assay on L929 mouse fibroblasts.

Results: FT-IR spectra of the modified chitosan samples presents characteristic bands for acyl

chains and amide bonds whereas bands typical for pyranose ring as well as glycosidic bonds

are also visible which confirms chitosan N-acylation. Biodegradation study showed that CS-

80 sample was degraded in 68%, CS-85 in 69, CS-90 in 72% and CS-95 in 77%. MTT assay

results showed that % of viable fibroblasts in the all cases was above 100%.

Conclusions: Proposed method of chitosan modification resulted in the obtainment of N-

acylated derivatives according to Green Chemistry principles. Prepared biomaterials were

biodegradable since all of the evaluated samples degraded in at least 60% during 28 days.

Moreover, all evaluated chitosan materials were non cytotoxic and had positive impact on

fibroblasts cells proliferation.

Key Words: chitosan, biomaterials, microwave-assisted reactions


2017/26/D/ST8/00979


30

P17

The potential role of ODAP (β-N-oxalyl-α, β-diaminopropionic

acid) in response of grass pea (Lathyrus sativus L.) to abiotic stress

in vitro

B. Piwowarczyk1, K. Tokarz

1, W. Makowski

1, A. Wysocka

1, M. Hanula

1


Biology and Biotechnology, Unit of Botany and Plant Physiology, Al. 29 Listopada 54, 31-

425 Krakow, Poland

Abstract

Purpose: Grass pea (Lathyrus sativus L.) is a leguminous plant characterized by high

resistance to drought and tolerance to salinity. The role and significance of β-N-oxalyl-α,β-

diaminopropionic acid (ODAP), commonly occurring in grass pea, remains poorly understood

despite the studies carried out in recent years. ODAP is a non-protein amino acid, causing

irreversible limb paralysis in humans and animals, after prolonged consumption of grass pea

seeds. Few literature data on ODAP role in plants indicate its contribution to the transport of

zinc ions, scavenging of hydroxyl radicals and the protection of photosynthetic apparatus at

high light intensity (Jiao et al. 2011, Xu et al. 2017). The aim of the presented research was to

determine the level of ODAP accumulation in grass pea plants depending on the type and the

level of abiotic stress (drought and salinity).

Experimental: Plant material comprised of two polish grass pea cultivars: Derek and Krab.

Sterile seeds were laid on control medium and media with addition of PEG (12,5; 17,5 i 22,0

mM) or NaCl (50, 100, 150 mM). After 14 days of culture, shoots and roots were harvested,

freeze-dried and ODAP content were analysed according to Addis i Narayan (1994) method.

Results: In Derek shoots, accumulation of ODAP increased on NaCl media regardless of the

concentration used but on PEG media increased with increasing concentration. A similar

relation was also noted in Derek roots. In turn, in Krab shoots ODAP content did not change

or decreased in seedlings cultivated on media with NaCl and increased only on the medium

with the highest PEG concentration. Whereas, in Krab roots, ODAP accumulation increased

with increasing PEG concentration and on the medium with highest NaCl dose.

Conclusions: Increased ODAP content in shoots and roots of both tested cultivars treated

with PEG, and also in Derek seedlings growing under salt stress, may indicate the

participation of this non-protein amino acid in the grass pea acclimation strategy for both

osmotic and chemical stress. The exact mechanism of this compound action requires further

research.

References: Addis G, Narayan RKJ. 1994. Ann Bot 74.3, 209-215; Jiao C-J, et al. 2011. Food Chem Toxicol

49.3:543-549; Xu Q, et al. 2017. Int J Mol Scis 18.3:526.

Key Words: grass pea, NaCl, PEG, osmotic stress, salinity stress, stress response,


Republic of Poland


Plant Physiology, Al. 29 Listopada 54, 31-425 Krakow, [email protected]

31

P18

Microwave-assisted synthesis and characterization of

antibacterial chitosan derivatives for biomedical applications

J. Radwan-Pragłowska1, Ł. Janus

1, M. Piątkowski

1, D. Bogdał

1



Poland

Abstract

Purpose: The main aim of the research was to develop chitosan derivatives with antibacterial

properties by eco-friendly method with enhanced mechanical properties applicable in wound

healing.

Experimental: Chitosan modification was performed by O-acylation in the field of

microwave radiation. For the reactions, chitosan with average molecular mass of 5, 20 and

300 kDa was used. As an acylation agent oleyol chloride was applied. All reactions were

carried out in non-aquatic conditions. Reaction time was 45 min. Ready products were

purified using acetone. Acylated chitosan was verified over chemical structure by FT-IR

method. Antibacterial activity was determined by inhibition zone test. Tensile strength, elastic

modulus and elongation at break values were determined. Chitosan derivatives were also

evaluated over water vapor permeability.

Results: FT-IR spectra showed bands of various intensity corresponding to ester bonds

between acyl chains and chitosan OH groups. All of the obtained samples inhibition zones

were in the range between 21-23 mm. TS values were in the range between 2.6 and 3.4 MPa,

Eb values were between 62 and 69 %, whereas EM values were in the range between 34 and

40 MPa. Water vapor permeability was in the range from 1200 up to 1940 g·m−2

·d−1

.

Conclusions: Microwave irradiation enabled fast and efficient O-acylation of chitosan.

Thanks to the preservation of the free NH2 groups chitosan maintained its antibacterial

properties against E. coli and S. aureus. Chitosan modification resulted in the significant

increase of mechanical durability while maintaining very good water vapor permeability

therefore increasing the applicability of the derivatives as antimicrobial biomaterials

especially in wound healing. The results showed that properties of the modified polymer were

average molecular mass dependent. Performed analyses demonstrated that the best candidate

for biomedical applications is O-acylated chitosan prepared from polymer with 20 kDa

average molecular mass.

Key Words: biopolymers, antibacterial materials, chitosan


2016/23/N/ST8/01273


32

P19

Evaluation of liposomes containing vitamin C incorporated into

the hydrogel base

B. Sarecka-Hujar1, H. Józefiak

1, B. Dolińska

1

1Medical University of Silesia in Katowice, Faculty of Pharmacy with the Division of

Laboratory Medicine, Department of Pharmaceutical Technology, Kasztanowa 3, 41-200

Sosnowiec, Poland

Abstract

Purpose: Liposomes are spherical vesicles, composed of a phospholipid bilayer with the

aqueous phase in the core area. Liposomes show biocompatibility with cell membranes and

are considered as non-toxic. Since the character of liposomes is both, hydrophobic and

hydrophilic various active substances may be placed inside them along with their optimal

transport through the skin. L-ascorbic acid (vitamin C) is a biomolecule playing a crucial role

in number of biologically important pathways. In cosmetology it is used to reduce wrinkles,

discolorations or to improve skin tone and its elasticity. The aim of the present study was

preparation and assessment of liposomes containing L-ascorbic acid suspended in hydrogel

base. Release analysis of vitamin C was also performed.

Experimental: Liposomes containing vitamin C were prepared using azolectin (a dry soybean

extract containing lecithin, cephalin and phosphatidyl inositol) with a method of dry lipid film

hydration. Obtained liposomes were then suspended in the 5% and 10% hypromellose

(HPMC) gels. The size of liposomes was measured with the use of an optical microscope (MT

4200 Series Meiji Techno Co. LTP, Japan) equipped with a digital camera. Rheological

properties of the preparations were carried out in a Reometer RM 200 Touch (Lamy-

Rheology) using a DIN-33 measuring set consisting of an application tube and a spindle.

Spreadability test was performed using an extensometer. The release tests were conducted

using Extraction Cells and paddle apparatus Erweka DT600 and dialysis membrane

Spectra/Por 2 (MWCO 12000-14000 Da).

Results: We observed that after 6 hours 88.60% and 89.90% of vitamin C released from 5%

and 10% HPMC gels, respectively. These results were found to be lower compared to

hydrogel with vitamin C in the solution (95.75%). The preparation containing liposomes with

vitamin C suspended in 10% HPMC was characterized by higher viscosity, while the

preparation with liposomes in 5% HPMC had better spreadability.

Conclusions: Encapsulation of vitamin C in liposomes causes its slower release compared to

vitamin C in the solution and in turn it may prolong action of vitamin C in subsequent skin

layers.

Key Words: liposomes, pharmaceutical availability, vitamin, ascorbic acid

Correspondence: Beata Sarecka-Hujar, Department of Pharmaceutical Technology, Medical University of

Silesia in Katowice, Kasztanowa 3, 41-200 Sosnowiec, e-mail: [email protected]


33

P20

Extracellular DNA as an important component of biofilms formed

by the opportunistic pathogens from Candida species

M. Smolarz1, M. Zawrotniak

1, M. Rąpała-Kozik

1

1 Department of Comparative Biochemistry and Bioanalytics, Faculty of Biochemistry,

Biophysics and Biotechnology, Jagiellonian University in Krakow

Abstract

Purpose: The aim of the study was to investigate the release of extracellular DNA (eDNA)

during the biofilm formation by fungal pathogens belonging to Candida spp.

Experimental: The experiments were performed using various species of Candida:

C albicans, C. glabrata, C. tropicalis and C. parapsilosis. In order to form biofilms, the serial

dilutions of Candida cells were cultured in RPMI 1640 medium in 96-well polystyrene plates

at 37C for 48 hours. Extracellular DNA present in biofilm was stained with the SYTOX

Green dye and the cell wall was labeled with Calcofluor White. The amount of extracellular

DNA was visualized by fluorescence microscopy and quantified by spectrofluorimetry. In

order to determine the localization of DNA, enzymatic degradation of the cell wall was

performed using: lyticase (β-1,3-glucanase), westase (β-1,6-glucanase, β-1,3-glucanase),

chitinase, α-1,6- mannosidase and α-1-2,3-mannosidase.

Results: On the basis of the experimental data we can conclude that the amount of eDNA in

biofilms presents an inverse dependence on the number of cells. The largest amount of eDNA

was identified in biofilms created by C. albicans and C. tropicalis. The analysis of eDNA

released by Candida spp. indicated that major part of eDNA is located on the fungal cell

surface. Treatment of biofilms with enzymes that degrade the main components of the

Candida cell wall pointed out that β-1,3-glucanase and chitinase are the most effective in

eDNA removal from the surface of biofilm.

Conclusions: Extracellular DNA is a structural component of the Candida biofilm, that due to

its physical properties provides stability to the entire structure, affect the fungal cell adhesion

and could be involved in triggering and development of infection. At lower cell density

Candida exists in the hyphal form that favors the release of DNA into the biofilm matrix. The

release of DNA is related to the activity of enzymes such as glucanases and chitinases, which

the highest activity was observed during the changes of the fungal cell morphology.

Key words: DNA, Candida, biofilm


34

P21

The changes in proteome and phytohormones level in oak after

forming of gall

P. Waligórski1, A. Kalandyk

1, L. Jankiewicz

2, P. Mielczarek

3,4, F. Dubert

1, P. Kędra

5

1The Franciszek Górski Institute of Plant Physiology Polish Academy of Sciences in Kraków,

ul. Niezapominajek 21, 30-239 Kraków, Poland 2Research Institute of Horticulture in Skierniewice, ul. Konstytucji 3 Maja 1/3, 96-100

Skierniewice, Poland 3AGH University of Science and Technology, Faculty of Materials Science and Ceramics,

Department of Biochemistry and Neurobiology, al. Mickiewicza 30, 30-059 Kraków, Poland 4Polish Academy of Sciences, Institute of Pharmacology, Laboratory of Proteomics and Mass

Spectrometry, ul. Smętna 12, 31-343 Kraków, Poland 5University of Agriculture in Krakow, Faculty of Biotechnology and Horticulture, al. 29

Listopada 54, 31-425 Kraków

Abstract

Purpose: The aim of our studies is to find specific changes in proteome of oak leaves during

forming of galls. We are strongly interrested in factor and mechanisms responsible for

transformation of normal leaves tissue into plant tumor tissue of galls. Two opponent theses

are considered: transfection by insect’s DNA and production of phytohormones by gall wasp

larva.

Experimental: Samples of leaves and galls were collected from the same individuals. The

proteins were extracted from samples with the TRIS-HCl buffer containing CHAPS and

mercaptoethanol. Proteomic analyses were performed with 2D electrophoresis and spot

identification by MALDI TOF apparatus, the second proteomic analyses were done with

ShotGun technique. We examined changes between leaves and galls.

The other part of our experiments were LCMS analyses of endogenous hormones in galls and

leaves. We analysed cytokinines, IAA, ABA, jasmonic acid, salicylic acid and free proline

content.

Results: We have found fundamental changes of proteome between leaves and galls. The

phytohormone levels were also changed, with the most of analysed compound decreased in

galls, the only exception was ABA.

Conclusions: We have not found increased levels of any growth stimulating hormones, so the

factor responsible for galls forming may not be phytohormone, or may be different type of

substance. The alternative is different kind of factor, for example plasmide. We have found

limited premises that the factor can be plasmide. The other conclusion is that many proteins

are represented in galls by their mitochondrial/cytoplasmatic isoforms, not by chloroplastic.

Correspondence: Piotr Waligórski, The Franciszek Górski Institute of Plant Physiology of the Polish Academy

of Sciences in Krakow, ul. Niezapominajek 21, 30-239 Kraków, Poland. [email protected]

35

P22

Aldehyde:ferrodoxin oxidoreductase from Aromatoleum

aromaticum

A. Winiarska1,

, F. Arndt2 , M. Szaleniec

1, J. Heider

2, A. Bodzoń-Kułakowska

3

1Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,

Niezapominajek 8, 30239 Krakow, Poland

2Laboratory of Microbial Biochemistry, Philipps-Universitat Marburg, Karl-von-Frisch-

Strasse, 35043 Marburg, Germany

3Department of Biochemistry and Neurobiology Faculty of Materials Science and Ceramics

AGH University of Science, Al. A. Mickiewicza 30, 30-059 Kraków, Poland

Abstract

Purpose: Aromatoleum aromaticum (EbN1) is a denitrifying facultative anaerobic bacterium,

capable of degradation of a number of organic compounds (i.a. considered environmental

pollutants: benzene, toluene). One of the A. aromaticum enzymes, a tungsten-dependent

aldehyde: ferrodoxin oxidoreductase (AOR), catalyzes the conversion of aldehydes to

carboxylic acids. In comparison with known archaeal AORs, the examined enzyme has a

more complex structure ( 2 2 2) and higher resistance to inactivation by oxygen [1]. We

conducted an initial biochemical characterization of the AOR from EbN1 to examine enzyme

activity and substrate spectra.

Experimental: Anaerobic kinetic assays were conducted by means of UV-Vis spectroscopy,

to identify substrate spectrum and apparent kinetic parameters of AOR in reaction with either

benzyl viologen or NAD+ as electron acceptors. Moreover, homology modelling using

MODELLER protocol was conducted to get insight into the still unknown structure of

bacterial AOR, especially the active center of the enzyme.

Results: The assay showed a broad substrate specificity of AOR both for aromatic and

aliphatic aldehydes. Benzyl viologen and NAD+ are suitable electron acceptors for AOR, with

highest activities at pH 8 and 7,5 for BV and NAD+,respectively. Non-linear regression fitting

of the obtained kinetic data suggests Hill kinetic model with a cooperativity coefficient of 2

for phenylacetaldehyde.

Conclusions: The value of the Hill coefficient may suggest, that two active sites of the

dimeric enzyme are involved in reaction. AOR from A. aromaticum has perspective as a

model enzyme in elucidation of catalytic mechanism of bacterial AORs. Moreover, the

relative resistance to oxygen and low temperature optimum of the reaction have been

confirmed, contrasting this enzyme to archaeal AOR.

Key Words: anaerobic bacteria, tungsten enzymes, kinetic assays

Acknowledgements: Research has been partly supported by the EU Project POWR.03.02.00-00-I004/16 and PL

Grid (CYFRONET)

Correspondence: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,

Niezapominajek 8, 30239 Krakow, Poland, [email protected]

[1] MK Chan, S Mukund, A Kletzin, MW Adams, DC Rees, Structure of a hyperthermophilic tungstopterin

enzyme, aldehyde ferredoxin oxidoreductase, Science, 267, 1995, 1463-1469

[2] C Debnar-Daumler, A Seubert, G Schmitt, J Heider, Simultaneous Involvement of a Tungsten-Containing

Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine

Metabolism, Journal of Bacteriology, 196(2), 2013, 483-92

36

P23

The potential role of mGluR1 antagonist in enhancement of the

anti-tumor response of BRAF kinase inhibitor in a malignant

melanoma cell model

M. Woźniak1, K. Pogoda

1, C. Paluszkiewicz

1, W. Kwiatek

1

1Institute of Nuclear Physics, Polish Academy of Sciences, PL-31342, Kraków, Poland

Abstract

Purpose: One of the causes of many diseases, including neoplasms, is an excessive level of

glutamate, which leads to excitotoxicity and affects the uncontrolled cells proliferation. The

mGlu1 receptor deserves on a special attention, because is responsible for glutamate

overexpression and in consequence it can induce the pro-oncological changes.

The main purpose of the study was to investigate the potential antitumor synergic therapy

involving simultaneous administration of low doses of antagonist of mGlu1 metabotropic

glutamate receptor (JNJ16259685) with a BRAF kinase inhibitor (dabrafenib) in the SK-

MEL-2 cell model of malignant melanoma.

Experimental: To detect the molecular changes in cells (before and after treatment) the pro-

and anti-apoptotic genes expression was investigated by polymerase chain reaction (Real-time

PCR). For analysis were taken two biomarkers participated in the tumor process, respectively:

Bcl-2 and Bax factors which determine the apoptosis and cell proliferation. Moreover, cells

metabolism activity was measured by cytotoxic test (MTT).

Results: Our study showed that simultaneous administration of low doses of mGluR1

antagonist with a BRAF kinase inhibitor significantly decreased the anti-apoptotic gene

expression (Bcl-2) in melanoma cells line. What is more, the expression of pro-apoptotic

factor (Bax) was increased after using of both compounds. The MTT test confirmed the

reduced survival of cancer cells after treatment of tested drugs.

Conclusions: Currently, the huge defect of chemotherapy is its significant toxicity, which is

associated with the occurrence of dizziness, nausea, failure kidneys or skin changes.

Therefore, conducted in vitro studies on SK-MEL-2 melanoma model and use of two

compounds at low doses (involving glutamate regulation based on affinity to mGluR1 and

BRAF kinase inhibitor) may be an alternative and effective way to reduce the side effects

while maintaining the therapeutic effect.

Key Words: melanoma, tumor, apoptosis, proliferation, mGluR1, BRAF kinase inhibitor, PCR, MTT



37

P24

Response of the grass pea (Lathyrus sativus L.) photosynthetic

apparatus to salt stress

A. Wysocka1, K. Tokarz

1, B. Piwowarczyk

1, W. Makowski

1, R. J. Jędrzejczyk

2


Biology and Biotechnology, Unit of Botany and Plant Physiology, Al. 29 Listopada 54,

31-425 Krakow, Poland

2Plant-Microorganism Interactions Group, Małopolska Centre of Biotechnology, Jagiellonian

University Kraków, Poland

Abstract

Purpose: Grass pea (Lathyrus sativus L.) is one of the most promising crop plants in arid and

semi-arid regions due to its high protein content in the seeds and resistance to abiotic stress

conditions – mainly drought, but also salinity. The aim of the experiment was to evaluate

physiological mechanisms of grass pea response, at the level of photosynthetic apparatus to

salt stress.

Experimental: Seeds of Polish grass pea cultivar ‘Krab’ were germinated in the presence of

medium containing salt (NaCl) in different concentrations: 0, 50 and 100 mM. 3 day-old

seedlings were transferred to hydroponic conditions of the same treatments. After 4 weeks of

growth, the efficiency of photosystem II was estimated using chlorophyll a fluorescence

measurements on fully developed leaves. In harvested plants (separately in leaves and roots),

estimation of Na+ ions concentration and superoxide dismutase (SOD) and catalase (CAT)

activity were conducted.

Results: Plants treated with 50 mM NaCl showed no visible symptoms of sensitivity, while

plants treated with 100 mM NaCl died after 4 weeks of treatment. The activity of CAT and

SOD decreased along with increasing NaCl concentration both in leaves and roots. However,

SOD isoform which was present in leaves showed decrease only in the presence of the highest

salt concentration (100 mM NaCl). Content of sodium ions increased in leaves and roots

along with increasing salt concentration in the medium, but it remained unchanged comparing

shoots of plants treated with 50 mM and 100 mM NaCl. Chlorophyll a fluorescence

measurements showed decreased number of reaction centers (RCs) in plants treated with 100

mM NaCl while this parameter remained unchanged in plants treated with 50 mM NaCl.

Conclusions: We believe that acclimation to moderate salinity (50 mM NaCl) is associated

with efficient photosynthetic apparatus protection by diminish of LHCII complexes size and

linear electron flow maintaining, what preserves plants from oxidative stress. Moreover,

effective sodium ions sequestration in vacuoles may take place. Decreased activity of SOD

and CAT suggests existence of other mechanisms which protect plants from reactive oxygen

species (ROS) production or which provide effective ROS scavenging.

Key Words: antioxidant enzymes, grass pea, NaCl, photosystems, salinity stress


Republic of Poland


Plant Physiology, Al. 29 Listopada 54, 31-425 Krakow,

[email protected]; [email protected]

38

P25

Identification method of XRCC5 protein expression level in

gastric tumor tissue

A. Żołynia1, A. Drabik

1

1AGH University of Science and Technology, Faculty of Materials Science and Ceramics,

Department of Biochemistry and Neurobiology, Mickiewicza 30, 30-059 Krakow, Poland

Abstract

Purpose: The XRCC5 X-Ray Repair Cross Complementing 5 protein is a subunit of the

Ku80 heterodimer protein. The XCCR5 protein might be a potential candidate for cancer

biomarker. The coexpression of the XCCR5/XRCC6 pair has been found to play role in

multiple cancers. The main goal of the research was to measure the expression level of

XRCC5 protein in gastric tumor tissue and compare with a healthy tissue from the same

patient.

Experimental: Cancer and margin tissues were homogenized, followed by the measuring the

total protein concentration using Bradford protein assay. The expression level of XRCC5

protein was analyzed using indirect ELISA test. The study group was 5 gastric carcinoma

patients. The tissue was collected intraoperatively from carcinoma tumor, while surgical

margin was treated as a control group.

Results: The results show that the expression level of the XRCC5 protein is upregulated in

cancer tissue in 60% of the cases.

Conclusions: The study confirms that the expression level is higher in cancer tissues. The

XRCC5 might be a potential biomarker of gastric cancer, however the extended research is

required in larger study group for detailed statistical evaluation.

Key Words: XRCC5 protein, gastric tumor

Acknowledgements: This work was supported by grant 2013/09/B/NZ4/02531



39

Autor index

40

Arndt F. P22

Bąchor R. P10

Bialczyk J. P02, P11

Bieniek W. L06

Bober B. P02, P11

Bodzoń-Kułakowska A. P22

Bogdał D. P08, P18

Bryniarska N. P01

Cebrat M. P10

Chmiela M. P05

Chmielewski M. K. L02

Chrapusta E. P02, P09, P11

Chrobak Ł. P03

Dolińska B. P13, P19

Drabik A. P04, P25

Dubert F. P21

Duchnik K. P02

Ducongé F. L04

Edwards C. P09

Franczyk M. P03

Gonciarz W. P05

Guć M. P06, P15

Hanula M. P07, P17

Heider J. P22

Hinc K. P05

Jankiewicz L. P21

Jankowski J. L03

Janus Ł. P08, P16, P18

Jędrzejczyk R. J. P07, P24

Józefiak H. P19

Jóźwiak A. P14

Kalandyk A. P21

Kaliszewski P. L05

Kaminski A. P02, P09

Kankofer M. P03

Kędra P. P21

Kijewska M. P10

Kluczyk A. P10

Kubiak A. P01

Kwiatek W. P23

Kwiatkowska D. L05

Latkowska E. P11

Lawton L. P09

Łabędź-Masłowska A. P01

Makowski W. P07, P12, P17, P24

Malgieri G. L01

Malinowski Z. P14

41

Mielczarek P. P21

Ner-Kluza J. P04

Obuchowski M. P05

Ostróżka-Cieślik A. P13

Paluszkiewicz C. P23

Pasternak B. M. P14

Pawlaczyk M. P06, P15

Pianka D. L03

Piątkowski M. P08, P16, P18

Piwowarczyk B. P07, P12, P17, P24

Pogoda K. P23

Radwan-Pragłowska J. P08, P16, P18

Rąpała-Kozik M. P20

Ryszka F. P13

Sarecka-Hujar B. P19

Schroeder G. P06, P15

Setner B. P10

Sierakowska A. P16

Silberring J. P04

Smolarz M. P20

Stefanowicz P. P10

Szaleniec M. P22

Szewczuk Z. P10

Tokarz K. P07, P12, P17, P24

Ulmaniec B. P16

Walencka M. P05

Waligórski P. P21

Wawrzykowski J. P03

Winiarska A. P22

Witek K. P12

Woźniak M. P23

Wysocka A. P17, P24

Zawrotniak M. P20

Zuba-Surma E. P01

Żołynia A. P25