Laboratory rotations

2017-2018

Life and Health Sciences Research InstituteSchool of MedicineUniversity of Minho

Post-Graduation

1.Surgical Sciences Research Domain1.1 Molecular insights into the response of cervical cancer cells to molecular targeted therapies1.2 Raf kinase inhibitor protein (RKIP): A modulator of RTKs molecular targeted therapies response in solid tumors?1.3 Exploitation of Monocarboxylate transporters (MCTs) as therapeutic targets in solid tumours1.4 Lung development orchestration by tissue macrophages1.5 Extracellular vesicles in the modulation of macrophage-driven lung differentiation and repair1.6 Characterization of intercellular signaling pathways involved in chick lung development1.7 Functional role of the tumour suppressor gene SPINT2 in Melanoma1.8 Clinical relevance of tumour suppressor gene SPINT2 in Cervical Cancer1.9 Lactate as a key player in the metabolic symbiosis between tumour cells and tumour-associated macrophages

2. Microbiology and Infection Research Domain2.1 Host genetic variability of IL-10 and its impact on antifungal immunity 2.2 Dissecting immunometabolism traits of antifungal immunity activation 2.3 Impact of IL-10 on the plasticity of the spleen 2.4 Dissecting the regulatory crosstalk between the gut microbiome and anti-tumor immune responses 2.5 Immunogenetics in Buruli Ulcer: the role of TNF-α (Tumour Necrosis Factor Alpha) polymorphisms 2.6 The interplay between autophagy and DNA replication stress during aging: the glutaminolysis contribution 2.7 The impact of exophagy in aging: studies in yeast cells model 2.8 Metabolic state relevance for in Acute Myeloid Leukemia2.9 Molecular characterization of genetic alterations involved in acute myeloid leukemia 2.10 IL-17A and IL-17F: one family, different effects on macrophage metabolism2.11 Immunometabolic characterization of mice intestinal mucosa upon chemically-induced colitis2.12 Characterization of the genetic diversity of LTA4H locus in TB patients2.13 Implication of amino acid metabolism in monocyte differentiation2.14 Gene editing: the cross-kingdom system for genohacking2.15 Impact of strain variability on the response to Paracoccidioides spp2.16 The contribution of PTX3 to the human immune response to Trichosporon spp.2.17 Phylogenetic analysis of HIV-1 genomic sequences2.18 Rapid Diagnostic Tests and PCR for Detection of Malaria Infection in Dominican Republic

Neurosciences Research Domain3.1 Searching for therapies for Machado-Joseph disease. 3.2 Temporal modulation of the subventricular zone neural stem cell niche by choroid plexus-cerebrospinal fluid. 3.3 Exploring the Therapeutic Potential of the Secretome of Mesenchymal Stem Cells and Bioreactors in Parkinson’s Disease Animal Models. 3.3 Pharmacotherapies and Extracellular Matrix like Hydrogels as tools for Spinal Cord Injury Regeneration: a combinatory Approach. 3.5 Modulation of Mesenchymal Stem Cells Secretome through Peptide Grafted 3D Culture Environments: A Focus on Spinal Cord Injury Repair. 3.6 Sorting Nexin 3 role in neuronal development and behavior. 3.7 Exploring Sorting Nexin 27 role in Stress. 3.8 Exploring Sorting Nexin 27 role in Pain. 3.9 A Poly-pharmacological Therapy to Restore the Injured Spinal Cord.

2

3.10 Neural circuits of reward and motivation. 3.11 Linking stress with brain cancer: effects on glioma initiation, aggressiveness and prognosis. 3.12 Regulation of WNT signaling by HOXA9 in Glioblastoma: mechanistic and therapeutic insights.3.13 Interplay between HOXA9 and P-Cadherin in Glioblastoma: Functional and Clinical Implications. 3.14 The role of IL10 in depression and antidepressant treatment. 3.15 Impact of chronic pain in decision-making - the role of dopamine. 3.16 Asymmetries in rodents’ brain function. 3.17 Asymmetries in human brain function.3.18 Social or anti-social? The disruptive power of a peripheral neuropathic lesion. 3.19 Is memory formation dependent solely on neuronal function? Dissecting the role of astrocytes in cognitive processes.3.20 Adult neuroplasticity as a pathological trigger to recurrence in depression. 3.21 Exploring the role of the transcription factor AP2gamma in the control of post-natal glutamatergic neurogenesis. 3.22 Drug screening for chronic pain disorders. 3.23 Stress-driven changes in synaptic interactome: a link between depression and Alzheimer’ s disease.3.24 Synaptic Tau protein: an unknown target for anesthetics malfunction? 3.25 Pain-triggered synaptic plasticity: identifying the mechanistic involvement of Tau protein. 3.26 “Stressed” proteostasis: the chronic stress impact on the orquestration of proteasomal and autophagic pathways in Alzheimer´s disease pathology. 3.27 Coaching strategies to prevent stress anxiety in test performance.3.28 Inflammatory response in AD: the role of interferons.3.29 The impact of lipocalin 2 in the central nervous system homeostasis: friend or foe? 3.30 Behavioral and brain histological characterization of phospholipase D knock-out mice. 3.31 Stress impact on the brain: neuropsychiatric and physiological consequences.3.32 Implication of RNA splicing for Machado Joseph Disease.3.33 Functional correlates of adult hippocampal cytogenesis: a matter of sex and time? 3.34 Immunological aging as a trigger for multiple sclerosis.3.35 Exploring the involvement of striatum in the pathology of Machado-Joseph disease: study in a mouse model.3.36 Exploring Sorting Nexin 27 role in Mycobacterium avium infection.3.37 Tau function and dysfunction: characterization of a neuronal cell line.

Skills to be trained in each project are listed in an excel file associated to this document

3

1.Surgical Sciences Research Domain1.1 Molecular insights into the response of cervical cancer cells to molecular targeted therapies1.2 Raf kinase inhibitor protein (RKIP): A modulator of RTKs molecular targeted therapies response in solid tumors?1.3 Exploitation of Monocarboxylate transporters (MCTs) as therapeutic targets in solid tumours1.4 Lung development orchestration by tissue macrophages1.5 Extracellular vesicles in the modulation of macrophage-driven lung differentiation and repair1.6 Characterization of intercellular signaling pathways involved in chick lung development1.7 Functional role of the tumour suppressor gene SPINT2 in Melanoma1.8 Clinical relevance of tumour suppressor gene SPINT2 in Cervical Cancer1.9 Lactate as a key player in the metabolic symbiosis between tumour cells and tumour-associated macrophages

4

1.1Molecular insights into the response of cervical cancer cells to molecular targeted therapies

SummaryCervical cancer (CC) is a common malignancy which kills 288 000 women annually. However, despite technological advances, up to 35% of all CC patients will develop a metastatic disease. Infection of human keratinocytes by oncogenic HPV subtypes is critical for cervical carcinogenesis, however, it is not sufficient for cancer development. Among other molecular cofactors investigated by our group, receptor tyrosine kinases (RTKs), mainly the Erbb family, and MAPK pathway modulators (such as RKIP) are frequently altered in CC, constituting in some cases predictors of poor prognosis.In the past two decades we have seen the successful development of RTKs signaling targeted drugs, expanding treatment options for cancer patients. However, actually there is no FDA approved molecular targeted therapies in CC mainly because the preclinical studies are scarce.

AimsThe aim of this study is firstly to screen for effective molecular targeted therapies in cervival cancer cell lines and secondly to find out potential predictive factors of therapy response.

References-Martinho O, Silva-Oliveira R, Miranda-Gonçalves V, Clara C, Almeida JR, Carvalho AL, Barata JT, Reis RM. In Vitro and In Vivo Analysis of RTK Inhibitor Efficacy and Identification of Its Novel Targets in Glioblastomas. Transl Oncol. 2013 Apr;6(2):187-96.-Martinho O, Pinto F, Granja S, Miranda-Gonçalves V, Moreira MA, Ribeiro LF, di Loreto C, Rosner MR, Longatto-Filho A, Reis RM. RKIP inhibition in cervical cancer is associated with higher tumor aggressive behavior and resistance to cisplatin therapy. PLoS One. 2013;8(3):e59104

Supervisor(s)Olga Martinho

5

1.2Raf kinase inhibitor protein (RKIP): A modulator of RTKs molecular targeted therapies response in solid tumors?

SummaryCancer treatment is being revolutionized by the translation of knowledge achieved in cancer biology studies in the development of new drugs that act by molecular recognition. However, the understanding of cancer molecular genetics suggests that the complex heterogeneity of human malignancies is a major limitation for the application of these novel cancer treatment approaches, and the prognosis of the majority of solid tumors remains largely dismal. Similarity to chemotherapy, the main challenge of targeted therapy in cancer treatment is drug resistance. In particular, deregulation of one signaling pathway can sometimes alleviate or bypass the “addiction” to another pathway. For example, disruption of the intracellular signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade can modulate the response to anti-RTKs inhibitors. Raf kinase inhibitor protein (RKIP), firstly described as an inhibitor of MAPK pathway, is involved in other intracellular signaling pathways. Thus, RKIP downregulation is associated with prognosis and malignant progression in several tumor types. Moreover, downregulation of RKIP led to tumor cells resistance to chemotherapy. Due to the important role of RKIP in the regulation of important intracellular signaling pathways, and its involvement in some tumors malignancy, we believe that RKIP can also modulate tumor cells response to RTKs molecular targeted therapies. Surprisingly, besides our preliminary and exciting results, there are no studies screening for RKIP expression as a possible predictor or modulator of cancer patients response to targeted therapies.

AimsThus, the major aim of this project is to shed light on the mechanisms of cancer cells response to RTK targeted therapies, by studying the role of RKIP expression in the modulation of tumors response to both established and emergent anti-RTKs therapies in solid tumors.

References-Martinho O, Silva-Oliveira R, Miranda-Gonçalves V, Clara C, Almeida JR, Carvalho AL, Barata JT, Reis RM. In Vitro and In Vivo Analysis of RTK Inhibitor Efficacy and Identification of Its Novel Targets in Glioblastomas. Transl Oncol. 2013 Apr;6(2):187-96.- Martinho O, Pinto F, Granja S, Miranda-Gonçalves V, Moreira MA, Ribeiro LF, di Loreto C, Rosner MR, Longatto-Filho A, Reis RM. RKIP inhibition in cervical cancer is associated with higher tumor aggressive behavior and resistance to cisplatin therapy. PLoS One. 2013;8(3):e59104-Martinho O, Granja S, Jaraquemada T, Caeiro C, Miranda-Gonçalves V, Honavar M, Costa P, Damasceno M, Rosner MR, Lopes JM, Reis RM. Downregulation of RKIP is associated with poor outcome and malignant progression in gliomas. PLoS One. 2012;7(1):e30769.

Supervisor(s)Olga Martinho and Rui M. Reis

6

1.3Monocarboxylate transporters (MCTs) as therapeutic targets in cancer and mediators of response to therapy

SummaryMost solid tumours rely on glycolysis for energy, which results in an increased production of acids, namely lactic acid. To reduce the intracellular level of acids and maintain the physiological pH, tumour cells upregulate several membrane transporters, of which MonoCarboxylate Transporters (MCTs). Therefore, acid efflux through MCTs constitutes an important mechanism involved in the maintenance of tumour intracellular pH. Recent evidence demonstrates that MCTs are upregulated in some in solid tumours. MCTs are putative targets for cancer therapy, which are now starting to be explored in the clinical context (first clinical started in 2011, AstraZeneca).We are currently evaluating the role of MCT expression and activity in several types of solid tumours and, including colorectal, cervix, breast and brain tumours. However, the dependence of tumour cells on MCT activity is still poorly understood. Besides studying the expression of the different MCT isoforms and other metabolism-related proteins in human tumors, we are also characterizing the effect of MCT inhibition on several “in vitro” and “in vivo” models, by exposing cancer cells to known MCT inhibitors or with specific inhibition of MCT expression. Additionally, we are characterizing the role of MCTs in the response to cancer therapy and in the crosstalk among the cells which are part of the tumor microenvironment.

AimsCharacterize the effect of MCT inhibition in cancerCharacterize the role MCTs in the response to cancer therapy

ReferencesMorais-Santos F, Granja S, Miranda-Gonçalves V, Moreira AHJ, Queirós S, Vilaça Jl, Schmitt FC, Longatto-Filho A, Paredes J, Baltazar F, Pinheiro C. Targeting lactate transport suppresses in vivo breast tumour growth, Oncotarget 2015, 6(22):19177-89.Miranda-Gonçalves V, Honavar M, Pinheiro C, Martinho O, Cordeiro M, Bebiano G, Costa P, Reis RM, Baltazar F. Monocarboxylate transporters (MCTs) in gliomas: Expression and exploitation as therapeutic targets, Neuro-Oncology 2013, 15(2):172-88.

Supervisor(s)Fátima Baltazar and Sara Granja

7

1.4Lung development orchestration by tissue macrophages

SummaryLung dysfunction represents a great challenge, since current therapies simply provide symptomatic relief, without reversing organ damage. Strong evidences support that developmental genes and pathways are commonly associated with disease pathogenesis. Thus, it is essential to deeply understand lung developmental processes.Importantly, macrophages are one of the cell populations present throughout foetal and postnatal lung development (1,2). They are a plastic and heterogeneous cell population that shape their cellular neighbourhood, in different pathological situations (3,4). Interestingly, growing evidences demonstrate developmental morphogenesis regulation by macrophages, namely in vasculature and branching, key processes in lung organogenesis (6). Although, these data highlights a promising role for macrophages in lung development, nothing is known about it. Our unpublished data show that macrophage deficient fetus present impairment in lung morphology, vasculature and alveolar differentiation.

AimsUnderstand the consequences of tissue macrophage ablation in lung morphogenesis (e.g. microvasculature, proliferation and apoptosis, differentiation) and in lung developmental molecular pathways;Identification of molecules induced by tissue macrophages regulating lung development;Understand the contribution of macrophages-derived molecules to lung epithelial differentiation and repair.

ReferencesStouch AN, Zaynagetdinov R, Barham WJ, et al. IkB kinase activity drives fetal lung macrophage maturation along a non-M1/M2 paradigm. J Immunol, 2014; 193(3):1184–1193.Blackwell TS, Hipps AN, Yamamoto Y, et al. NF- B Signaling in Fetal Lung Macrophages Disrupts Airway Morphogenesis. J Immunol, 2011; 187(5):2740–2747.Takeda Y, Costa S, Delamarre E, et al. Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis. Nature, 2011; 479(7371):122-64.. Rolny C, Mazzone M, Tugues S, et al. HRG Inhibits Tumor Growth and Metastasis by Inducing Macrophage Polarization and Vessel Normalization through Downregulation of PlGF. Cancer Cell, 2011; 19(1):31–44.Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature, 2014; 496(7446):445–455.

Supervisor(s)Sandra Costa

8

1.5Extracellular vesicles in the modulation of macrophage-driven lung differentiation and repair

SummaryLung dysfunction represents a great challenge with current therapies not reversing organ damage. The lung is a complex organ composed by dozens of different cells (1), among them macrophages. Macrophages are present throughout lung development (2-3). Our unpublished data show that tissue macrophage deficient foetus present lung morphology and alveolar differentiation impairment, probably through paracrine mechanisms. Paracrine signaling through conditioned medium (CM) has an instrumental role in the regulation of cellular functions, including lung repair (4), through bioactive extracellular vesicles (EVs). Recent evidences demonstrated that cells tweet lung repair messages through EVs (5-6). Thus, we hypothesize that lung tissue macrophages drives pulmonary differentiation, repair and regeneration through EVs. The main goal of this project is to elucidate the crosstalk between lung cells and the EVs-carried molecules crucial on it and empower the knowledge on the use a new promising therapy agent delivery vehicle in the respiratory system.

AimsEstablish primary lung cells culture;Characterize foetal lung EVs population(s);Identify the role of lung tissue macrophage-derived EVs on foetal lung differentiation and lung epithelial repair in a in vitro approach;Reveal lung tissue macrophage-derived EV content, namely proteins and miRNA, mediating lung differentiation and repair.

ReferencesRock, J.R. & Hogan, B.L.M. Epithelial Progenitor Cells in Lung Development, Maintenance, Repair, and Disease. Annu. Rev. Cell Dev. Biol. 27, 493–512 (2011).Blackwell, T.S. et al. NF- B Signaling in Fetal Lung Macrophages Disrupts Airway Morphogenesis. The Journal of Immunology 187, 2740–2747 (2011).Jones, C.V. et al. M2 macrophage polarisation is associated with alveolar formation during postnatal lung development. Respiratory Research 14, 41 (2013).Fujita Y, et al. Extracellular vesicles in lung microenvironment and pathogenesis. Trends in molecular medicine. 2015;21(9):533-42.Kovach MA, et al. IL-36gamma is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components. Journal of leukocyte biology. 2016. doi: 10.1189/jlb.4A0315-087R.Moon HG, et al. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway. Cell death & disease. 2015;6:e2016. doi: 10.1038/cddis.2015.282.

Supervisor(s)Sandra Costa

9

1.6Characterization of intercellular signaling pathways involved in chick lung development

SummaryLung development is directed by intrinsic epithelial-mesenchymal interactions that regulate cell proliferation, fate, migration and differentiation, leading to branching morphogenesis and ultimately to proper lung formation. Pulmonary development is controlled by genetically conserved intercellular signaling pathways also known to be involved in body pattern formation, and that include bone morphogenetic proteins, fibroblast growth factors, sonic hedgehog, retinoic acid and Wnt signaling pathways, insoluble extracellular matrix proteins and their receptors, as well as various transcription factors.The intricate mechanisms that determine a correct lung development depend on the involvement of several paths. Aiming to untangle an association between these signaling pathways in chick lung morphogenesis, inhibitors or activators of these signaling pathways will be added to chick lung explants culture to evaluate their effect in lung morphology. Moreover, the expression pattern of the genes belonging to these pathways will be characterized as well as the intracellular mechanisms involved in these processes.With this project, we expect to confirm the relationship between regulators of lung development (FGF, Notch, SHH, retinoic acid members), disentangling its potential role in the overall process of fetal lung morphogenesis.

AimsTo characterize the expression pattern of the members of known molecular players involved branching, in the chick modelTo dissect the links between different signaling pathways

ReferencesMoura RS, Silva-Gonçalves C, Vaz-Cunha P, Correia-Pinto J. 2016. Expression analysis of Shh signaling members in early stages of chick lung development. Histochem Cell Biol, doi:10.1007/s00418-016-1448-1.Moura RS, Vaz-Cunha P, Silva-Gonçalves C, Correia-Pinto J. 2015. Characterization of miRNA processing machinery in the embryonic chick lung. Cell Tissue Res, doi:10.1007/s00441-015-2240-6Moura RS, Carvalho-Correia E, daMota P, Correia-Pinto J. 2014. Canonical Wnt signaling activity in early stages of chick lung development. PLoS ONE, 9(12): e112388. doi:10.1371/journal.pone.0112388.Moura RS, Coutinho-Borges JP, Pacheco AP, daMota PO, Correia-Pinto J. 2011. FGF signaling pathway in the developing chick lung: expression and inhibition studies. PLoS ONE, 6(3): e17660. doi:10.1371/journal.pone.0017660.

Supervisor(s)Rute Moura

10

1.7 Functional role of the tumour suppressor gene SPINT2 in Melanoma

SummarySerine protease inhibitor Kunitz type 2 (SPINT2) has been identified as a tumour suppressor gene in various solid tumours. SPINT2 inhibits serine proteases involved in the extracellular matrix degradation and HGFA, regulating the HGF/MET signalling. Still, few mechanistic studies have explored SPINT2 deregulation and an extensive characterization of its clinical relevance is needed. We have screened SPINT2 promoter methylation on 77 cancer cell lines, from 13 different solid tumours and found that it was a frequent event in several tumour types. Further, we have extensively characterized series of colorectal, brain and prostate cancer, regarding SPINT2 methylation and protein expression and found correlations between promoter methylation, and absence or dislocation of protein expression, and tumour aggressiveness, particularly in brain and colorectal cancers. Recent findings from our group suggest that SPINT2 is highly frequently down-regulated in advanced stages of melanoma and promoter methylation is the epigenetic mechanism responsible for silencing the gene. Thus, we aim to determine the clinical relevance of the tumour suppressor SPINT2 in melanoma and its functional role in the genesis and progression of this tumor type.

AimsGrowth and maintenance of melanoma cell lines for assessment of cell viability and tumorigenic behaviour after SPINT2 expression modulation (downregulation of SPINT2 in cells previously expressing the protein or SPINT2 transfection and expression in cells without protein expression).Understand how SPINT2 expression modulation can affect therapeutic response of the cells.

ReferencesPereira MS, de Almeida GC, Pinto F, Viana-Pereira M, Reis RM. SPINT2 Deregulation in Prostate Carcinoma. Journal of Histochemistry and Cytochemistry, 2016, 64(1):32-41. Kongkham, P.N., et al., An epigenetic genome-wide screen identifies SPINT2 as a novel tumor suppressor gene in pediatric medulloblastoma. Cancer Res, 2008. 68: 9945-53.Dong, W., et al., Epigenetic inactivation and tumor suppressor activity of HAI-2/SPINT2 in gastric cancer. International Journal of Cancer, 2010. 127(7): 1526-34.Tung, E.K., et al., HAI-2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti-invasive functions. International Journal of Cancer, 2009. 124: 1811-9.

Supervisor(s)Marta Viana-Pereira and Rui Manuel Reis

11

1.8 Clinical relevance of tumor suppressor gene SPINT2 in cervical cancer

SummarySerine protease inhibitor Kunitz type 2 (SPINT2) has been identified as a tumor suppressor gene in various solid tumors. SPINT2 inhibits serine proteases involved in the extracellular matrix degradation and HGFA, regulating the HGF/MET signaling. Still, few mechanistic studies have explored SPINT2 deregulation and an extensive characterization of its clinical relevance is needed. We have screened SPINT2 promoter methylation on 77 cancer cell lines, from 13 different solid tumors and found that it was a frequent event in several tumor types. Further, we have extensively characterized series of colorectal, brain and prostate cancer, regarding SPINT2 methylation and protein expression and found correlations between promoter methylation, and absence or dislocation of protein expression, and tumor aggressiveness, particularly in brain and colorectal cancers. Little is known about the protein expression and gene methylation status of SPINT2 in cervical carcinoma, with a single report characterizing a small series of 52 tumors limited to FIGO stage I and II.

AimsDetermine the clinical relevance of the tumor suppressor SPINT2 in cervical carcinoma by evaluating the protein expression and gene promoter methylation status in a series of 350 tumors. Correlate the presence of SPINT2 methylation with clinical-pathological features of the patients.

ReferencesPereira MS, de Almeida GC, Pinto F, Viana-Pereira M, Reis RM. SPINT2 Deregulation in Prostate Carcinoma. Journal of Histochemistry and Cytochemistry, 2016, 64(1):32-41. Kongkham, P.N., et al., An epigenetic genome-wide screen identifies SPINT2 as a novel tumor suppressor gene in pediatric medulloblastoma. Cancer Res, 2008. 68: 9945-53.Dong, W., et al., Epigenetic inactivation and tumor suppressor activity of HAI-2/SPINT2 in gastric cancer. International Journal of Cancer, 2010. 127(7): 1526-34.Tung, E.K., et al., HAI-2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti-invasive functions. International Journal of Cancer, 2009. 124: 1811-9.

Supervisor(s)Marta Viana-Pereira and Adhemar Longatto

12

1.9Lactate as a key player in the metabolic symbiosis between tumour cells and tumour-associated macrophages

SummaryTumour cells are characterized by high rates of glycolysis, with abundant expression of glucose transporters, glycolytic enzymes and monocarboxylate transporters (MCTs). However, little is known about how the expression/activity of MCTs mediate the metabolic symbiosis between tumour cells and tumour associated-macrophages (TAMs). TAMs are one of the most common stromal cell types, and their presence correlates with the outcome of several cancers. Macrophages can be differentiated in the classically activated type 1 macrophages (M1) and the alternatively activated type 2 macrophages (M2). There is evidence suggesting that M1 macrophages act as tumour suppressors, promoting an immune response that eliminates tumour cells, while M2 macrophages promote tumour growth, increasing tissue remodeling, angiogenesis and extracellular matrix degradation. Thus, we aim to understand the role of lactate on the metabolic symbiosis between the two cell populations.

AimsEvaluate the role of MCT1 and MCT4 to the modulation of tumor/macrophage interaction using an in vitro co-culture system Understand how inibiting MCTs in tumor cells affects in vivo TAM recruitment.

References-

Supervisor (s)Sara Granja and Fátima Baltazar

13

2. Microbiology and Infection Research Domain2.1 Host genetic variability of IL-10 and its impact on antifungal immunity 2.2 Dissecting immunometabolism traits of antifungal immunity activation 2.3 Impact of IL-10 on the plasticity of the spleen 2.4 Dissecting the regulatory crosstalk between the gut microbiome and anti-tumor immune responses 2.5 Immunogenetics in Buruli Ulcer: the role of TNF-α (Tumour Necrosis Factor Alpha) polymorphisms 2.6 The interplay between autophagy and DNA replication stress during aging: the glutaminolysis contribution 2.7 The impact of exophagy in aging: studies in yeast cells model 2.8 Metabolic state relevance for in Acute Myeloid Leukemia2.9 Molecular characterization of genetic alterations involved in acute myeloid leukemia 2.10 IL-17A and IL-17F: one family, different effects on macrophage metabolism2.11 Immunometabolic characterization of mice intestinal mucosa upon chemically-induced colitis2.12 Characterisation of the genetic diversity of LTA4H locus in TB patients2.13 Implication of amino acid metabolism in monocyte differentiation2.14 Gene editing: the cross-kingdom system for genohacking2.15 Impact of strain variability on the response to Paracoccidioides spp2.16 The contribution of PTX3 to the human immune response to Trichosporon spp.2.17 Phylogenetic analysis of HIV-1 genomic sequences2.18 Rapid Diagnostic Tests and PCR for Detection of Malaria Infection in Dominican Republic

14

2.1Host genetic variability of IL-10 and its impact on antifungal immunity

SummaryInvasive aspergillosis (IA) is an infectious disease caused mainly by the fungus Aspergillus fumigatus. This infection typically affects immunocompromised patients such as hematological patients or recipients of hematopoietic stem cell transplants. Owing to a constantly increasing incidence, IA is a leading cause of death among transplant recipients with a 1-year mortality reaching 75%, despite the availability of several antifungal drugs.IL-10 is an important immunoregulatory cytokine with a wide variety of functions across multiple cell types. These include the restraining of T-cell activation and proinflammatory cytokine production, and the suppression of macrophage function, proliferation and activation, leading to impaired pathogen control and/or reduced immunopathology. Although the role of IL-10 during infection appears however to vary for different microorganisms, a largely detrimental role has been attributed to this cytokine during aspergillosis. Elevated levels of circulating IL-10 were observed in patients with chronic pulmonary aspergillosis and in non-neutropenic patients with IA. Thus, it not surprising that patients with IA also display noticeably higher frequencies of A. fumigatus-specific T-cells producing IL-10 compared to uninfected controls and that, in contrast, the numbers of these cells are markedly reduced in the airways of IL-10–deficient mice.The initial investigation of the genetic variability in the IL10 locus led to the identification of three common single nucleotide polymorphisms (SNPs) in the upstream region of the gene. Thus, IL-10 may be a reasonable candidate for the genetic regulation of differential responses to A. fumigatus in high-risk patients. Significant relationships between these promoter variants and aspergillosis have been found in patients with cystic fibrosis, hematological patients, and recipients of allogeneic HSCT. However, besides the limited sample size of these early studies, the biological consequences of genetic variability in IL-10 to antifungal immunity remain elusive.

AimsCharacterize the role of IL-10 in antifungal immunity by resorting to cellular models of fungal infection;Elucidate the functional consequences of genetic variants in the IL10 locus to antifungal immunity.

References-

Supervisor(s)Cristina Cunha and Agostinho Carvalho

15

2.2Dissecting immunometabolism traits of antifungal immunity activation

SummaryInvasive aspergillosis (IA) is an infectious disease caused mainly by the fungus Aspergillus fumigatus. This infection typically affects immunocompromised patients such as hematological patients or recipients of hematopoietic stem cell transplants. Owing to a constantly increasing incidence, IA is a leading cause of death among transplant recipients with a 1-year mortality reaching 75%, despite the availability of several antifungal drugs.Alterations in cellular metabolism represent a fundamental mechanism by which immune cells maintain homeostasis. In response to pathogens or tissue damage, immune cells must rapidly adapt their metabolic programs to meet specialized host defense needs. This adaptation is bioenergetically expensive, requiring precise control of cellular metabolic pathways relying on the consumption of substrates such as glucose, fatty acids or amino acids. By fueling the cell fate decisions and effector functions of immune cells, metabolic pathways of oxidative metabolism, glycolysis and glutaminolysis are critical during immunity and inflammation processes.Although several evidences have implicated immunometabolic dynamics in shaping immunity to several pathogens, nothing is known about these processes in defining antifungal immune responses. Our laboratory is now focussing on investigating how immunometabolism dynamics converge to influence the function of macrophages and ultimately the immune response against A. fumigatus. To do so, we are using in vitro models of infection and clinical specimens of human patients suffering from IA.

AimsCharacterize the immunometabolism dynamics and its modulation in primary human macrophages infected with live conidia of A. fumigatus;Define the peripheral and lung metabolome of stem-cell transplanted patients suffering from IA.

References-

Supervisor(s)Cristina Cunha and Agostinho Carvalho

16

2.3Impact of IL-10 on the plasticity of the spleen

SummaryThe spleen is a lymphoid organ extremely well organized to answer to blood born infections. The spleen organization is mediated by cytokines and chemokines. We have an experimental model were mice, over expressing IL-10 for 30 days show a disorganization of the spleen. Interestingly, as IL-10 levels are set normal the spleen acquires what seems to be a normal organization. These observations raise important questions.

AimsWhat are the molecular mediators, regulated by IL-10, that are involved in spleen disorganization?Does the, apparently normal, organization of the spleen upon exposition to high levels of IL-10 is really normal?

References-

Supervisor(s)Gil Castro and Bruno Silva

17

2.4Dissecting the regulatory crosstalk between the gut microbiome and anti-tumor immune responses

SummaryThere is growing evidence that alterations in the composition of the gut microbiome (dysbiosis), can disrupt intestinal homeostasis, promote inflammation, and increase susceptibility to cancer. However, the functions of specific members of the gut microbiota and the mechanisms wereby they modulate tumor susceptibility have not been clearly defined. We have recently shown that alteration in dietary habits, including the use of supplements, may have important consequences for cancer development. Using a novel pre-clinical model, we found that dietary zinc (Zn) supplementation promotes tumor growth, by modulating the gut microbiome. Accordingly, mice depleted of their endogenous microbiome did not become susceptible to tumor growth when their diet was supplemented with Zn. We also found that the immunoregulatory cytokine IL-10 counteract the pro-tumor role of Zn-induced dysbiosis. Since the primary interaction between microbiota and the immune system is the gut mucosa, our working hypothesis is that alterations in gut microbiome will modulate immunity in the gut mucosa, where IL-10 has a key role in maintaining homeostasis, with impact in the anti-tumor immune response.

AimsWe aim to define how the composition and diversity of the gut microbiome associate with the tumor immune response. To this end, we will characterize the effects of dysbiosis in gut immune responses and use metabolomic analysis of intestinal components to design a regulatory map linking microbiota with immune responses in the gut and how these impact tumor control.

References-

SupervisorsEgídio Torrado and Ana Margarida Barbosa

18

2.5Immunogenetics in Buruli Ulcer: the role of TNF-α (Tumour Necrosis Factor Alpha) polymorphisms

SummarySusceptibility to an infectious agent and variability in disease outcomes are dependent on the interaction between environmental and host genetic factors. Nevertheless, little is known about the role of host genomics in susceptibility to Buruli Ulcer (BU), a neglected necrotizing skin disease caused by Mycobacterium ulcerans infection. Data from a population-based genetic association study performed by our group using samples from a BU endemic population in Benin report that a single nucleotide polymorphism (SNP) rs1800629 located in the promoter region of the TNFA (Tumour Necrosis Factor Alpha) gene is associated with the development of BU.TNF- α is a proinflammatory cytokine produced by a wide range of cell types, including macrophages, and induce different cellular responses. The relevance of this cytokine in the control low and intermediate virulence M. ulcerans infection was previously shown by our lab, since TNF-α absence led to an increased bacterial proliferation.Therefore, we propose to uncover if this polymorphism impacts TNFA gene transcription during M. ulcerans infection, leading to a differential production of TNF-α depending on the genotype. To accomplish our goal, macrophages will be differentiated from human Peripheral Blood Mononuclear Cells (PBMCs). Cells with different genotypes for the SNP will be challenged with a mycolactone-negative M. ulcerans strain in the presence/absence of non-cytotoxic doses of mycolactone. At different time-points post-infection, TNF-α expression and protein production will be measured by Quantitative Real-Time PCR (qRT-PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), respectively.

AimDetermine the impact of TNFA rs1800629 polymorphism in TNF-α production during M. ulcerans infection.

References-

SupervisorsAlexandra Fraga and Jorge Pedrosa

19

2.6The interplay between autophagy and DNA replication stress during aging: the glutaminolysis contribution

SummaryAging is characterized by the progressive accumulation of damaged macromolecules/organelles associated with the decline of housekeeping processes. The inefficient removal of nonfunctional cellular components due to autophagy impairment has been proposed to be the main cause of the biological “waste” accumulation that appears to be critical in the progression of aging. How autophagy regulation is coordinated with other cellular processes during aging is still matter of intensive research. DNA replication stress, caused by DNA replication forks that delay the repair of damages, is also an important factor in aging progression. Our previous work demonstrated that under a proteotoxic context, elicited by the heterologous expression of the human alpha-synuclein, aged yeast cells presented increased autophagy activity linked to the promotions of DNA replication stress. This connection is reflected by lower ATP intracellular levels associated with the autophagic-dependent Rnr1 (ribonucleotide reductase 1, responsible for the dNTPs synthesis) degradation and concomitantly decreased dNTPs pool. Nucleotides are essential for a wide variety of biological processes and are constantly synthesized in cells, either de novo or via the nucleoside salvage pathways. Glutaminolysis is a mitochondrial pathway that comprises a series of biochemical reactions by which glutamine is degraded to glutamate that is further metabolized by conversion to alpha-ketoglutarate and then into malate. During this reactions, it is produced NADPH that among other processes is used in the nucleotide metabolism. On the other hand, a crosstalk between glutaminolysis and autophagy was already established. Therefore, the present project aims to elucidate mechanisms underlying the crosstalk among DNA replication stress, glutaminolysis and autophagy in yeast aged cells. The understanding of the molecular mechanisms coordinating DNA replicative stress and autophagy is essential to understand aging and age-related diseases.

Aims-To monitorize autophagy activity by the GFP-Atg8 processing assay and cell cycle during chronological lifespan (CLS) of wild-type cells submitted or not to conditions of DNA replication stress, induced by proteotoxic stress;-To determine glutamine, NADH and glutamate concentrations during the cells CLS;To determine intracellular amino-acids, particularly serine and glycine, by HPLC.

References-

SupervisorsPaula Ludovico and Belém Sampaio-Marques

20

2.7The impact of exophagy in aging: studies in yeast cells model

SummaryAging is a multifactorial and complex process determined by the combination of genetic, environmental and stochastic factors that lead to the progressive decline of multiple physiological functions. Several hallmarks of aging that represent age-common denominators in different model organisms have been proposed, including telomere attrition, epigenetic alterations, cellular senescence, stem cell exhaustion, altered intercellular communication, mitochondrial dysfunction, genomic instability, loss of proteostasis and deregulated nutrient-sensing. To face the stress posed by the progressive decline of cellular function, cells attempt to mount a response involving different quality control mechanisms, like autophagy, which is responsible for the degradation of damage and obsolete proteins and organelles. Recently, a novel autophagic secretory process, called exophagy, supports the existence of a coordinated intercellular transmission of signals and molecules, which appears to impact on aging progression. However, the nature of the secreted molecular signals and how exophagy influences their release and its regulation during aging are still unknown. This project aims to further elucidate the regulation of exophagy and its impact on cellular intercommunication during aging in the yeast Saccharomyces cerevisiae.

AimsTo evaluate the chronological lifespan, autophagy activity (by the GFP-Atg8 processing assay), accumulation of reactive oxygen species (ROS) (by flow cytometry) and cell cycle of yeast wild type cells cultivate in conditioned medium produced by yeast aged cells harboring the vector control or heterologous expressing alpha-synuclein.

References-

SupervisorsPaula Ludovico and Belém Sampaio-Marques

21

2.8Metabolic state relevance for in Acute Myeloid Leukemia

SummaryAcute myeloid leukemia (AML) is the most common type of acute leukemia in adults, yet has the lowest survival rate. Such prognostic has not changed over the past decades as well as the therapeutic strategy that has also not been significantly modified over the last 40 years. Although the therapeutic regimen results in a positive response in young patients, intensified therapy is highly toxic, has limited application and poor outcomes among old individuals, the most affected population, resulting in high mortality. Mounting evidence has demonstrated the role of the metabolic pathways, like LKB1-AMPK-mTOR and PI3K-AKT-mTOR, in the survival/proliferation and chemoresistance. Autophagy has been defined as a central player in the AML cells response to chemotherapy. Nevertheless, the role of those metabolic pathways, and autophagy, in the AML context, is still not well understood and controversial. Our preliminary results suggest that the functioning and implications of autophagy and the metabolic pathways in leukemia is dependent of the AML subtype. Thus the present project aims to characterize the basal metabolic state, focusing in the LKB1-AMPK-mTOR and PI3K-AKT-mTOR pathways and autophagy, of cells from different AML subtypes.

AimsTo evaluate the basal activation levels of LKB1-AMPK-mTOR and PI3K-AKT-mTOR pathways and autophagy in HL-60 e KG-1 cell lines, which represent two different AML subtypes;To evaluate the cells metabolic response to modulation of the metabolic pathways.

References-

SupervisorsPaula Ludovico and Belém Sampaio-Marques

22

2.9Molecular characterization of genetic alterations involved in acute myeloid leukemia

SummaryAcute myeloid leukemia (AML) is the most common form of adult acute leukemia. It comprises a genetic heterogeneous group of neoplasias characterized by impaired differentiation and proliferation of myeloid progenitor cells and consequent accumulation of the immature myeloid progenitors in the bone marrow (BM). Despite continuous advances in the understanding of AML biology, its prognosis remains severe, associated with high mortality. Still half of young adults and about 90% of elder individuals with AML die from the disease, even if complete remission is initially achieved, showing the limitations of today’s AML treatment approach. Interestingly, the presence of several hallmarks of metabolic syndrome, including diabetes mellitus (DM), obesity and increased body mass index (BMI), has been recently suggested to be associated with the occurrence (or poor prognostic) of different hematological malignancies including AML. Specifically, epidemiological evidence suggests that DM is associated with increased incidence of AML and poor outcomes within AML patients. Nevertheless, the biological mechanisms underlying these observations and the increased susceptibility of those individuals to AML remain elusive. Importantly, data from our group shows that expression of well-established autophagy regulators is consistently decreased in BM from individuals with DM, suggesting that DM negatively impairs cellular basal mechanisms. Given these preliminary results, the present projects aims to explore if any genetic alteration can underlay the decreased expression levels of the autophagic players and its correlation with AML progression.

AimsTo evaluate if any single nucleotide polymorphism (SNP), in central autophagic players, can be associated with the higher prevalence of AML in DM patients.

References-

SupervisorsPaula Ludovico and Belém Sampaio-Marques

23

2.10IL-17A and IL-17F: one family, different effects on macrophage metabolism

SummaryMacrophages are innate immune cells that play a central role in inflammation and host defense. Upon polarization, macrophages undergo a metabolic reprogramming, crucial for their correct functioning in immunity. IL-17 family members have been described as major players in inflammatory conditions, particularly the members IL-17A and IL-17F. Although their functions have previously been thought to be redundant, recent works have shown that these cytokines may induce distinct outcomes.

AimsHolding the premise that these cytokines can differentially modulate macrophage effector functions, we aimed to explore the link between immune function and metabolic reprogramming responsible for the long-term effects of IL-17A and IL-17F-stimulation on macrophages.

References-

SupervisorsInês Mesquita and Ricardo Silvestre

24

2.11Immunometabolic characterization of mice intestinal mucosa upon chemically-induced colitis

SummaryInflammatory Bowel Disease (IBD) is a complex set of inflammatory disorders comprising Crohn’s Disease and Ulcerative Colitis. Although the precise etiology of IBD is unknown, the pathogenesis of IBD has been defined as an immune-mediated condition with a strong genetic predisposition, triggered by environmental factors that affect mucosal immune system development and function. In the steady-state intestinal mucosa, the recruited Ly6Chi monocytes experience a differentiation process through a number of intermediate subsets, each one evidencing a gradual functional specialization. This is particularly relevant since the disruption of the differentiation pattern of monocytes in this tissue plays a critical role in IBD. Alterations in lymphoid populations in the intestine, such as regulatory T cells (Treg) and T helper 17 cells (Th17), are also correlated with the pathogenesis of IBD. Moreover, recent studies have revealed an intricate relationship between immune cell biology and extracellular or intracellular metabolite profiles. In this context, we aim to clarify the environmental metabolic factors and pathways accountable for immune cell training in the mucosa.

AimsOptimization of a leukocyte enrichment protocol from gut samples from mice, both colitic or healthy.Development of a flow cytometry strategy, using specific surface markers, to identify the different myeloid and lymphoid cell populations in gut samples from mice, both colitic or healthy.

References-

SupervisorsJoana Gaifem and Ricardo Silvestre

25

2.12Characterization of the genetic diversity of LTA4H locus in TB patients

SummaryTuberculosis (TB) ranks among the deadliest infectious diseases worldwide and Portugal presents one of the highest TB incidences among Western Europe.Susceptibility to TB is often attributed to an inadequate immune response. A growing body of evidence has shown that eicosanoids, important host lipid mediators that regulate inflammation, modulate the susceptibility to infection by Mycobacterium tuberculosis (Mtb).Polymorphisms in the leukotriene A4 hydrolase (LTA4H) locus are associated with mycobacteria susceptibility in humans and it is a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. Modulation of this locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility: inadequate inflammation caused by excess lipoxins and hyper-inflammation driven by excess leukotriene B4.The goal of this research line is to characterise the gene LTA4H in a specific cohort of patients, composed by healthy individuals, as well as TB patients. This characterization will enable further insight into transcriptional regulation of this bifunctional enzyme and its role in various inflammatory processes.

AimsTo identify the extent of polymorphic variability in the LTA4H gene implied in inflammatory differences and response to Mtb. The specific aims of this proposal are to identify variants or haplotypes, new or previously reported that could be linked to an increment of the susceptibility to TB in the general population.The work plan includes: DNA extraction and PCR amplification of the gene LTA4H in a group of individuals with or without TB and assessment of the genetic differences in the sample sets following NGS performed externally.

References-

SupervisorsTeresa Rito and Margarida Correia-Neves

26

2.13Implication of amino acid metabolism in monocyte differentiation

SummaryMonocytes represent a pool of myeloid precursors that ultimately originate tissue macrophages and dendritic cells. These cells act as sentinels, being simultaneous coordinators and effectors of homeostasis and inflammation in peripheral tissues. The development of specific immune responses relies on the accurate utilization of nutrients, thus determining distinct metabolic phenotypes and consequently its effector functions. Although several reports state the importance of the metabolic environment for the correct function of macrophages, the impact of amino acids during monocyte differentiation remains unclear.

AimsWe aimed to characterize the monocyte amino acid metabolic requirements during the differentiation process and their impact on the development of macrophage effector functions.

References-

SupervisorsRicardo Silvestre

27

2.14Gene editing: the cross-kingdom system for genohacking

SummaryHerein, we aim to use the trans-kingdom RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 [nuclease]/[nickase; Cas9n] technology to edit genomes from human cell lines to fungal cells. We will develop and apply the programmable guide sequences to direct cleavage by the Cas9/Cas9n and produce either double (DSB)- or single (SSB)- strand breaks at target sites triggering the formation of small insertions/deletions (INDELs) or allele-specific substitution via exogenous donor template. It is currently known that the introduction of DSB at specific loci allows modification of genomes with high precision and efficiency, where HDR increases in several orders of magnitude. Our main goal is to use the CRISPR/Cas9 system to perform genetic manipulation by 1) the insertion of mutations and 2) targeted homologous recombination, which represent a step towards the comprehension of cell and molecular biology aspects revealed by gene function in its own genetic context. This system offers a promising way of circumvent the resilience of Paracoccidioides spp. to target homologous molecular manipulation, allowing the generation of knock-out strains and also fine-genomic-editing.

AimsTaking advantage of the resolution offered by this newly developed technology, we expect to successfully manipulate the genome of Paracoccidioides spp., thus moving the field beyond the state of art. So, the generation of targeted loss/gain of function mutants in Paracoccidioides spp. is still both a huge gap and a challenge in the field. In addition, we will apply this system to human primary cell lines targeting pre-known Single Nucleotide Polymorphisms associated to increased susceptibility to immune response.

ReferencesShalem O, Sanjana NE, Zhang F 2015 High-throughput functional genomics using CRISPR-Cas9.Nat Rev Genet.16(5):299-311.Cunha C, Aversa F, et al. 2014. Genetic PTX3 deficiency and aspergillosis in stem-cell transplantation. N Engl J Med. 370(5):421-32.Menino JF, Saraiva M, et al. 2013. P. brasiliensis virulence is affected by SconC, the negative regulator of inorganic sulfur assimilation. PLoS One. 8(9):e74725.Menino JF, Almeida AJ, Rodrigues F. 2012. Gene knockdown in Paracoccidioides brasiliensis using antisense RNA. Methods Mol Biol.;845:187-98.

SupervisorsCristina Cunha and Fernando Rodrigues

28

2.15Impact of strain variability on the response to Paracoccidioides spp

SummaryThe mammalian immune system has innate and adaptive components, which cooperate to protect the host against infection. Pathogenic fungi can subvert the immune response to prevent elimination by the host. Genetic variability of the Paracoccidioides spp clade have also been recently acknowledge. In the context of this project we aim at defining how different clinical isolates of Paracoccidioides spp modulate the inflammatory response and how does that impact the ability of the host to control infection. To this end, we will use a model of bone-marrow derived macrophages to probe the inflammatory response of these cells to different clinical isolates of Paracoccidioides spp and correlate this information with the ability of mice to control infection.

AimsDefine immune response variability associated with Parcoccidioides spp strain variability

ReferencesBagagli E, Theodoro RC, Bosco SM, McEwen JG. 2008 Paracoccidioides brasiliensis: phylogenetic and ecological aspects.Mycopathologia. Apr-May;165(4-5):197-207. ReviewMenino JF, Saraiva M, et al. 2013. P. brasiliensis virulence is affected by SconC, the negative regulator of inorganic sulfur assimilation. PLoS One. 8(9):e74725.Menino JF, Almeida AJ, Rodrigues F. 2012. Gene knockdown in Paracoccidioides brasiliensis using antisense RNA. Methods Mol Biol.;845:187-98.

SupervisorsEgídio Torrado and Fernando Rodrigues

29

2.16The contribution of PTX3 to the human immune response to Trichosporon spp.

SummaryPentraxin (PTX)3 is a member of a family of multimeric pattern-recognition proteins with known proinflammatory activity. PTX3 acts as a non-redundant component of the humoral branch of innate immunity being also involved in fine-tuning inflammation. It has been shown that peritoneal macrophages derived, from mice overexpressing PTX3, have an improved opsonin-independent phagocytosis of yeast form of Paracoccidioides brasiliensis. Human genetic variants that conditioning the expression levels of PTX3 were shown to be associated with increased susceptibility to aspergillosis.The manipulation the host response with a protective effect of PTX3 by recombinant-PTX3-treating may reveal PTX3 as a potential therapeutic tool in paracoccidioidomycosis, either alone or in combination with antifungals therapy.Trichosporon species include basidiomycetes with a yeast-like morphology and that are able to colonize the skin and gastrointestinal tract. In the recent decades, these species emerged as important opportunistic fungal pathogens in immunocompromised patients. Although the increased prevalence of these fungal species in the clinical setting has been acknowledged, the mechanisms underlying their recognition by the immune system and the establishment of protective responses are far from being understood.Pentraxin 3 (PTX3) is an inflammatory glycoprotein with a pivotal role in the innate immune response against multiple pathogens, including fungi. The synthesis of PTX3 is stimulated in endothelial cells, macrophages, myeloid cells, and dendritic cells by cytokines and endotoxins such as bacterial products, interleukin-1, and TNF. PTX3 binds with the high-affinity complement component C1q to initiate the pathway of complement activation and facilitate pathogen recognition by macrophages. Importantly, data from our group has demonstrated that genetic variants in the human PTX3 gene compromise the expression of proposer levels of the molecule and render carriers highly susceptible to fungal infection. Because the role of PTX3 to the immune response to Trichosporon spp. remains unknown, we aim to evaluate its contribution to antifungal effector functions of macrophages and, in parallel, to assess the impact of genetic variation in the immune response.

AimsEvaluate the role of the long pentraxin 3 in the antifungal immune response of macrophages to Trichosporon spp.Unravel the functional consequences of host genetic variants in PTX3 to macrophage immunity to Trichosporon spp.

References-

SupervisorsCristina Cunha and Fernando Rodrigues

30

2.17Phylogenetic analysis of HIV-1 genomic sequences

SummaryGlobally, more than 30 million people are estimated to be infected with HIV-1. Phylogenetically, HIV-1 is divided into four genotype groups: M, O, N, and P. The vast majority of HIV-1 infections in the world are caused by M group viruses that can be further divided into at least nine genetic subtypes, A to D, F to H, J, and K, as well as different circulating and unique recombinant forms (CRFs and URFs, respectively). The geographic patterns of the M group subtypes are dynamic and can change in response to human population migrations and active transmission networks. Presently, HIV-1 B subtype is the most prevalent subtype in Western Europe, however in a part of the Iberian peninsula (including the provinces of Minho, Portugal and Galicia, Spain) there is a higher HIV-1 subtype diversity. The in detail study of this region is of great importance since it may be considered a “hotspot” of viral recombination and might also constitute a starting point for the geographic expansion in Western Europe of non-B HIV-1 genotypes. The introduction of non-B HIV-1 subtype in the Iberian peninsula is likely a consequence of migratory movements with African and South American countries. However, it is unclear if the transmission of non-B subtypes is becoming established among local individuals and if it has the potential to influence the geographic pattern of HIV-1 subtype diversity in Western Europe. In order to address these questions, this project focus on the monitoring of the phylodynamics of HIV-1 viruses in the high subtype diversity region of the Iberian peninsula and its comparison to HIV-1 viral sequences isolated from other locations. Although current antiretroviral regimens appear to have comparable efficacies across all HIV-1 subtypes, there is evidence suggesting that particular HIV-1 subtypes have a transmission advantage, higher replicative efficiency or a higher predisposition to drug resistance reinforcing the need for continuous monitoring of HIV-1 subtype diversity.

AimsPerform molecular epidemiologic characterization of HIV-1 sequences.Identify viral transmission networks and possible relationships with other globally described transmission clusters.

ReferencesCarvalho A, Costa P, Triunfante V, Branca F, Rodrigues F, Santos CL, Correia-Neves M, Saraiva M, Lecour H, Castro AG, Pedrosa J, Osório NS. Analysis of a local HIV-1 epidemic in portugal highlights established transmission of non-B and non-G subtypes. J Clin Microbiol. 2015 May;53(5):1506-14. doi:10.1128/JCM.03611-14.

SupervisorNuno S. Osório

31

2.18Rapid Diagnostic Tests and PCR for Detection of Malaria Infection in Dominican Republic

SummaryRapid diagnostic test (RDT) remains an efficient tool for Plasmodium falciparum case detection among fever patients in Dominican Republic. Nevertheless, there are concerns regarding the usefulness of RDT, both with regards to case detection with differences in test performances in various epidemiological settings and to adherence to negative test results among health care workers. Considering that high quality Plasmodium falciparum DNA can reliably be extracted from malaria RDTs, in this lab rotation we intend to evaluate 200 negative RDT for its false-negativeness by highly sensitive PCR assay. Results from this lab rotation will provide knowledge on the prevalence of false negative RDTs predicting the reliability of the use of RDTs as a diagnostic tool for malaria in Dominican Republic.

AimsOptimize Plasmodium falciparum DNA extraction from the dipstick of the RDT card.Perform molecular diagnosis of presence of malaria parasite DNA into the negative RDTs.Check for species identification, screening of molecular markers of resistance and multiplicity of infection into the falce-negative RDTs.

References-

SupervisorMaria Isabel Veiga

32

Neurosciences Research Domain3.1 Searching for therapies for Machado-Joseph disease. 3.2 Temporal modulation of the subventricular zone neural stem cell niche by choroid plexus-cerebrospinal fluid. 3.3 Exploring the Therapeutic Potential of the Secretome of Mesenchymal Stem Cells and Bioreactors in Parkinson’s Disease Animal Models. 3.3 Pharmacotherapies and Extracellular Matrix like Hydrogels as tools for Spinal Cord Injury Regeneration: a combinatory Approach. 3.5 Modulation of Mesenchymal Stem Cells Secretome through Peptide Grafted 3D Culture Environments: A Focus on Spinal Cord Injury Repair. 3.6 Sorting Nexin 3 role in neuronal development and behavior. 3.7 Exploring Sorting Nexin 27 role in Stress. 3.8 Exploring Sorting Nexin 27 role in Pain. 3.9 A Poly-pharmacological Therapy to Restore the Injured Spinal Cord. 3.10 Neural circuits of reward and motivation. 3.11 Linking stress with brain cancer: effects on glioma initiation, aggressiveness and prognosis. 3.12 Regulation of WNT signaling by HOXA9 in Glioblastoma: mechanistic and therapeutic insights.3.13 Interplay between HOXA9 and P-Cadherin in Glioblastoma: Functional and Clinical Implications. 3.14 The role of IL10 in depression and antidepressant treatment. 3.15 Impact of chronic pain in decision-making - the role of dopamine. 3.16 Asymmetries in rodents’ brain function. 3.17 Asymmetries in human brain function.3.18 Social or anti-social? The disruptive power of a peripheral neuropathic lesion. 3.19 Is memory formation dependent solely on neuronal function? Dissecting the role of astrocytes in cognitive processes.3.20 Adult neuroplasticity as a pathological trigger to recurrence in depression. 3.21 Exploring the role of the transcription factor AP2gamma in the control of post-natal glutamatergic neurogenesis. 3.22 Drug screening for chronic pain disorders. 3.23 Stress-driven changes in synaptic interactome: a link between depression and Alzheimer’ s disease.3.24 Synaptic Tau protein: an unknown target for anesthetics malfunction? 3.25 Pain-triggered synaptic plasticity: identifying the mechanistic involvement of Tau protein. 3.26 “Stressed” proteostasis: the chronic stress impact on the orquestration of proteasomal and autophagic pathways in Alzheimer´s disease pathology. 3.27 Coaching strategies to prevent stress anxiety in test performance.3.28 Inflammatory response in AD: the role of interferons.3.29 The impact of lipocalin 2 in the central nervous system homeostasis: friend or foe? 3.30 Behavioral and brain histological characterization of phospholipase D knock-out mice. 3.31 Stress impact on the brain: neuropsychiatric and physiological consequences.3.32 Implication of RNA splicing for Machado Joseph Disease.3.33 Functional correlates of adult hippocampal cytogenesis: a matter of sex and time? 3.34 Immunological aging as a trigger for multiple sclerosis.3.35 Exploring the involvement of striatum in the pathology of Machado-Joseph disease: study in a mouse model.3.36 Exploring Sorting Nexin 27 role in Mycobacterium avium infection.3.37 Tau function and dysfunction: characterization of a neuronal cell line.

33

3.1Searching for therapies for Machado-Joseph disease

SummaryMachado-Joseph disease (MJD) is a neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) tract within the C-terminal of the ataxin-3 protein. Ataxin-3 is known to interact with polyubiquitin chains and to have a deubiquitylase (DUB) activity in vitro, however the cellular and physiological role(s) of this protein remain unknown. The leading hypothesis concerning the pathogenesis of polyQ diseases is that the expanded polyQ tract confers a toxic gain of function to the mutant proteins. These disease proteins acquire the ability to self associate and form aggregates. The lack of therapeutic strategies that effectively prevent neurodegeneration in MJD patients prompted us to search for compounds that modulate mutant ataxin-3 aggregation and neurological dysfunction. Recent data from our lab reveal that many aspects of MJD can be properly modeled in the round worm Caenorhabditis elegans, and others have shown that this animal provides a suitable platform for both the discovery of new bioactive compounds and therapeutic target identification. This project is based on the idea that the finding of effective drugs can be accomplished by looking simultaneously at protein aggregation (conformational disorder) in the live neuronal cells, and on its impact on neuronal-regulated behavior of the whole-animal (neurodegenerative disorder). With this in mind, the student will test some small molecules for their ability to prevent or delay the formation of mutant ataxin-3 aggregates by feeding them to our MJD C. elegans model. Additionally, we will address their effect on motor neuron dysfunction by performing motility analysis. The drugs thus identified will in the future be tested in a mouse model of the disease.

Aims-To evaluate the effect of pharmacological compound(s) on the aggregation of mutant ATXN3, using imaging-based criteria;-To evaluate the effect of the same compound(s) on mutant ATXN3-mediated motor neuron dysfunction (motility assay).

References-Teixeira-Castro A*, Jalles A*, Esteves S*, Kang S, Silva Santos L, Silva-Fernandes A, Neto MF, Brielmann RM, Bessa C, Duarte-Silva S, Miranda A, Oliveira S, Neves-Carvalho A, Bessa J, Summavielle T, Silverman RB, Oliveira P, Morimoto RI, -MACIEL P. “Serotonergic signaling suppresses ataxin-3 aggregation and neurotoxicity in animal models of Machado-Joseph disease”. Brain, in press.-Teixeira-Castro A , Ailion M, Jalles A, Brignull HR, Vilaça JL, Dias N, Rodrigues P, Oliveira JF, Neves-Carvalho A, Morimoto RI, Maciel P. “Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF1 pathways”. Human Molecular Genetics, 20(15):2996-3009, 2011.-http://www.wormbook.org/-Voisine C, Varma H, Walker N, Bates EA, Stockwell BR, et al. 2007 “Identification of Potential Therapeutic Drugs for Huntington's Disease using Caenorhabditis elegans.” PLoS ONE 2(6): e504. doi:10.1371/journal.pone.0000504.

SupervisorsAndreia Teixeira-Castro and Patrícia Maciel

34

3.2Temporal modulation of the subventricular zone neural stem cell niche by choroid plexus-cerebrospinal fluid derived factors

SummaryWith this project we aim at determining the contribution of molecules secreted from the choroid plexus (CP) towards the cerebrospinal fluid (CSF) in the modulation of the subventricular zone (SVZ) neural stem cell population during brain maturation. Lining the lateral walls of the CSF-filled brain ventricles and in the proximity of the CP, the subventricular zone (SVZ) is one of the main neural stem cell niches in the postnatal brain. Several studies suggest that the SVZ functions as a reservoir of progenitor cells for brain repair. Adult neural progenitor cells in the SVZ are derived from radial glia, the embryonic neural stem cells; during development, radial glia generates neurons and glial cells for the assembly of the mature brain. Interestingly, at birth, the lateral wall of the brain ventricles is still comprised of radial glial cells similarly to what is observed in the ventricular zone in early embryonic stages. Then, in the initial postnatal days, radial glia cells are converted into adult neural stem cells; the latter are the neural progenitors at juvenile and adult stages. The mechanism through which this cellular transition occurs and is regulated in such a short time window period is largely unknown. We propose that factors secreted by the CP towards the CSF play a role in this timely transition of brain morphology. In this project we also aim at understanding how adult CP-derived factors impact in the adult SVZ.

AimsWe currently aim at exploring the specific contribution of CP-born CSF molecules in the homeostasis of the SVZ neural stem cell niche by:Determining the temporal changes in the CP transcriptome in specific milestones of SVZ development (pre-natal, early post-natal and adult);Study in vitro and in vivo the impact of identified CP-derived proteins/molecules in the SVZ; in vitro by using a transwell co-culture system of CP epithelial cells and SVZ neurospheres; in vivo with viral vectors that specifically target CP epithelial cells and thus abrogate or overexpression of CP proteins secretion towards the CSF; the impact of this changes in the SVZ is analyzed by using cell specific markers; cell fate of SVZ born cells is analyzed in the olfactory bulb and in the corpus callosum.

ReferencesFalcão, A. M. et al. The path from the choroid plexus to the subventricular zone: go with the flow! Front Cell Neurosci 6, 34 (2012).Lun, M.P et al. Development and functions of the choroid plexus-cerebrospinal fluid system. Nat Rev Neurosci 16, 445-57 (2015).

SupervisorsJoão Carlos Sousa and Diana Afonso

35

3.3The role of N-glycans in cell-cell communication in neural cells

SummaryThis project aims at gaining insight in the role of cell surface and extracellular matrix glycans in the modulation of neural cells communication. Glycans are complex carbohydrate molecules composed of variable length chains of simple sugars that are associated with intracellular and cell surface proteins and lipids. Glycosylation is an important post-translational modification with a strong impact in organism development and homeostasis. In the brain glycans are relevant for neural cells differentiation and function, and were recently implicated in synapse formation and synaptic transmission. Nevertheless how different glycosylation patterns affect nervous system wiring and function is still largely unknown. We are currently using animal models (C.elegans and rodents) to address how N-glycans modulate neuronal function. Specifically, we are exploring the effects of disturbing specific enzymatic pathways of glycosylation on the nervous system.

AimsWe are using specific C.elegans loss-of-function mutants for key enzymes in the synthesis of N-glycans to evaluate: neuronal dependent animal behaviour;structure of the nervous system; the effects on glycosylated proteins of the synapse; andvariations in the type and levels of glycans.Furthermore we explore the effects of the stress response in the glycosylation process in the neural cells. Knowledge obtained will be of value to understand pathophysiological processes such as the stress response and neuronal regeneration.

ReferencesKleene, R. & Schachner, M. Glycans and neural cell interactions. Nat. Rev. Neurosci. 5, 195–208 (2004).

SupervisorJoão Carlos Sousa and Ana Veloso

36

3.4Exploring the Therapeutic Potential of the Secretome of Mesenchymal Stem Cells and Bioreactors in Parkinson’s Disease Animal Models

SummaryHuman Mesenchymal stem cells (hMSCs) have been proposed as possible therapeutic agents for central nervous system (CNS) disorders. Nowadays it is suggested that their effects are mostly mediated through their secretome, which contains several neuroregulatory molecules capable of increasing cell proliferation, differentiation and survival (1). In light of the actual knowledge of the MSCs therapeutic potential is extremely relevant to establish the best culture parameters of MSC populations because little is known about the secretome of MSCs and their applications in the CNS (2). Additionally, as MSCs are highly responsive to dynamic culturing environments, one could expect to modulate and possibly increase the level of the above referred neuroregulatory factors in the secretome through the use of bioreactors. Thus, it is logical to hypothesize that when subjected to different dynamic culturing conditions, the secretome of these cells might change. Moreover, as high yields of cells will be obtained, the possibility of having higher concentrations of neuroregulatory factors in their conditioned media (CM) will also increase. We have previously shown that the secretome of MSCs cultured in bioreactors is able to induce higher differentiation rates in populations of human neural stem cells. Following this initial results the goal is to test its therapeutic properties in a Parkinson’s Disease rat model. For this purpose the 6-OHDA unilateral model (4) will be used, and the secretome of MSCs locally injected in the striatum and substantia nigra and compared with MSCs and NSCs transplanted groups. Follow up will be assessed trough behavioral and histological analysis.

Aims Unveil how the dynamic cultured obtained secretome of MSCs modulates: 1) the motor symptoms of PD in the 6-OHDA model; 2) the non-motor symptoms in the 6-OHDA model; 3) Neuronal cell survival and differentiation in the affected areas. Experimental Techniques: Cell and Tissue Culture, Behavioral Analysis, Immunohistochemitry, Histology, Neurostructural Analysis, Fluorescence/Confocal Microscopy.

ReferencesSalgado AJ et al., Stem Cells & Development, 2010, 19(7): 1067-74Teixeira FG et al., Stem Cells Reviews and Reports, 2015, 11(2): 288Teixeira FG et al., Stem Cell Research and Therapy, 2015, 6(1): 133Salgado AJ et al., Frontiers in Cellular Neuroscience, 2015, 9: 249

SupervisorsAntónio Salgado and Fábio Teixeira

37

3.5Pharmacotherapies and Extracellular Matrix like Hydrogels as tools for Spinal Cord Injury Regeneration: a combinatory Approach

SummarySpinal cord injury (SCI) is a major medical problem world-wide that affects 11,000 people/year in EU only and usually results in devastating and permanent loss of function (paraplegia and quadriplegia). Therefore it is urgent to find novel strategies that can lead to the regeneration of SCI affect sites and individuals, as the present ones (mainly pharmacological agents) do not elicit regeneration. Due to the complexity of SCI, only regenerative strategies based on multidisciplinary and integrative approaches such as those presented by tissue engineering concepts, will adequately tackle the problem. Tissue engineering, a field of science that has been developed through the last 15 years, stands on the interface between materials science, biology and medical sciences, and aims at developing tissue hybrids that induce tissue regeneration [1]. By following these concepts we have previously developed extracellular matrix like hydrogels l with enhanced cell adhesion and proliferation properties (mimicking the natural extracellular matrix) [2-4]. The role of the hydrogel is to promote axonal migration, in order to restore the cord’s functional properties. Therefore we have modifier it with peptides that are involved in this process, namely GRGRDS (fibronectin) and YIGSR (laminin). On the other hand, while the regenerative step can be fostered by these structures (with or without the combination with stem cells), it is also need to render the injured spinal cord more amenable characteristics for reparative processes to happen, namely by partially inhibiting processes such as inflammation and glial scar formation. Such processes can be achieved by using drugs that target them. Therefore the objectives of this rotation will be to test different combination of drugs and ECM-like hydrogels (with or without cells) and their impact on in vitro and in vivo models of axonal degeneration/regeneration.

Aims and TechniquesTest different combination of drugs and ECM-like hydrogels (with or without cells) and their impact on in vitro and in vivo models of axonal degeneration/regeneration.Experimental techniques: cell and explant culture; biomaterials modification; immunohistrochemistry; neurostructural analysis; fluorescence/confocal microscopy; in vivo models of SCI; behaviour analysis

ReferencesLanger R, Vacanti J. Tissue engineering. Science. 1993;260:920-6.- Silva NA, et al. Tissue Engineering Part A. 2010;16:45-54.Silva NA, et al., Biomaterials, 2012, 33(27):6345-54Assunção-Silva RC et al., Biomedical Materials, 2015, in press

SupervisorsAntónio Salgado and Nuno Silva

38

3.6Modulation of Mesenchymal Stem Cells Secretome through Peptide Grafted 3D Culture Environments: A Focus on Spinal Cord Injury Repair

SummarySpinal Cord Injury (SCI) is a chronic condition for which there is still no clinical treatment. In the last decade, the transplantation of Mesenchymal Stem Cells (MSCs) has been suggested as a possible therapy for SCI. This pro-regenerative capacity of MSCs has been linked to the secretion of bioactive molecules that provide trophic support to the damaged tissues, known as the secretome (1,2).In spite of the aforementioned beneficial roles of MSC transplantation, very low numbers of cells survive within the lesion site, which represents a drawback for their clinical application. A possible alternative is to combine them with biodegradable biomaterials. They can protect encapsulated MSCs, while stimulating the production of growth factors by them, which will enhance the regeneration of SCI (3). Moreover by using peptides from proteins present in MSCs ECM (e.g. fibronectin, laminin and collagen) involved in processes such as cell survival, adhesion and proliferation, to modify these hydrogels, it will be possible to further modulate MSCs secretome, and thus increase the regenerative potential of these cells (4). Thus, it is possible to envision the development of tissue responsive/inductive GG based hydrogels that modulate the action of encapsulated MSCs in the injured spinal cord [11]. The aims of this rotation will be focused on testing a number of different peptides, grafted into hydrogel matrix based on gellan gum, a naturally occurring hydrogel, and assesse their effects on the regenerative potential of MSCs

Aims and Experimental techniquesTest a number of different peptides, grafted into hydrogel matrix based on gellan gum, a naturally occurring hydrogel, and assesse their effects on the regenerative potential of MSCsExperimental techniques: cell and explant culture; biomaterials modification; immunohistrochemistry; neurostructural analysis; fluorescence/confocal microscopy;

ReferencesTeixeira FG et al., Cellular and Molucar Life Sciences, 2013, 70(20): 3871Silva NA et al., Progress in Neurobiology, 2014, 114: 25Assunção-Silva RC et al., Stem Cells International, 2015, 948040Silva NA et al., Biochimie, 2013, 95(12): 2314

SupervisorsAntónio Salgado and Nuno Silva

39

3.7Sorting Nexin 3 role in neuronal development and behavior

SummaryProtein assembly and turnover abnormalities are hallmarks of several neurodegenerative disorders [1]. The Sorting Nexins family of proteins (SNXs) plays pleiotropic functions in protein trafficking and intracellular signal in neuronal and non-neuronal cells, and has been associated with several human diseases that result from abnormal endosomal function, namely, Alzheimer’s disease [2]. Despite the reported roles of SNXs in protein homeostasis in neurodegeneration, not much is known about SNXs function in the nervous system. The aim of this project was to use the nematode Caenorhabditis elegans that encodes in its genome eight SNXs orthologs, and is a reference model organism to study the function and malfunction of the nervous system, to functionally characterize SNXs. We found that SNX3 gene mutation led to an array of developmental defects, namely, reduced brood size, embryonic lethality, delayed hatching, and to a decreased life span. Additionally, SNX3 mutant worms presented distinct behavioral deficits, such as, increased motor uncoordination, impaired chemotaxis and susceptibility to osmotic, thermo and oxidative stresses, which implies perturbed neuronal functions. Altogether, our data supports a prominent role of SNX3 in nervous system development. In this manner, we would like to pursuit with C. elegans SNX-3 characterization, by (i) quantifying in the wild-type (WT) worm SNX-3 expression during the developmental cycle (egg to adulthood); (ii) quantifying, in the snx-3 deletion mutant, the expression level of several receptors and neurotransmitters by qRT-PCR and Western-blot analysis; (iii) microinject the human SNX-3 and SNX-12 genes into the C. elegans snx-3 mutant background; and (iv) silence SNX-3 expression in the WT worm using siRNA. We will finally perform behavioral characterization of these snx mutant worms.

AimsCharacterization of C. elegans SNX-3 mutant expression during development.Evaluate the expression of distinct receptors in the C. elegans snx-3 mutant background.Evaluate the behavior of the C. elegans snx-3 mutant and of the reintegrated clones (C. elegans SNX-3; Human SNX-3 and SNX-12).

ReferencesDouglas, P.M. and A. Dillin, Protein homeostasis and aging in neurodegeneration. J Cell Biol, 2010. 190(5): p. 719-29.Cullen, P.J., Endosomal sorting and signalling: an emerging role for sorting nexins. Nat Rev Mol Cell Biol, 2008. 9(7): p. 574-82.

SupervisorNeide Vieira

40

3.8Exploring Sorting Nexin 27 role in StressSummarySorting nexins (SNXs) are a family of proteins that play pleiotropic roles in protein trafficking and that have been associated with endocytic events underlying neurodegeneration, synaptic plasticity and cognition [1-4]. Despite this, not much is known about SNXs role in the nervous system and how their function is modulated by aging, age-related pathologies and stress; conditions that are inextricably linked to endocytic dysregulation, cognitive decline and synaptic malfunction. Previous data from the host lab demonstrated that SNX27 expression is significantly reduced in the PFC (a brain region severely affected by stress exposure [5]) of rats exposed to a mild stress protocol. Interestingly, a C. elegans snx-27 deletion mutant strain displayed an increased susceptibility to heat shock, osmotic or oxidative stresses, which implies a role for SNX-27 in stress tolerance. Taking into consideration the recent findings that SNX27 is tightly associated with Down's syndrome [3] and Alzheimer's disease [6], where its expression is markedly reduced; that stress is a major trigger in neurodegenerative disorders; and that stress-related pathologies display cognitive impairments, synaptic malfunction/atrophy, and decreased proteostasis [7-8], we aim to dissect SNX27 role in stress. For that, SNX27 heterozygous mice (KO mice are not viable) and littermate controls will be exposed to a 6-week Chronic Mild Stress (CMS) protocol. Animals will then be analysed for several behavioral dimensions: locomotion, emotion and cognition. Behavioral assessment will be performed in the following order: elevated plus maze (EPM; anxiety-like behavior), open field (OF; locomotor and exploratory behavior), forced-swim test (FST; depressive-like behavior), working memory water maze (WM; spatial short-term memory), Morris water maze (MWM; spatial reference memory) and spatial reversal (behavioral flexibility). Following behavioral characterization, animals will be anesthetized, CSF samples will be collected, as well as blood serum samples (for posterior analysis of metabolites, cytokines, among others). Tissue samples will also be collected (liver, adrenal glands, thymus, spleen and brain). Half of the brain will then be macrodissected for further RNA or protein analysis, and the remaining brain processed for Golgi staining (for posterior stereological analysis and 3D neuron reconstructions).

AimsCharacterize SNX27 expression by RT-PCR and western-blot analysis in control and stress exposed wild-type and Snx27+/- mice.Evaluate the subcellular localization and membrane-association of SNX27 in control and stress exposed wild-type and Snx27+/- mice.Evaluate distinct behavioral dimensions in control and stress exposed wild-type and Snx27+/- mice.Analyze neuronal volumes, cell numbers and neuronal morphology.

ReferencesCullen, P.J. Nat Rev Mol Cell Biol, 2008. 9(7): p. 574-82.Teasdale, R.D. and B.M. Collins Biochem J, 2012. 441(1): p. 39-59.Wang, X., et al. Nat Med, 2013. 19(4): p. 473-80.Cerqueira, J.J., et al. Cereb Cortex, 2007. 17(9): p. 1998-2006.Wang, X., et al. Cell Rep, 2014. 9(3): p. 1023-33.Powers, E.T., et al. Annu Rev Biochem, 2009. 78: p. 959-91.

SupervisorNeide Vieira and Susana Roque

41

3.9Exploring Sorting Nexin 27 role in Pain

SummarySorting nexins (SNXs) are a large family of phosphoinositide-binding proteins that play fundamental roles in orchestrating cargo sorting through the endosomal network. SNX27 is one of the most studied members of this family and has a well-established role in glutamate receptors trafficking regulation [1, 2]. Glutamatergic transmission is critical for nociception (acute pain) and underlies the onset and maintenance of hypersensitivity in chronic pain conditions [3] but surprisingly, no previous study assessed SNX27 potential as a therapeutical target for pain. Based on this rationale we propose to study the manifestation of acute and chronic pain in a mice model with reduced expression of SNX27. Male SNX27 -/+ mice and littermate controls will be used to assess pain-related behaviors in models of acute noxious thermal (tail- and paw-flick), tonic inflammatory (formalin) and chronic neuropathic pain (spared nerve injury, SNI). In the later, emotional and cognitive behaviors will also be characterized as these are frequently affected in chronic pain conditions [4], using the following paradigms: elevated plus maze (EPM; anxiety-like behavior), open field (OF; locomotor and exploratory behavior), forced-swim test (FST; depressive-like behavior), working memory water maze (WM; spatial short-term memory), Morris water maze (MWM; spatial reference memory) and spatial reversal (behavioral flexibility). Animals will then be anesthetized, spinal cord will be removed for c-fos immunohistochemistry and density estimation. Sciatic nerve ultrastructure will also be studied for g-ratio calculation [axon diameter/ (axon diameter+myelin thickness)]. A- and C-fiber conduction velocity and compound action potential will also be followed.

AimsEvaluate the impact of decreased SNX27 expression in distinct behavioral dimensions.Analyze nociception of wild-type and Snx27+/- mice.Assess Snx27+/- mice tolerance to peripheral neuropathic pain.Evaluate neuronal volumes, cell numbers and neuronal morphology of wild-type and Snx27+/- mice exposed to SNI.Quantify spinal cord neuronal activation by c-fos immunostaning in wild-type and Snx27+/- mice exposed to SNI.

References1.Wang, X., et al., Loss of sorting nexin 27 contributes to excitatory synaptic dysfunction by modulating glutamate receptor recycling in Down's syndrome. Nat Med, 2013. 19(4): p. 473-80.2.Loo, L.S., et al., A role for sorting nexin 27 in AMPA receptor trafficking. Nat Commun, 2014. 5: p. 3176.3.Tao, Y.X., J. Gu, and R.L. Stephens, Jr., Role of spinal cord glutamate transporter during normal sensory transmission and pathological pain states. Mol Pain, 2005. 1: p. 30.4.Leite-Almeida, H., et al., Differential effects of left/right neuropathy on rats' anxiety and cognitive behavior. Pain, 2012. 153(11): p. 2218-25.

SupervisorNeide Vieira and Hugo Almeida

42

3.10A Poly-pharmacological Therapy to Restore the Injured Spinal Cord

SummarySpinal cord injury leads to devastating neurological deficits that have a strong impact in the physiological, psychological and social behavior of patients. For these reasons, it is urgent to develop therapeutic strategies that can specifically target this problem. When the spinal cord suffers a mechanical trauma it begins a cascade of cellular and biochemical reactions that leads to further damage. This cascade of reactions, also known as "secondary injury", it is characterized by a strong inflammatory response, glutamate excitotoxicity, release of myelin-derived inhibitors and the formation of a glial scar. These events are known to have a crucial contribution for axon regeneration failure after a SCI. The modulation of the secondary events will most likely play a central role in future clinical therapy. Several authors already demonstrated that the neutralization of a single secondary event leads to some motor recovery and higher neurite extensions in SCI animals. For instance, the modulation of inflammation using demonstrated to promote behavioral and histological improvements in injury rats. Moreover, the administration of Mg or Riluzole revealed to reduce the glutamate excitotoxicity and to promote motor improvements. In addition, the glial scar degradation with has shown to enhance axon regeneration and improve motor function in SCI rat. It was also demonstrated that blocking myelin inhibitory proteins, such as Nogo, MAG or OMgp, facilitates the regeneration of the injured spinal cord. Finally, it was previously shown that the prevention of cAMP hydrolysis stimulates axonal regeneration and motor improvements. These are promising results, however it is missing an integrative approach that combines the activation of growth promoting programs; while at the same time attenuate growth inhibitory pathways and promotes neuroprotection. For this reason, we are studying a poly-pharmacotherapy that can tackle most of the molecular issues responsible for the failure of axon regeneration upon SCI.

AimsDetermination of the bioactivity of the selected drugs when combined into a single pharmacotherapy approach;Establish a contusion model of SCI in rats.

References-

SupervisorNuno Silva

43

3.11Neural circuits of reward and motivation

SummaryThe mesolimbic circuit, comprising dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), is involved in processing emotionally salient stimuli of both positive and negative valence, being crucial for reward and aversion modulation. The NAc is mainly composed of dopamine receptor D1 and D2-type medium spiny neurons (MSNs) that have been proposed to have an opposing role in behavior; whereas D1 signaling promotes reinforcement and is associated with positive valence, D2 activation mediates aversion and encodes negative valence. However, recent evidence from our team suggests that this canonical perspective is oversimplistic and that both types of MSNs are important for reward and reinforcement. In this work, we will use optogenetic and electrophysiological tools in order to dissect what is the role of the NAc MSNs in reward and motivation.

AimsOptogenetic manipulation/inhibition of D1 and/or D2 neurons in reward/motivation-related tasksElectrophysiological measurements

ReferencesRodrigues, A. J., Leao, P., Carvalho, M., Almeida, O. F., and Sousa, N. (2010) Potential programming of dopaminergic circuits by early life stress, Psychopharmacology (Berl).Rodrigues, A.J., Leão, P., Pêgo, J.M., Cardona, D., Carvalho, M.M., Oliveira, M., Costa, B.M., Carvalho, A.F., Morgado, P., Araújo, D., et al. (2012). Mechanisms of initiation and reversal of drug-seeking behavior induced by prenatal exposure to glucocorticoids. Mol. Psychiatry (2012)Borges, S., Coimbra, B., Soares-Cunha, C., Miguel Pêgo, J., Sousa, N., and João Rodrigues, A.. Dopaminergic modulation of affective and social deficits induced by prenatal glucocorticoid exposure. Neuropsychopharmacology (2013).Soares-Cunha C, Coimbra B, Borges S, Carvalho MM, Rodrigues AJ, Sousa N.The motivational drive to natural rewards is modulated by prenatal glucocorticoid exposure”. Translational Psychiatry (2014).

SupervisorAna João Rodrigues

44

3.12Linking stress with brain cancer: effects on glioma initiation, aggressiveness and prognosis

SummaryThe existence of a clear link between psychological stress and cancer is still a matter of debate. Despite some correlative studies suggesting that stress may influence cancer onset and the prognosis of patients, a truly causative relationship has yet to be shown. This is particularly challenging as stress is not easy to measure objectively, the levels of perceived stress are highly subjective among individuals, and there are different stressors in nature and exposure periods. In this project, we aim to understand the effects of chronic stress in glioma initiation and aggressiveness/prognosis. Gliomas are particularly relevant as the etiologic factors are largely unknown, they are very aggressive cancers for which curative treatments do not exist, and they originate in the brain, where stress induces remarkable alterations. To achieve our goal, and taking advantage of the expertise of many researchers at ICVS, merging the key research areas of neurosciences and oncology, we will induce glioma formation in mice by orthotopic implantation of tumor cells in the brain, and evaluate how the exposure of these mice to established chronic unpredictable stress (CUS) protocols may affect tumor initiation, progression, and mice overall survival. Additionally, the influence of pharmacological approaches that modulate stress-related phenotypes (e.g., cortisol or β-adrenoceptor agonists) in these mice models of glioma will also be tested. After sacrifice, mice brains will be collected for histological and molecular characterization of tumor and surrounding brain tissue. The levels of corticosteroid in the peripheral blood will also be measured and used as a biological correlate of stress.

AimsEvaluate blood corticosteroid levels and assess putative associations with the survival of mice implanted with glioma.Evaluate the effect of stress in:the histological and molecular characteristics of glioma in mice brains;tumor volume, and lymphatic and blood vessel density within and around tumors.the recruitment of relevant immune cells into the tumor.Test if pharmacological approaches (e.g., cortisol or β-adrenoceptor agonists) can modulate stress-related phenotypes, in these mice models of glioma.

References-

SupervisorBruno M. Costa and Ana João Rodrigues

45

3.13Regulation of WNT signaling by HOXA9 in Glioblastoma: mechanistic and therapeutic insights

SummaryGlioblastoma (GBM) is the most common and most malignant type of glioma, a heterogeneous group of primary brain tumors. While the clinical outcome of GBM patients is unpredictable, patients are equally treated with a standardized approach. Thus, the identification of new biomarkers is crucial. HOXA9 overexpression in GBM is associated with poor prognosis and a more aggressive tumor phenotype. We recently found that HOXA9 transcriptionally activates components of the WNT pathway. This pathway is hyper-activated in several tumors and associated with cancer stem cell-like features, as well as increased proliferation and therapy resistance. Interestingly, we observed that WNT6, a WNT ligand/activator, is overexpressed in glioma in a grade-dependent manner, and that this overexpression is associated with the poor prognosis of GBM patients. In vitro, WNT6 overexpression was associated with increased viability, invasion, stem cell capacity and resistance to TMZ. In vivo, mice bearing WNT6-positive tumors presented faster glioma-related symptomatology and a significantly shorter overall survival. In this context, we aim to i) understand the molecular mechanisms regulated by Wnt signaling / WNT6 that may contribute to glioma aggressiveness; ii) test novel therapeutic approaches combining the current chemotherapeutic used in the clinics (temozolomide) with an inhibitor of this pathway to improve the therapeutic response of glioma in vitro and in vivo.

AimsStudy intracellular signaling pathways regulated by Wnt6 in glioma (e.g., phosphorylation state of components of PI3K, JNK, and MAPK pathways using phospho-arrays).Assess the therapeutic potential of a new combinatorial treatment including an inhibitor of the Wnt pathway and temozolomide on glioma models.

References-

SupervisorBruno M. Costa

46

3.14Interplay between HOXA9 and P-Cadherin in Glioblastoma: Functional and Clinical Implications

SummaryGlioblastoma (GBM) is a highly malignant and the most common primary brain tumor, for which curative therapies are not available. We previously showed that HOXA9, a critical transcription factor during development, is overexpressed in a subset of glioblastoma samples, and is associated with increased tumor resistance to therapy and shorter patient survival. A recent study demonstrated that HOXA9 affects the aggressiveness of ovarian cancer cells by affecting the expression of P-Cadherin, which encodes a transmembrane protein with roles in cell adhesion. Since this HOXA9/P-Cadherin link has not been described in other cancer types, and nothing is currently known on the importance of P-Cadherin in the context of malignant brain tumors, we will study: i) how HOXA9 may affect P-Cadherin levels in glioblastoma; ii) the functional roles of P-Cadherin in glioblastoma, particularly at the levels of cell migration, invasion, viability, and response to chemotherapy; iii) the roles of P-Cadherin in in vivo intracranial models of glioblastoma; iv) the clinical value of P-Cadherin as a prognostic marker in glioblastoma. These approaches will clarify for the first time the relevance of a critical Cadherin in the context of malignant brain tumors.

Aims-Evaluate P-Cadherin expression in a panel of glioblastoma tumor samples from patients and in cell lines, and correlate them with HOXA9 levels;-Evaluate how the levels of P-Cadherin influence the aggressiveness of glioblastoma using in vitro models (cell lines) and orthotopic in vivo mice models.

References-

SupervisorBruno M. Costa

47

3.15The role of IL10 in depression and antidepressant treatment

SummaryPrevious studies from our laboratory showed that female mice lacking IL10 expression show a depressive-like behavior, a phenotype that is rescued by IL10 administration. In accordance, mice overexpressing IL10 present decreased learned helplessness. Interestingly antidepressant treatment, both in patients with depression and in animal models, increases IL10 production. In fact, the reversion of the inflammatory milieu has been shown to play a role for the efficacy of antidepressant treatment. Indeed, treatment-resistant patients have been shown to present a persistent inflammatory profile after antidepressant treatment. In accordance, the concomitant treatment of those patients with antidepressants and anti-inflammatory drugs improved the efficacy of antidepressant therapy. Thus, the goal of this work is to unravel the mechanisms underlying the role of the anti-inflammatory cytokine IL10 in the etiology of depressionand in response to antidepressant therapy. Since two of the most widely discussed mechanisms underlying depression are the altered HPA axis and the imbalance production of cytokines, based respectively, on the frequently observed increased levels of glucocorticoids and pro-inflammatory cytokines in depressed patients, respectively, we will investigate these two mechanisms in mice lacking IL-10 expression. Moreover, preliminary data suggest that IL10KO mice seem to be resistant to antidepressant therapy (both with fluoxetine and imipramine), suggesting that IL10 expression is crucial for the reversion of the depressive-like phenotype. Thus, further studies will be performed to clarify if this animal model could help to better understand the causes of treatment-resistant depression.

Aims-Assess whether colon inflammation and corticosterone levels could be associated with depressive-like behavior;-Clarify the role of IL10 in antidepressant treatment

References-

SupervisorsSusana Roque and Margarida Correia-Neves

48

3.16Impact of chronic pain in decision-making - the role of dopamine

SummaryChronic pain impacts brain structure and function. Indeed, besides sensory abnormalities, chronic pain is frequently accompanied by alterations in emotional and cognitive behavior. The neurotransmitter dopamine emerges here as an interesting link between chronic pain, depression and decision-making as it is individually related with each of these dimensions.Recently we demonstrated in a rat model of chronic pain that i) chronic pain impacts in a lateralized way decision-making and ii) there are side-specific alteration in the expression of dopamine receptors in the prefrontal cortex and nucleus accumbens, areas involved in decision-making.In this project we intend to further explore these observations, unraveling the lateralized impact of chronic pain in the dopaminergic system and its posterior involvement in the observed impairments in decision-making.

AimsStudy the impact in decision-making of nucleus accumbens unilateral dopamine depletion;Manipulate the dopaminergic system unilaterally and study side-specific effects on behavioural readouts in a chronic neuropathic pain model;

ReferencesLeite-Almeida, H., Cerqueira, J.J., Wei, H., Ribeiro-Costa, N., Anjos-Martins, H., Sousa, N., Pertovaara, A., Almeida, A., 2012. Differential effects of left/right neuropathy on rats' anxiety and cognitive behavior. Pain 153, 2218-2225.

SupervisorsHugo Leite-Almeida and Margarida Cunha

49

3.17Asymmetries in rodents’ brain function

SummaryBrain functional asymmetry in a nearly symmetrical body remains paradoxical. Nevertheless, since the 1861’s Paul Broca description of a left dominance for language, many side-biased brain processes and behaviors have been described. Such functional asymmetry is not exclusively human; it has also been described in various vertebrate and invertebrate classes, suggesting that laterality is highly conserved across species. The relevance of brain functional asymmetry for cognition and executive function is not understood. For instance, brain imaging studies in older individuals report bilateral activity during the execution of cognitive tasks that in younger subjects were associated with lateralized activity. It remains an open question if the lack of asymmetry is a compensatory mechanism operating from the non-dominant hemisphere and therefore rendering individuals with lower asymmetry functional advantage or if it simply is a correlate of poorer executive function.The present project proposal is designed to study these questions. We aim to characterize corticostriatal activity in a behavioral paradigm of intertemporal decision-making (impulsivity) and to correlate task-solving efficiency with activity side-biases particularly, its magnitude and directionality (left>right or left<right).

AimsCharacterize trait impulsivity in rats;Obtain bilateral local-field potentials recordings in relevant areas (e.g. prefrontal cortex) during the execution of the paradigm;

ReferencesLeite-Almeida, H., Guimaraes, M.R., Cerqueira, J.J., Ribeiro-Costa, N., Anjos-Martins, H., Sousa, N., Almeida, A., 2014. Asymmetric c-fos expression in the ventral orbital cortex is associated with impaired reversal learning in a right-sided neuropathy. Mol Pain 10, 41.SupervisorsHugo Leite-Almeida and Madalena C. Esteves

50

3.18Asymmetries in human brain function

SummaryBrain functional asymmetry in a nearly symmetrical body remains paradoxical. Nevertheless, since the 1861’s Paul Broca description of a left dominance for language, many side-biased brain processes and behaviors have been described. Such functional asymmetry is not exclusively human; it has also been described in various vertebrate and invertebrate classes, suggesting that laterality is highly conserved across species. The relevance of brain functional asymmetry for cognition and executive function is not understood. For instance, brain imaging studies in older individuals report bilateral activity during the execution of cognitive tasks that in younger subjects were associated with lateralized activity. It remains an open question if the lack of asymmetry is a compensatory mechanism operating from the non-dominant hemisphere and therefore rendering individuals with lower asymmetry functional advantage or if it simply is a correlate of poorer executive function.The present project proposal is designed to study these questions. We aim to characterize both structural and functional hemispheric asymmetries in humans. In order to achieve this we are currently taking advantage of ICVS’ large database of MRI and fMRI data from several different cohorts.

AimsCharacterize structural hemispheric asymmetries and find influencing factors;Determine the importance of functional and structural asymmetries for cognitive function;

ReferencesCorballis MC. The evolution and genetics of cerebral asymmetry. Philos Trans R Soc Lond B Biol Sci 2009;364:867-879.Sun T, Walsh CA. Molecular approaches to brain asymmetry and handedness. Nat Rev Neurosci 2006;7:655-662.Toga AW, Thompson PM. Mapping brain asymmetry. Nat Rev Neurosci 2003;4:37-48.

SupervisorsHugo Leite-Almeida and Madalena C. Esteves

51

3.19Social or anti-social? The disruptive power of a peripheral neuropathic lesion

SummaryMood disorders are frequently accompanied by alterations in the amount and quality of social interactions. While it is well-established that in experimental models of chronic pain animals develop anxiety- and depressive-like phenotypes, no study has been carried to understand how the social environment contributes to pain manifestation and to the emergence of associated comorbid emotional disorders. Recently, we have observed that in chronic neuropathic pain conditions the rodent’s natural ability to interact with an unfamiliar animal conspecific (with or without pain) is disrupted. This proof-of-concept prompted us to further explore these findings. In a series of experiments we will study mutual influences in pain- and mood-related behaviors between cage-mate pairs pain/no-pain, pain/pain and control no-pain/no-pain. Also, social interactions will be studied between familiar and unfamiliar conspecifics in the conditions above. The spared nerve injury (SNI) model of neuropathic pain will be used and putative side-specific effects in left- (SNI-L) or right (SNI-R) –lesions will be analysed as previous studies demonstrated that SNI-L is more anxiogenic than SNI-R. Finally, analgesic pharmacotherapy will be employed attempting to rescue the phenotypes.

AimsStudy mutual influences between cage mates on different conditions of painCharacterize social behaviour between familiar and (un)familiar animals with and without painStudy possible lateralized effects of pain (SNI-L vs SNI-R) on social behaviour.

ReferencesLeite-Almeida, H., Cerqueira, J.J., Wei, H., Ribeiro-Costa, N., Anjos-Martins, H., Sousa, N., Pertovaara, A., Almeida, A., 2012. Differential effects of left/right neuropathy on rats' anxiety and cognitive behavior. Pain 153, 2218-2225.Zhou W, Dantzer R, Budac DP, Walker AK, Mao-Ying QL, Lee AW, Heijnen CJ, Kavelaars A: Peripheral indoleamine 2,3-dioxygenase 1 is required for comorbid depression-like behavior but does not contribute to neuropathic pain in mice. Brain Behav Immun 2015, 46:147-153.

SupervisorsHugo Leite-Almeida

52

3.20Is memory dependent solely on neuronal function? Dissecting the role of astrocytes in cognitive processes.

SummaryThe classical paradigm that brain information processing is exclusively neuronal has been challenged in the past ten years by an exciting body of evidence. Indeed, the importance of glial cells is rising due to emerging data supporting dynamic neuron-glia interactions. These inter-cellular interactions are nowadays widely accepted. Nevertheless, little is known about effects of glial cells, namely astrocytes, in complex behaviour outputs. Astrocytes are able to sense, integrate and respond to neurons. In the lab we have currently mice strains that incorporate genetic modifications to impair specifically each of these 3 functions. The goal of this rotation is to test their performance in cognitive tasks and correlate this performance with electrophysiological or molecular/morphological alterations. This way, the applicant may choose between different approaches to solve the same problem.By disclosing the net consequence of astrocytic modulation of the neuronal networks we expect to better understand behaviour computation and therefore understand the possible therapeutic approaches in diseases characterized by cognitive dysfunction (e.g. Alzheimer’s, Major Depression…).

Aims-To test the performance of mice with astrocytic dysfunction in working memory and long term memory paradigms (hole board and Morris Water Maze).-To correlate the performances obtained in the tests with the observed expression of the transgenes in the specific areas by microscopic observation of brain slices containing the targeted areas (prefrontal cortex and hippocampus, respectively), as well as quantification of altered protein levels.-To analyse the morphological changes in both neurons and astrocytes by means of immunohistochemistry and unbiased stereological techniques.

ReferencesOliveira JF, Sardinha VM, Guerra-Gomes S, Araque A, Sousa N (2015). Do stars govern our actions? Astrocyte involvement in rodents’ behavior. Trends in Neurosciences 38:535–549M.M. Halassa, C. Florian, T. Fellin, J.R. Munoz, S. Lee, T. Abel, P.G. Haydon, e M.G. Frank, Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss, Neuron, vol. 61, Jan. 2009, pp. 213-219

SupervisorJoão Oliveira

53

3.21Adult neuroplasticity as a pathological trigger to recurrence in depression

SummaryDepression is a highly prevalent neuropsychiatric disorder, frequently associated with the occurrence of relapses and recurrences. Although the vast majority of studies focus the onset and treatment of depression, knowledge regarding recurrence into a new depressive episode remains scarce. Giving the high rate of recurrence in depressive patients, it is of great relevance to understand the molecular mechanisms behind this phenomenon. According to recent studies, the impact of depression on neuronal morphology and adult cytogenic processes namely, hippocampal neurogenesis and gliogenesis may determine the progression to a further depressive episode.Modeling recurrent depression using an animal model of depression, we intend to unravel the behavioral and molecular response to a repeated depressive episode, determine impact of treatment with typical antidepressants and characterize detectable molecular alterations in biological fluids. Unveiling these mechanisms may pave the way to the development of efficient treatments to prevent recurrent depression.

AimsDetermine the behavioral and neuroplastic alterations associated to the recurrence into a new depression episode;Characterize the therapeutic relevance of initial antidepressants treatment to promote resilience to recurrent depression;Identify on peripheral fluids, physiological and neuromolecular correlates of recurrence in depression

ReferencesMateus-Pinheiro A, Pinto L, et al., Sustained remission from depressive-like behavior depends on hippocampal neurogenesis. Transl Psychiatry. 2013 Jan 15;3:e210.Mateus-Pinheiro A, Patricio P, Bessa J, Sousa N and Pinto L, Cell genesis and dendritic plasticity: a neuroplastic pas de deux in the onset and remission from depression, Mol Psychiatry. 2013, Jul;18(7):748-50Patrício P, et al., Differential and Converging Molecular Mechanisms of Antidepressants' Action in the Hippocampal Dentate Gyrus. Neuropsychopharmacology. 2015 Jan;40(2):338-49

SupervisorLuísa Pinto

54

3.22Exploring the role of the transcription factor AP2gamma in the control of post-natal glutamatergic neurogenesis

SummaryThe view of the central nervous system (CNS) as an immutable system in now outdated, and several lines of evidence demonstrate that the CNS is endowed with significant regenerative and neuroplastic potential. In fact, it is now well established that new cells are continuously generated during adulthood in specific brain regions, a process called neurogenesis. The current challenge relies on dissecting the regulatory mechanisms of the adult neurogenic process. Recently, we have identified AP2gamma (AP2𝛾) as an important transcription factor involved in developmental corticogenesis. We now seek to explore whether AP2𝛾 has a role on the regulation of adult neurogenesis and its importance for different emotional and cognitive modalities.

AimsAnalyze different behavioral and molecular parameters in WT and AP2gamma conditional knockout mice.Evaluate the impact of AP2gamma deletion on adult hippocampal neurogenesis and neuronal morphology.

ReferencesPinto L. et al, “AP2γ regulates basal progenitor fate in a region- and layer-specific manner in the developing cortex”, Nat Neurosci. 2009 Oct;12(10):1229-37

SupervisorLuísa Pinto

55

3.23Drug screening for chronic pain disorders

SummaryChronic pain and depression are enormous health and financial burdens on our society, with depression also being the major comorbidity of chronic pain disorders. Chronic pain induces a profound change in the expression of peptides and their receptors that leads to changes in brain wiring (neuronal plasticity) of central pathways mediating pain [1].In this work, chronic pain will be induced in Wistar han adult rats for a period of 4 weeks followed by the administration of drugs for an additional 3 weeks. At the end of this period, behavioural analysis to evaluate anxiety- (acoustic startle, open field and elevated plus maze), depressive-like (forced swimming test and sucrose preference test) behaviour as well as nociception [pain behaviour - tail and paw-flick tests (heat noxious stimulation), cold allodynia (acetone test), the Randall-Selitto and the pressure application measurement tests (mechanical noxious stimulation)] will be performed.At the end of the behavioural task the animals will be sacrificed and brains will be removed for the evaluation of changes in neurotransmitters pathways, either through RT-PCR or immunohistochemistry. Emotional and nociceptive behavioural data will be correlated to the expression of neurotransmitters and its receptors in several areas of the brain involved in pain modulation.

AimTo evaluate the ability of different types of drugs to reverse the impact of chronic inflammatory pain upon mood disorders in rodents;To correlate drug efficiency with changes in the neurochemistry of brain areas involved in pain modulation.

ReferencesNeugebauer V, Galhardo V, Maione S, Mackey SC. Forebrain pain mechanisms. Brain Res Rev. 60(1):226-242.

SupervisorsDiana Amorim and Filipa Pinto-Ribeiro

56

3.24Stress-driven changes in synaptic interactome: a link between depression and Alzheimer’ s disease

SummaryWorld Health Organization estimates that the leading cause of mental disability in the coming years will be depression and Alzheimer’s disease (AD), raising these two diseases as significant public health problem. Focusing on risks factors of these diseases, previous clinical and experimental studies suggest a causal role of environmental parameters e.g. chronic stress and the subsequent elevation of stress hormones, glucocorticoids (GC), in pathogenesis of depression while recent findings involve stress in the onset/progression of AD. Neuronal atrophy and synaptic failure have been suggested to play an essential role in stress-related pathologies such as depression as well as in AD. Furthermore, important clues of synaptic disruption mechanism(s) previously implicated in pathophysiology of AD have been recently suggested to also contribute in stress-driven brain pathology involving, for the first time, Tau missorting in mechanism(s) of synaptic damage beyond Alzheimer’s disease (Pinheiro et al., 2015; Sotiropoulos et al., 2011). Based on the recently suggested role of Tau in synaptic structure and function interacting with NMDA receptors, PSD-95 and Fyn proteins, this project examines the alterations of synaptic interactome underlying stress-induced neuronal atrophy and synaptic loss searching for molecular targets with neuro- and synapto-protective properties.

AimsMolecular characterization of stress/GC impact on postsynaptic vs extra-synaptic/presynaptic interactome In vitro and/or in vivo monitoring of intracellular and synaptic trafficking of receptors and related cell signalling under stressful conditions.

ReferencesPinheiro S., et al., (2015) Mol Neurob, Sotiropoulos et al., (2015) JAD; Kimura et al., (2014) Phil Trans Roy Soc: Biol Scienc; Sotiropoulos I., et al., (2011) J Neurosc; Catania C., Sotiropoulos et al., (2009) Mol Psychiatry 14: 95–105; Sotiropoulos I., et al., (2008) Neurosc Biobeh Rev 32: 1161-1173.

SupervisorsIoannis Sotiropoulos and Nuno Sousa

57

3.25Synaptic Tau protein: an unknown target for anesthetics malfunction?

SummaryGeneral anesthetics (GA) are widely used drugs with various clinical applications but many studies report various cognitive problems (some persistent) after GA exposure. Previous studies have shown that different anesthetics induced Tau hyperphosphorylation whereas Tau hyperphosphorylation is related to memory deficits through brain synaptic loss. Based on the recently suggested role of Tau in synaptic structure and function interacting with various proteins such as NMDA receptors (Ittner and Gotz, 2011), we hypothesize that synaptic Tau may have a unique involvement in anesthetics action. First results of this project have shown that NMDA-related anesthetics modifies neuronal structure altering dendritic arborization and synapses in hippocampus accompanied by cognitive impairment in WTs while this effect was attenuated in absence of Tau. Thus, this project aims to clarify the role of Tau and its interaction with NMDA receptors on synaptic mechanisms underlying anesthetic-induced neuroremodeling and cognitive deficits.

AimPerform detailed molecular and structural analysis of synaptic cell signalling and synaptic interactome of Tau

References-

SupervisorsIoannis Sotiropoulos and Hugo Almeida

58

3.26Pain-triggered synaptic plasticity: identifying the mechanistic involvement of Tau protein

SummaryTau protein hyperphosphorylation and consequent malfunction is a hallmark of Alzheimer’s disease pathology; importantly, pain perception is diminished in these patients. In physiological conditions, Tau contributes to cytoskeletal dynamics and in this way, influences a number of cellular mechanisms including axonal trafficking, myelination and synaptic plasticity. While it is known that these cellular processes are also implicated to pain perception, it was just recently that we demonstrated the direct in vivo role of Tau in nociception (Sotiropoulos et al., 2014) showing that Tau-deficient mice display altered pain perception but the the underlying mechanisms remain unknown. Interestingly, Tau has an important role in the post-synaptic density, where, via interaction with Fyn, contributes to NMDA receptor/PSD95 complex formation the latter has also been implicated in nociception (Ittner and Gotz, 2011; D'Mello et al., 2011). Thus, this project will analyze the molecular alterations of pain-stimulated synapses with particular focus on the suggested interaction of Tau with synaptic receptors responsible for synaptic plasticity in both spinal cord and brain

AimsMonitor the effect of pain stimuli on synaptic TAU phosphorylation;-Identify the influence of Tau on synaptic plasticity mechanisms (e.g. trafficking of synaptic receptors, interaction with scaffold proteins)

ReferencesSotiropoulos et al., (2014) Exper Neurol; Ittner and Gotz, (2011) Nat Rev Neurosci; D'Mello et al., (2011) Mol Ther

SupervisorsIoannis Sotiropoulos and Hugo Leite-Almeida

59

3.27“Stressed” proteostasis: the chronic stress impact on the orquestration of proteasomal and autophagic pathways in Alzheimer´s disease pathology

SummaryTau aggregation is a common feature in Alzheimer’s disease (AD), frontotemporal dementia (FTD) and other tauopathies. Consistent with suggestions that lifetime stress may be a clinically-relevant precipitant of AD pathology, we previously showed that chronic stress and the main stress hormones, Glucocorticoids (GC), trigger Tau aggregation affecting molecular chaperones such as Hsp90 which is known to be involved in both GC signaling and Tau degradation (Sotiropoulos et al., 2015); still, the underlying mechanisms of the above stress/GC effects remain unclear. This project focuses on how stressful conditions converge on cellular mechanisms underlying Tau aggregation in AD brain monitoring tress/GC-triggered downstream degradation mechanisms (proteasome- and lysosome-related ones) mechanisms using both animal and cell culture experimental approaches.

Aims-Molecular and cellular characterization of ubiquin-proteasome and autophagic pathways under chronic stress and/or GC treatment.-Clarification of stress/GC-driven molecular mechanisms relating molecular chaperones such as Hsp90 to Tau degradation pathways.

ReferencesSotiropoulos et al., (2015) J Alz Dis; Sotiropoulos et al., (2011) J Neurosc; Sotiropoulos et al, (2008) J Neurochem

SupervisorsJoana Silva and Ioannis Sotiropoulos

60

3.28Coaching strategies to prevent stress anxiety in test performance

SummaryModern lifestyle exerts an enormous burden on individuals by pushing towards increasing productivity and longer working hours. This feeling of persistent underachievement, that modern working conditions exert, leads to burnout and stress-related mental disorders such as anxiety. Additionally, workplace environment is often inappropriate to provide a stress-free environment due to poor lighting, temperature or noise conditions.Higher education is a transition period before students reach the working environment. Students are subjected to increasing periods of work with a progressive focus on autonomy and continuous assessment as mandated by current educational policies. The increasing workload is perceived as stressful and commonly leads to mental disorders and perception that their cognitive performance is bellow their expected standards. This is corroborated by the high prevalence of anxiety disorders among higher education students. Stress is known to affect an individual’s cognitive performance in a biphasic mode. Too little stress impairs adequate performance, increasing with physiological levels of acute stress and followed by a decrease in performance again with prolonged or disproportioned levels of stress. When an individual is exposed to stressful stimuli (physical or psychological), the organism perceives it as a threatening event and mounts an adequate biological and behavioral responses. The main biological agents are the release of cathecolamines, like epinephrine (adrenaline), and corticosteroids (cortisol). The positive effects of acute stress are attributed mainly to the effect of catecholamines while exposure to chronic stress results in repeated activation of the hypothalamic-pituitary-adrenal axis, leading to cortisol secretion, and elevated circulation of pro-inflammatory cytokines, affecting glucocorticoid sensitivity, brain function and behavior.Assessment is a fundamental phase in the training and certification process that a higher education student is submitted to. It is also one of the strongest stress factors due to the high-stake implications in the academic progress and self-perceived image. Stress is a risk factor for anxiety and may lead to worsening of performance in assessment tasks. The present proposal will use cohorts of higher education students as a model in order to study the effect of stress/anxiety in the performance of high demand tasks. We intend to correlate stress/anxiety markers with the response pattern in high-stakes exams and develop coaching strategies in order to improve the students’ performance.

AimsSpecifically we aim to:-characterize how anxiety affects medical student’s performance in their assessment, correlating basal levels of self-perceived stress with anxiety levels and test performance; -address potential biological mechanisms underlying the adverse effects of stress/anxiety on cognitive overload and overall performance of high demand tasks. We hypothesize that these effects are mediated by elevations in stress hormones and pro-inflammatory signals during challenging cognitive tasks;-evaluate if a directed coaching strategy will improve performance in exams by reducing the level of anxiety and/or stress.

ReferencesDavide Carneiro, Paulo Novais, José Miguel Pêgo, Nuno Sousa, and José Neves. Using Mouse Dynamics to Assess Stress During Online Exams. E. Onieva et al. (Eds.): HAIS 2015, LNAI 9121, pp. 1–12, 2015.

SupervisorJosé Miguel Pêgo

61

3.29Inflammatory response in AD: the role of interferons

SummaryAlzheimer's disease (AD) is the most prevalent form of dementia. Cognitive decline and the incapacity to form new memories are the major constrains of AD patients. Herein we intend to target cognitive decline by addressing the impact of the levels of interferons (IFNs) on cognitive performance in AD. Here we propose to explore how peripheral and central interferons (IFNs) impact on Alzheimer's disease (AD). Our motivation is triggered by our recent observation of a shift in the expression levels from the IFN type II to type I response during aging, both in the liver and in the choroid plexus of an AD mouse model. In face of publish data and our preliminary results, the present project is biologically relevant in proposing to (i) explore the novel concept that the modulation of peripheral and central IFNs in vivo may hamper cognitive decline in AD, and (ii) unravel how IFNs influence pathology, namely in amyloid precursor protein processing/Aβ levels, neurotrophins expression, inflammation and synapse functioning.

Aims1-Investigate the impact of IFNs, on AD-related cognitive decline and pathology.2-Determine to what level cognitive impairment is preventable and/or (fully or partially) reversible by targeting central and/or peripheral IFNs.

ReferencesMesquita SD, Ferreira AC, Gao F, Coppola G, Geschwind DH, Sousa JC, Correia-Neves M, Sousa N, Palha JA, Marques F. The choroid plexus transcriptome reveals changes in type I and II interferon responses in a mouse model of Alzheimer's disease. Brain Behav Immun. 2015 Oct;49:280-92. doi: 10.1016/j.bbi.2015.06.008.

SupervisorFernanda Marques

62

3.30The impact of lipocalin 2 in the central nervous system homeostasis: friend or foe?

Summary Lipocalin 2 (LCN2), an acute-phase protein that, by binding to iron-loaded siderophores, is a potent bacteriostatic agent since it participates in the iron-depletion strategy of the immune system to control pathogens. The recent identification of a mammalian siderophore also suggests a physiological role for LCN2 in iron-homeostasis, specifically through iron delivery to cells via a transferrin-independent mechanism. LCN2 participates, as well, in a wide variety of cellular processes, including apoptosis and proliferation. In the central nervous system, less is known about the processes involving LCN2, namely which cells are producing this molecule or its impact on cell proliferation and death or on emotional behaviours. Importantly, LCN2 has recently emerged as a relevant clinical biomarker, namely in multiple sclerosis; again, there are conflicting views on the role of LCN2 in pathophysiological processes, with some studies pointing to its neurodeleterious effects, while others revealing it as neuroprotective.

Aims Determine the role of LCN2 in cognition and cell proliferation

ReferencesFerreira AC, Dá Mesquita S, Sousa JC, Correia-Neves M, Sousa N, Palha JA, Marques F. From the periphery to the brain: Lipocalin-2, a friend or foe? Prog Neurobiol. 2015 Aug;131:120-36. doi: 10.1016/j.pneurobio.2015.06.005

SupervisorFernanda Marques

63

3.31Behavioral and brain histological characterization of phospholipase D knock-out mice

SummarySince lipids are the major constituent of the brain, the modulation of its levels can potentially have an impact in its functioning. One the enzymes that can modulate the levels of signaling lipids is phospholipase D (PLD). Specifically, PLD is responsible for the generation of phosphatidic acid (PA) from phosphatidylcholine. PA is a central signalling lipid with membranar fusogenic properties. Consequently, the modulation of its levels can potentially alter cellular/neuronal functioning.In mammals there are two main PLD isozymes: PLD1 and PLD2. They both catalyse the same reaction, but they differ in their regulatory properties and cellular location. Thus the genetic ablation of either PLD1 or PLD2 has a differential impact. To date there are only four published studies with PLD knock-out mice, which show a role for PLD1 in platelet functioning and autophagy and the ablation of PLD2 was shown to be protective in a Alzheimer’s disease mouse model. However the precise role of PLD1 and PLD2 in brain functioning is still elusive. We propose to study the impact of both PLD1 and PLD2 ablation in mice cognitive-associated behavior and brain hippocampal organization.

Aims-To characterize the impact of PLD1 and PLD2 ablation in mice behavior.-To characterize the impact of PLD1 and PLD2 ablation in mice brain histological organization.

References-Comparative lipidomic analysis of mouse and human brain with Alzheimer disease. Chan RB, Oliveira TG, Cortes EP, Honig LS, Duff KE, Small SA, Wenk MR, Shui G, Di Paolo G. J Biol Chem. 2012 Jan 20;287(4):2678-88.-Phospholipase D2 ablation ameliorates Alzheimer's disease-linked synaptic dysfunction and cognitive deficits. Oliveira TG, Chan RB, Tian H, Laredo M, Shui G, Staniszewski A, Zhang H, Wang L, Kim TW, Duff KE, Wenk MR, Arancio O, Di Paolo G. J Neurosci. 2010 Dec 8;30(49):16419-28.-Phospholipase D in brain function and Alzheimer's disease. Oliveira TG, Di Paolo G. Biochim Biophys Acta. 2010 Aug;1801(8):799-805. Epub 2010 Apr 23. Review.

SupervisorTiago Gil Oliveira

64

3.32Stress impact on the brain: neuropsychiatric and physiological consequences

SummaryStress affects decision-making and prolonged stress exposure can lead to depression, posttraumatic stress disorders and other deleterious health effects. Under stressful situations, glucocorticoids are released by the body to act in the brain, restoring homeostasis and promoting behavioral adaptation. Although this short-term stress is adaptive, long-term stress is clearly associated with disease vulnerability, including mental illness. Despite self-awareness from the affected individuals, the magnitude of stress-response is uncontrollable and the "tipping point" between adaptive and maladaptive stress remains unknown.How does stress remodels brain activity and switches-off cognitive control over actions? This project uses electrophysiology recordings and optical fibers implanted in the brain, to register and manipulate brain activity in stressed mice. Behavioral analysis and physiological parameters will also be collected to understand the global impact of stress.

Aims1-Investigate the neurological impact of stress on specific brain circuits.2-Determine if stress response can be modulated by stimulating defined regions in the brain.

ReferencesSousa, N. The dynamics of the stress neuromatrix. Mol Psychiatry. 2016 Mar;21(3):302-12.

SupervisorPatricia Monteiro and Nuno Sousa

65

3.33 Implication of RNA splicing for Machado Joseph Disease

SummaryMachado-Joseph disease (MJD) is a neurodegenerative disorder caused by a polyQ-tract expansion within Ataxin-3 (ATXN3), a deubiquitylating (DUB) enzyme. The precise physiological role of ATXN3 and the molecular mechanism causing selective neurodegeneration remain unknown. We recently found altered polyubiquitylation of several splicing regulatory factors (SRFs) in neuronal cells lacking ATXN3; consistently, these cells showed disturbed splicing profiles. We hypothesize that ATXN3 regulates the turnover of key SRFs through its DUB activity, and that polyQ-expansion may be compromising this interaction, resulting in splicing deregulation of key transcripts, contributing to neurodegeneration. Using biochemical/biophysical and cellular approaches the student will: i) study the interaction of SRFs with ATXN3 and ii) evaluate the regulation of SRFs by ATXN3 DUB activity. We expect to characterize this new ATXN3 biological function and its relevance for MJD, opening new possibilities for rational design of therapeutics.

Aims-To evaluate interaction of SRFs with non-expanded and mutant ATXN3;-To evaluate the relevance of ATXN3 DUB activity on the regulation of SRFs turnover.

ReferencesAlmeida, B., Fernandes, S., Abreu, I.A. and Macedo-Ribeiro, S. Trinucleotide repeats: a structural perspective, Front Neurol, 4 (2013) 76.C.A. Matos, S. de Macedo-Ribeiro, A.L. Carvalho, Polyglutamine diseases: the special case of ataxin-3 and Machado-Joseph disease, Prog Neurobiol, 95 (2011) 26-48.B.G. Burnett, R.N. Pittman, The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation, Proc Natl Acad Sci U S A, 102 (2005) 4330-4335.P.O. Bauer, N. Nukina, The pathogenic mechanisms of polyglutamine diseases and current therapeutic strategies, Journal of neurochemistry, 110 (2009) 1737-1765.

SupervisorBruno Almeida and Patrícia Maciel

66

3.34 Functional correlates of adult hippocampal cytogenesis: a matter of sex and time?

SummaryAlong with the subependymal-olfactory bulb system, the hippocampus is one of the two regions of the adult brain that retains the ability to generate new cells throughout life. Indeed, within the hippocampal dentate gyrus, neural stem cells (NSCs) have the ability to continuously generate new neuronal or astroglial cells, in a process called neurogenesis or astrogliogenesis, respectively. Understanding how these new cells in the adult hippocampus can reshape brain circuits and affect different behavioral functions is a complex task. This complexity emerges primarily from the difficulty to precisely segregate the function of newborn cells from the preexisting neuron-glia cell network in which they are integrated. Moreover, there is sufficient evidence to support the notion that cytogenesis functional roles are variable according to (i) factors that are intrinsic to the studied animals (eg. young adult vs old; female vs male), (ii) experimental interference (eg. physiological/basal conditions vs disease states or pharmacological interventions) and (iii) the time-window of the analysis (eg. newly-formed cells vs 6-weeks old, fully integrated new cells). Hence, to better dissect the functional roles of new hippocampal cells, here we will use the transgenic GFAP-Tk rat model to specifically ablate GFAP-expressing NSCs in the adult mammalian brain. We will then perform a longitudinal characterization of the impact of cytogenesis ablation in different emotional and cognitive behavioral dimensions, addressing the differential role of immature vs mature newborn cells. Furthermore, a comparison analyses betweens male and female animals will be perform to characterize putative sex-specific functional correlates of adult hippocampal cytogenesis

AimsInduce cytogenesis ablation in GFAP-Tk adult male and female ratsExplore the functional consequences of cytogenesis ablation in different behavioral test paradigms

ReferencesMateus-Pinheiro A, Pinto L, et al., Sustained remission from depressive-like behavior depends on hippocampal neurogenesis. Transl Psychiatry. 2013 Jan 15;3:e210.Mateus-Pinheiro A, Patricio P, Bessa J, Sousa N and Pinto L, Cell genesis and dendritic plasticity: a neuroplastic pas de deux in the onset and remission from depression, Mol Psychiatry. 2013, Jul;18(7):748-50

SupervisorAntónio Mateus-Pinheiro and Luísa Pinto

67

3.35Immunological aging as a trigger for multiple sclerosis

SummaryMultiple Sclerosis (MS) is an autoimmune disorder of the central nervous system mediated by T lymphocytes, and characterized by progressive demyelination, axonal damage and neuronal loss. MS develops in two major forms: the relapsing-remitting (RRMS), characterized by bouts of neurological impairment interleaved with recovery periods, followed by a progressive phase; and the primary progressive (PPMS) with a sustained disability accumulation from onset. PPMS onset tends to occur 10 years later than RRMS (mean 40 vs 29 years). We hypothesize that the development of one or the other MS form depends on inter-individual differences on the T cells aging. In accordance, children with MS revealed signs of premature T cells aging, a process that normally initiates at young adulthood, but that displays great inter-individual differences. The aim of the project is to clarify how T cells aging impacts on MS and its phenotypes. For this, in blood samples from MS patients, the distinct forms of the disease will be related to thymic output (quantification of recent thymic emigrants by flow-cytometry and of T-lymphocytes receptor excision circles by qPCR) and T-lymphocytes’ aging (telomere length determined by flow-FISH).

AimsTo assess how T cells aging impacts on MS and its phenotypes. To do so parameters that determine the degree of T cells aging will be dissected in 4 groups of patients according to their onset age: RRMS and PPMS with onset at <=40 and >40 years.

References-

SupervisorMargarida Correia-Neves e Cláudia Nóbrega

68

3.36Exploring the involvement of striatum in the pathology of Machado-Joseph disease: study in a mouse model

SummaryMachado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder for which no effective treatment is currently available. MJD is characterized by motor uncoordination and gait ataxia, difficulty with speech and swallowing, spasticity, altered eye movements, double vision, and frequent urination. Some patients have dystonia (sustained muscle contractions that cause twisting of the body and limbs, repetitive movements, abnormal postures, and/or rigidity), others have peripheral neuropathy and loss of muscle strength or symptoms similar to those of Parkinson's disease. The cerebellum and brainstem atrophy is well documented in MJD. Recent studies have shown that the neuropathology of this disease is not limited to cerebellum and brainstem, eventually affecting the integrity of several functional and neurotransmitter systems resulting in severe and widespread neuropathology throughout the brain, including the striatum. We have generated a new transgenic mouse model expressing human ataxin-3 with an expanded CAG tract ubiquitously and at near-endogenous levels – the CMVMJD135. This mouse model mimics the human disorder quite closely at the phenotypic level. The disease symptoms of this model appear gradually in life and progress as the mice age. Motor and gait deficits, loss of muscular strength and other neurological symptoms such as abnormal reflexes and tremors are present. This model also present intranuclear inclusions, as well as neuropathologic affection in key disease regions resembling the human disease such as (i) cellular loss in the pontine and dentate nuclei, in substantia nigra and spinal cord (ii) a reduction of calbindin positive Purkinje cells as well as a reduced thickness of the molecular layer of the cerebellum, (iii) reduced cholinergic neurons in the facial nuclei and spinal cord, (iv) a reduction of TH positive neurons and astrogliosis in the substantia nigra, (v) gross brain atrophy (late in life) and (vi) and alterations in the brain cytokine profile, as was described for MJD patients. Little is known the involvement of striatum in MJD, being the main goal of this lab rotation the study of its involvement in MJD pathology.

Aims- Perform immunohistochemistry in the striatum to:

- Quantify astrogliosis, using the GFAP astrocyte marker- Evaluate microgliosis using Iba-1 microglia marker- Measure the DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein) immunoreactivity- Measure the number of ChAt positive cells (cholinergic neurons marker)- Verify the presence/absence of ataxin-3 positive aggregates.

ReferencesSilva-Fernandes A*, Duarte-Silva S*, Neves-Carvalho A, Amorim M, Soares-Cunha C, Oliveira P, Thirstrup K, Teixeira-Castro A, Maciel P. “Chronic treatment with 17-DMAG improves balance and coordination in a new mouse model of Machado-Joseph disease”. Neurotherapeutics, 2014 Apr;11(2):433-49. doi: 10.1007/s13311-013-0255-9.

SupervisorsPatrícia Maciel and Sara Duarte-Silva.

69

3.37 Exploring Sorting Nexin 27 role in Mycobacterium avium infection

SummaryEndocytic dysregulation can affect the ability to mount an appropriate immune response, by impacting on antigen presentation and antigen receptor signaling [1]. Recently, the Sorting nexins (SNXs) emerged as a novel family of highly conserved proteins that facilitate protein trafficking and cell signaling [2,3], thus modulating the presence/abundance of proteins/receptors at the cell surface and also their pathological accumulation/clearance. Reports linking SNXs and endocytic events underlying distinct diseases and pathogen invasion, are growing in number [3]. In fact, distinct studies established a prominent role for SNX3 in the down-modulation of phagocytosis [4] and of SNX27 in immunological-synapses during T cell activation [5]. Specifically, SNX27 has been demonstrated to interact with diacylglycerol kinase z (DGKz), a negative regulator of T cell function and to regulate the Ras-ERK pathway, in T cells that encounter pulsed antigen-presenting cells [5]. Despite this, not much is known about SNX27 role in the immune system, namely on how its function is modulated during infection with a pathogenic agent. Taking advantage of the SNX27+/- mouse model we aim to dissect SNX27 role during infection with Mycobacterium avium. For that, SNX27 heterozygous mice (KO mice are not viable) and littermate wild-type controls will be infected with M. avium 2447. Animals will then be analysed for several behavioral dimensions: locomotion, emotion and cognition. Behavioral assessment will be performed at 4 and 12 weeks post infection in the following order: elevated plus maze (EPM; anxiety-like behavior), open field (OF; locomotor and exploratory behavior), forced-swim test (FST; depressive-like behavior), working memory water maze (WM; spatial short-term memory), Morris water maze (MWM; spatial reference memory) and spatial reversal (behavioral flexibility). Simultaneously, blood samples will be collected for flow-cytometry analysis of the immune cell population. Tissue samples will also be collected (liver, thymus, spleen, lungs and brain) for CFU quantification and flow-cytometry analysis. Aims- Evaluate distinct behavioral dimensions in control and M. avium infected wild-type and Snx27+/- mice.- Quantify CFUs in distinct organs of M. avium infected wild-type and Snx27+/- mice.- Analyze immune cell populations in control and M. avium infected wild-type and Snx27+/- mice.

References1. Gleeson, P.A., The role of endosomes in innate and adaptive immunity. Semin Cell Dev Biol, 2014. 31: p. 64-72.2.Cullen, P.J., Endosomal sorting and signalling: an emerging role for sorting nexins. Nat Rev Mol Cell Biol, 2008. 9(7): p. 574-82.3.Teasdale, R.D. and B.M. Collins, Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease. Biochem J, 2012. 441(1): p. 39-59.4. Chua, R.Y. and S.H. Wong, SNX3 recruits to phagosomes and negatively regulates phagocytosis in dendritic cells. Immunology, 2013. 139(1): p. 30-47.5. Rincon, E., et al., Translocation dynamics of sorting nexin 27 in activated T cells. J Cell Sci, 2011. 124(Pt 5): p. 776-88.

SupervisorsNeide Vieira and Susana Roque

70

3.38 Tau function and dysfunction: characterization of a neuronal cell line

SummaryThe microtubule-binding protein tau (MAPT) is predominantly expressed in neurons (1). Tau is a multifunctional protein, but its major known biological function is the assembly and stabilization of microtubules, contributing to morphogenesis, cell division, axonal extension and axonal transport (2). Much evidence has been accumulated pointing to the contribution of tau to Alzheimer’s disease (AD) pathology either by loss of function (such as stabilization of microtubules) as well as gain of toxic function (aggregation and deposition of neurofibrillary tangles) (3). Although these data provide a better understanding of tau pathogenesis, the disappointing clinical outcomes of therapeutic approaches suggest that continuing efforts towards understanding tau pathology in AD as well as unravel the physiological functions of tau are needed. Thus, this project aims to focus in tau’s function and dysfunction using a human neuronal model. This will provide an unique opportunity to improve our knowledge of the biological pathways tau belongs to in a human environment and to a better understanding of the AD-disease mechanisms, contributing to improved therapeutical strategies.

Aims1 – To characterize the phenotype of neuronal cell lines overexpressing Wild type and pathogenic tau2 – To study the effects of pathogenic tau on cell morphology, cytoskeleton, proliferation, migration and differentiation3 – To analyze the relevance of the findings for AD in a mouse model for the disease.

References1. Weingarten MD, Lockwood AH, Hwo SY, Kirschner MW. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975;72(5):1858-62.2. Drubin DG, Kirschner MW. Tau protein function in living cells. J Cell Biol. 1986;103(6 Pt 2):2739-46.3. Rosenmann H, Blum D, Kayed R, Ittner LM. Tau protein: function and pathology. Int J Alzheimers Dis. 2012;2012:707482.

SupervisorsAndreia Carvalho, Joana Silva, Ioannis Sotiropoulos and Patrícia Maciel

71