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L’anémie est un symptôme fréquent chez les patients atteints de cancer. La libération de la cytokine pro-inflammatoire TNFα, un inhibiteur connu de l’érythropoïèse, en est l’une des causes. L’érythropoïèse est un processus nécessitant l’arrêt de la prolifération et l’autophagie. Les résultats précédents ont montré que le TNFα inhibe l’expression des marqueurs érythroïdes et module l’expression de facteurs de transcription (FT) hématopoïétiques. Notre objectif est d’étudier l’implication de la voie TNFα/sphingomyélinase (SMase)/céramide dans l’inhibition de l’érythropoïèse en utilisant des cellules souches hématopoïétiques CD34+ induites à se différencier par l’érythropoïétine recombinante (Epo). Par l’utilisation de céramides exogènes, de SMase bactérienne et d’inhibiteurs de SMases, nous montrons l’implication de la voie SMase/céramide dans l’inhibition de l’expression des marqueurs érythroïdes mais également dans l’induction de la différenciation myéloïde avec une augmentation de l’expression du CD11b. Cet effet sur la différenciation est corrélé à la modulation du réseau FT/miR impliquant GATA-1, GATA-2 et PU.1 et les miR-144, 451, 155, 146a et 223. De plus, l’analyse par microscopie électronique à transmission, l’absence de formation de punctae GFP-LC3 et l’accumulation de SQSTM1/p62 montrent que le TNFα et les céramides inhibent l’autophagie induite par l’Epo. L’analyse des protéines impliquées dans la régulation de l’autophagie montre que le TNFα et les céramides activent mTOR. Son implication est confirmée par l’utilisation de rapamycine et l’inhibition de ULK1 et Atg13. De plus, le TNFα et les céramides inhibent l’expression de bécline 1 et de la formation du complexe Atg5-Atg12. Ces résultats démontrent que la voie TNFα/SMase/céramide joue un rôle dans l’homéostasie hématopoïétique par l’inhibition de l’érythropoïèse au profit de la myélopoïèse, en impactant les réseaux de régulation FT/miR et le processus d’autophagie / Anemia is a common symptom in cancer patients. It can be caused by the release of pro-inflammatory cytokines such as TNFα, a known inhibitor of erythropoiesis. Erythropoiesis involves proliferation arrest and autophagy. Our previous studies showed that TNFα inhibits the expression of erythroid markers as well as hematopoietic transcription factors (TF) expression. The aim is to study the involvement of TNFα/sphingomyelinase (SMase)/ceramide pathway in erythropoiesis inhibition using recombinant erythropoietin (Epo)-induced CD34+ hematopoietic stem cells. Using exogenous ceramides, a bacterial SMase and sphingomyelinase inhibitors, we show the involvement of SMase/ceramide pathway in the inhibition of erythroid markers as well as the induction of myeloid differentiation as shown by the increase in CD11b expression. This effect is correlated to the modulation of the TF/miR network involving GATA-1, GATA-2 and PU.1 as well as miR-144, 451, 155, 146a and 223. We show that TNFα and ceramides inhibit Epo-induced autophagy through transmission electron microscopy analysis, the absence of GFP-LC3 punctae formation and SQSTM1/p62 accumulation. Analysis of proteins involved in autophagy regulation showed that TNFα and ceramides activate mTOR, which is confirmed using rapamycin as well as the inhibition of ULK1 and Atg13. Moreover, TNFα and ceramides inhibit Beclin 1 expression and Atg5-Atg12 complex formation. These results demonstrate the role of TNFα/SMase/ceramide pathway in hematopoietic homeostasis through an erythropoiesis-myelopoiesis switch resulting from perturbation of TF/miR network and autophagy

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2017-06-22T17:47:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2017-06-26T18:42:58Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) / Made available in DSpace on 2017-06-26T18:42:58Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) Previous issue date: 2017-06 / CAPES / Introdução: A doença arterial coronariana (DAC) é a principal causa de morbidade e mortalidade no mundo, sendo caracterizada como uma doença inflamatória crônica, multifatorial, cuja fisiopatologia é a aterosclerose. Considerando a alta herdabilidade da doença, vários polimorfismos genéticos vêm sendo estudados e associados com o surgimento da DAC e, entre eles, destacam-se variantes nos genes CD14, TLR4, NFKB1 e TNFα, os quais regulam a via de sinalização celular do sistema imune inato e desencadeiam o processo inflamatório na DAC. Objetivo: O objetivo principal deste estudo é verificar a possível associação de polimorfismos nos genes CD14 (rs2569190), TLR4 (rs4986790 e rs4986791), NFKB1 (rs28362491) e TNFα (rs1800629 e rs361525) com a DAC. Metodologia: A amostra foi composta por 707 indivíduos adultos submetidos ao exame de cateterismo cardíaco no Hospital Bruno Born, de Lajeado, RS. Todos os indivíduos assinaram um termo de consentimento livre e esclarecido e responderam a um questionário semiestruturado. Os indivíduos foram classificados entre casos e controles, por um médico cardiologista, com base no seguinte critério: presença de estenose, com comprometimento maior do que 50%, em pelo menos uma das artérias coronárias. Foram também coletadas amostras de sangue periférico para análises bioquímicas e moleculares. A extração de DNA foi realizada pelo método de salting out. Os polimorfismos dos genes CD14, TLR4 e TNFα foram genotipados pelo sistema de discriminação alélica TaqMan, em equipamento de reação em cadeia da polimerase (PCR) em Tempo Real (StepOnePlus®). O polimorfismo rs28362491, no gene NFKB1, foi amplificado através da técnica convencional de PCR. Resultados: Identificamos uma associação dos polimorfismos rs2569190, localizado no gene CD14, e rs28362491, no gene NFKB1, com a DAC. Além disso, foram detectados efeitos dos polimorfismos dos genes TLR4 e TNFα nos níveis glicêmicos e dos polimorfismos nos genes TLR4, NFKB1 e TNFα no perfil lipídico. Conclusão: Nossos achados sugerem a participação de polimorfismos nos genes CD14 e NFKB1 no desenvolvimento da DAC na nossa amostra, corroborando evidências prévias do envolvimento de genes do processo inflamatório nessa patologia. / Introduction: Coronary artery disease (CAD) is the main cause of morbidity and mortality in the world, being characterized as a chronic, multifactorial inflammatory disease, whose pathophysiology is atherosclerosis. Considering the high heritability of the disease, several genetic polymorphisms have been investigated and associated with CAD and, among them, there are variants in the CD14, TLR4, NFKB1 and TNFα genes, which regulate cell signaling pathways of the innate immune system and inflammatory process in CAD. Objective: The main objective of this study is to verify the association beteween polymorphisms in the CD14 (rs2569190), TLR4 (rs4986790 and rs4986791), NFKB1 (rs28362491) and TNFα (rs1800629 and rs361525) genes and CAD. Methods: The sample group was composed of 707 adult individuals, recruited at the time when they were bought in for coronary angiography procedures at the Hemodynamic Center of the Hospital Bruno Born, City of Lajeado, Rio Grande do Sul. The individuals were classified between cases and controls by a cardiologist, based on the following criteria: presence of stenosis, greater than 50% of the luminal diameter, in at least one of the coronary arteries. Peripheral blood samples were also collected for biochemical and molecular analyzes. DNA extraction was performed using the salting out method. The polymorphisms in the CD14, TLR4 e TNFα genes were genotyped by Taqman® allelic discrimination assays. The rs28362491 polymorphism in the NFKB1 gene was amplified by polymerase chain reaction (PCR). Results: We identified an association between the polymorphisms rs2569190, located in the CD14 gene, and rs28362491, located in the NFKB1 gene, and CAD. In addition, there were detected significant effects of TLR4 and TNFα gene polymorphisms on glycemic levels and TLR4, NFKB1 and TNFα gene polymorphisms on the lipid profile. Conclusion: Our findings suggest a role for the polymorphisms in CD14 and NFKB1 genes in CAD susceptibility in our sample, corroborating previous evidence of the envolviment of inflamotory process genes in this patology.

- / Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((−)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1–1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

Ο σκοπός της παρούσας διπλωματικής εργασίας ήταν να διερευνήσει την ενδεχόμενη συσχέτιση του γενετικού πολυμορφισμού -308 (G→A) του προαγωγέα (promoter) του γονιδίου του TNFα με την κλινική υποτροπή ή αγγειογραφική επαναστένωση, σε 40 Έλληνες ασθενείς μετά από αγγειοπλα-στική των στεφανιαίων αρτηριών (PTCA). Στη μελέτη περιελήφθησε και ένας πληθυσμός 30 υγιών μαρτύ-ρων. Η παρούσα μελέτη αποσκοπούσε ακριβώς στο να διερευνήσει τη συχνότητα της κλινικής υποτροπής ή αγγειογρα-φικής επαναστένωσης 6-8 μήνες μετά από PTCA, σε πληθυσ-μούς της περιοχής μας σε σχέση με το γονότυπο των ασθενών αυτών για το παραπάνω γονίδιο. Η πλειοψηφία των ασθενών 95% (38/40) ήταν ομόζυγοι για τον άγριο τύπο W του αλληλομόρφου και είχαν τον γονότυπο WW, 5% (2/40) ήταν ετεροζυγώτες WM, ενώ δεν ανευρέθησαν ομοζυγώτες MM για τον πολυμορφισμό. Στο δείγμα των 30 υγιών μαρτύρων, 90% (27/30) ήταν ομόζυγοι για τον άγριο τύπο W του αλληλομόρφου και είχαν τον γονότυπο WW, 10% (3/30) ήταν ετεροζυγώτες WM, ενώ δεν ανευρέθησαν ομοζυ-γώτες MM για τον πολυμορφισμό. Αγγειογραφική επαναστέ-νωση συνέβη σε 7 (οι 6/7 ασθενείς έχουν WW γονότυπο και ο 1/7 έχει τον WM γονότυπο) από τους 40 ασθενείς. Στην παρούσα μελέτη, δεν παρατηρήθηκε στατιστικά σημαντική συσχέτιση του -308 γενετικού πολυμορφισμού του TNFα με την αγγειογραφική επαναστένωση ή την κλινική υποτροπή μετά από αγγειοπλαστική των στεφανιαίων αρτηριών (PTCA), δείχνοντας ότι ο -308 γενετικός πολυμορφισμός του TNFα δεν είναι από μόνος του ένας βασικός παράγοντας κινδύνου, τουλάχιστον στον Ελλαδικό πληθυσμό με στεφανιαία νόσο. Βέβαια, τα αρνητικά μας ευρήματα σχετίζονται άμεσα με τη χαμηλή συχνότητα εμφάνισης του συγκεκριμένου πολυμορφισμού στους διάφορους πληθυσμούς, αλλά και με τον πολύ μικρό αριθμό ατόμων των παρατηρήσεων. / A follow-up study was conducted to investigate whether -308 genetic polymorphism of TNF-α gene was associated with the increased risk of restenosis in 40 Greek coronary artery disease patients undergoing coronary angioplasty and stent implanta-tion. For comparison of genotype frequency, a control group of 30 asymptomatic individuals was also studied. The end-point of the current study was the incidence of restenosis at 6-8 months of clinical follow-up. The majority of patients (38/40) had the WW genotype (homozygous for the wild-no polymorphic type allele) and only 2/40 patients had the WM genotype (heterozygous). Patients homozygous for the polymorphism (MM genotype) were not found. The frequency distribution was not different from that of the control subjects. Restenosis occurred in 7 of the 40 patients. In the population studied, -308 genetic polymorphism of TNF-α gene was not found to predispose patients to an increased incidence of restenosis. Nevertheless, these findings should be considered as preliminary, taking into account the small number of patients that were studied and the rarity of the -308 genetic polymorphism of TNF-α gene.

Pregnancy toxaemia (ketosis) is a metabolic disease of ewes which occurs during the late gestation as a result of the inability of the pregnant ewe to maintain an adequate energy balance for the fast growing maternal fetal unit. As a result of energy defi-ciency mobilization of lipid reserves results in a doubling of the plasma free fatty acid (FFA) giving rise to fatty liver and increased ketone bodies β-hydroxybutyrate (BHB) in blood and urine. It is associated with a higher rate of mortality and causes severe economic losses. The objective of this study was directed at investigating the relationship between metabolic parameters and cytokine TNFα, to check the interaction between the TNFα and fat metabolism in late pregnant ewes of different breeds, and whether TNFα play a role in the pathogenesis of pregnancy toxaemia, which may serve as marker to early diagnosis of the disease. In this study, 29 pregnant and clinically healthy ewes (16 Merino, 13 Blackhead) were selected out of a flock of sheep. Blood samples were collected at 5, 3, and 1 week be-fore parturition (b.p.) and also 4 weeks after parturition (a.p.). The average numbers of lambs were 2.18 and 1.58 /ewe for Merino and Blackhead breeds respectively. The blood samples were analyzed for the following:  Concentration of metabolic parameters: glucose, insulin, free fatty acids (FFA), β-hydroxybutyrate (BHB), albumin, total protein (TP), iron (Fe), glutamat-dehydro-genase (GLDH), creatin kinase (CK), gamma-glutamyl-transferase (GGT), choles-terol, haptoglobin.  Haematological parameters: Haematocrite (HK), haemoglobin concentration (HB), erythrocyte count (EC), leukocyte count (LC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).  Cytokine TNFα by using ovine TNFα ELISA assay. The results of glucose concentration of pregnant ewes showed significant increase (3.8 mmol/l) in five weeks b.p. and declined with advancing gestation (2.6 mmol/l) one week b.p. Insulin concentration remained constant with an average of 0.11 nmol/l b.p., and then significantly increased to 0.22 nmol/l four weeks a.p. Maximal FFA concentrations were found at five weeks b.p. (976 µmol/l). The levels of FFA showed high levels b.p. compared with reference range (R.R. < 600 µmol/l), and the FFA levels significantly decreased postpartum (four weeks b.p.). while there was significant increasing (p<0.05) in the level of FFA in Merino sheep than in Black-head sheep b.p. On the other hand there was no significant difference a.p. The mean values of BHB in all periods of sampling, period 1(5 w.b.p.), period 2 (3 w.b.p.), period 3 (1 w.b.p.), and period 4 (4 w.a.p.) were 0.37 mmol/l, 0.23 mmol/l, 0.17 mmol/l and 0.3 mmol/l respectively. The mean of BHB indicated normal levels of BHB before and after parturition compared to subclinical ketosis (BHB > 1 mmol/l) and clinical ketoses (BHB > 1.6 mmol/l), and there was a significant difference (p<0.05) in the values of BHB between Blackhead and Merino breeds before parturi-tion while there was no significant difference after parturition. The concentration of TNFα showed elevated levels in all period of sampling before parturition. The TNFα values were 30.4 (17.2, 785.0) ng/ml (median, first, and third quartiles), 35.6 (13.6, 54.3), and 26.6 (13.0, 39.9) ng/ml in period 1(5 w.b.p.), period 2 (3 w.b.p.), and period 3 (1 w.b.p.) respectively. These values decreased to 19.1 (9.9, 33.8) ng/ml at 4 weeks after parturition. Statistical analysis showed that there was a positive correlation between free fatty ac-ids and TNFα. This correlation means that adipose tissue produces TNFα causing insu-lin resistance, which stimulates the lipolysis and leads to an increase of circulatory free fatty acids levels. It is concluded that fat mobilization occurs in the prepartum clinically healthy ewes with a significant increase in the levels of FFA, and also there is an increase in the proinflammatory cytokine TNFα at late gestation which predisposes ewes to pregnancy toxaemia and can aid in the diagnosis of the disease.

Les anticorps monoclonaux ont constitué une révolution dans le traitement desmaladies inflammatoires chroniques, mais ils présentent des inconvénients majeurs (effetssecondaires, coûts élevés, résistances).Notre équipe développe des inhibiteurs du TNFα par deux approches : immunisationactive contre des peptides de cytokine pour générer des anticorps neutralisants et petitesmolécules chimiques pouvant inhiber directement le TNFα.J’ai évalué in vitro les meilleurs composés d’un criblage de chimiothèque in silico, etnotamment identifié une petite molécule qui a protégé les animaux dans deux modèles demaladies in vivo (choc septique et colite au DSS). J’ai aussi réalisé l’analyse d’analogueschimiques des meilleurs composés identifiés in vitro.J’ai également évalué l’immunogénicité de plusieurs peptides de TNFα mais lesanticorps générés n’étaient pas neutralisants in vitro et nous n’avons donc pas testé lespeptides in vivo.Mon travail s’est situé à l’interface de la bioinformatique, de la chimie, et de labiologie et m’a permis de bien comprendre les enjeux du développement moderne dumédicament. / Monoclonal antibodies have been a revolution for the treatment of chronicinflammatory diseases but present several drawbacks (secondary effects, prohibitive costs,resistance)Our team develops TNFα inhibitors using two approaches : active immunizationagainst cytokine peptides and small compounds having a direct inhibition on TNFα.I have evaluated in vitro the best compounds selected after in silico screening of achemical library and I have identified a small molecule which was protective in two animalmodels (septic shock and DSS induced colitis). I have also analyzed chemical analogues ofthe best compounds found in vitro.I have also tested the immunogenicity of TNFα peptides but they did not yieldneutralizing antibodies in vitro, and we thus did not test them in vivo.My work was at the interface of bioinformatics, chemistry and biology, and this hasenabled me to understand the key issues in the modern development of drugs.

Depuis ces 10 dernières années, l’immunothérapie est à l’avant-garde de la thérapie anticancéreuse. Les cellules Natural Killer (NK) font partie du système immunitaire inné et possèdent la capacité unique de lyser les cellules tumorales sans activation préalable par un antigène spécifique. Elles jouent un rôle majeur dans le contrôle de plusieurs cancers hématologiques et solides dont les tumeurs stromales gastrointestinales (GIST). Leur activation dépend de l’équilibre entre leurs récepteurs activateurs et inhibiteurs. Les Natural Cytotoxicity Receptors (NCR) font partis des récepteurs activateurs les plus importants dans leur reconnaissance des cibles et comprennent le NKp30, NKp44 et NKp46. Le NKp30 possède 3 isoformes: NKp30a et NKp30b sont immunostimulantes induisant la sécrétion d’Interféron (IFN) γ et de Tumor necrosis factor (TNF) α alors que NKp30c est immunosuppressive favorisant la production d’interleukine 10 (IL-10). L’IFNγ est un puissant activateur des cellules immunitaires tandis que l’IL-10 est une cytokine anti-inflammatoire. Le TNFα a été décrit initialement comme un facteur sérique induisant la nécrose des tumeurs, cependant son rôle a depuis été élargi à des fonctions homéostatiques. De nombreuses études laissent à penser que les fonctions antitumorales des cellules NK ne se limitent pas à l’élimination des cellules tumorales. Malgré les progrès importants réalisés dans la compréhension des cellules NK, de nombreux travaux sont encore à mener pour exploiter pleinement leur potentiel antitumoral.Notre équipe a démontré l’importance capitale des cellules NK dans les GIST. Ainsi l’infiltrat NK prédit la survie sans progression des patients. De plus nous avons montré que l’expression préférentielle de l’isoforme immunosuppressive NKp30c impactait négativement le pronostic des patients GIST. Suite à ces résultats, nous avons cherché à mieux caractériser l’impact des isoformes du récepteur NKp30 chez les patients GIST en réponse à l’IM. Dans un premier temps, nous avons démontré qu’un haut ratio d’expression entre NKp30b et NKp30c prédisait une meilleure réponse à l’imatinib mesylate (IM, un inhibiteur de tyrosine kinase, traitement de référence des GIST) et que l’expression des isoformes de NKp30 impactait l’environnement cytokinique de la tumeur. De plus, nous avons établi pour la première fois le lien entre la présence de ligands solubles de NKp30, B7 Homolog 6 soluble (sB7-H6) et BCL2 Associated Athanogene 6 soluble (sBAG6), et la diminution de la survie sans évènement des patients GIST traités à l’IM.Malgré l’infiltration immunitaire de nombreuses tumeurs, les fonctions antitumorales des lymphocytes sont inhibées par le microenvironnement tumoral. Ainsi, nous avons étudié quelles voies de signalisation étaient associées à l’inhibition des cellules NK présentes dans cet environnement. Pour cela, nous avons réalisé un microarray à partir des cellules NK infiltrant les GIST et avons mis en évidence le rôle délétère de la voie TNFα/TNF Receptor 2/Baculoviral IAP Repeat Containing 3 (BIRC3)/TNF Receptor Associated Factor 1 (TRAF1) dans la fonctionnalité des cellules NK. En effet, l’activation de cette voie dans les cellules NK entraine la diminution de la transcription du gène du récepteur activateur NKp46 ainsi que son expression membranaire. Cette diminution était corrélée avec l’expression de l’isoforme NKp30c. Par ailleurs, nous avons pu mettre en évidence chez la souris que le TNFα facilitait la dissémination métastatique de la lignée tumorale sensible aux cellules NK B16F10.Nos résultats sur les cellules NK ont renforcé leur grand potentiel en tant que cible thérapeutique pour l’immunothérapie anticancéreuse. En effet, l’importance du récepteur NKp30 et de ses isoformes dans la prédiction de la réponse à l’IM dans les GIST et la mise en évidence d’un nouveau mécanisme inhibiteur des cellules NK par la voie TNFα/TNFR2/BIRC3/TRAF1 ouvrent la voie à de nouvelles stratégies dans le traitement des cancers. / Over the last 10 years, immunotherapy has been at the forefront of cancer therapy. Natural Killer (NK) cells are part of the innate immune system and have the unique ability to lyse tumor cells without any antigen specific priming. They have a key prognostic role in several hematological and solid cancers including gastrointestinal stromal tumors (GIST). A balance between activating and inhibitory receptors triggers NK cell activation. Natural cytotoxicity receptors (NCR) are among the most clinically relevant activating receptors and include NKp30, NKp44 and NKp46. NKp30 can be expressed in 3 different isoforms: NKp30a and NKp30b are both immunostimulatory, inducing interferon (IFN) γ and tumor necrosis factor (TNF) α secretion whereas NKp30c is immunosuppressive, producing interleukin 10 (IL-10). IFNγ is a potent activator of immune cells whereas IL-10 is an anti-inflammatory cytokine. TNFα was first described as a serum factor, inducing tumor necrosis but its role has since been broadened to homeostatic functions. Ample evidence suggests that anti-tumor functions of NK cells are tightly regulated and expand far beyond the simple killing of malignant cells. Despite the tremendous progress in understanding NK cell biology, further work is warranted to fully exploit the anticancer potential of these cells.Our group demonstrated the crucial role that NK cells have in GIST. Indeed, NK cell infiltrate positively correlates with progression-free survival. Moreover, we showed that the preferential expression of the immunosuppressive isoform NKp30c, negatively impacts the clinical outcome of GIST patients. To further extend these observations, we explored the influence of various NKp30 isoforms in GIST patients.Firstly, we revealed that a high ratio between the expression of NKp30b and NKp30c isoforms predicted a stronger imatinib mesylate (IM) response (a tyrosine kinase inhibitor, TKI – first line standard of care in GIST) and that tumor cytokine milieu is modified following NKp30 isoform expression. Furthermore, we demonstrated a link between the presence of soluble ligands of NKp30, soluble B7 Homolog 6 (sB7-H6) and soluble BCL2 Associated Athanogene 6 (sBAG6), and a decrease in event-free survival in IM-treated GIST patients.Despite the presence of immune infiltration in many tumors, antitumor functions of lymphocytes are inhibited by the tumor microenvironment. Thus, we explored which signaling pathways were associated with NK cell inhibition in the tumor microenvironment. To do so, we performed a microarray from GIST infiltrating NK cells which highlighted the deleterious effect of TNFα/TNF Receptor 2/Baculoviral IAP Repeat Containing 3 (BIRC3)/TNF Receptor Associated Factor 1 (TRAF1) pathway on the function of NK cells. Next, we demonstrated that activation of this pathway in NK cells decreased gene transcription and protein expression of the activating receptor NKp46 (also called Natural Cytotoxicity Triggering Receptor 1 NCR1). This decrease positively correlated with NKp30c isoform expression. Moreover we showed that in mice, TNFα increases the metastatic dissemination of the NK sensitive tumor cell line, B16F10.Results from our research on NK cells strengthen the potential of NK cells as a therapeutic target for anti-tumor immunotherapy. Taken together, this thesis demonstrates the key role of the NKp30 receptor and its isoforms in the IM therapy as predictive marker in GIST response and describes for the first time a new NK cell inhibitory mechanism via the TNFα/TNFR2/BIRC3/TRAF1 pathway, paving the way for novel therapeutic strategies in cancer treatment.

During inflammatory responses, macrophages make and secrete cytokines, including the proinflammatory cytokine TNF-alpha (TNF). TNF is a highly potent activator of immune responses with pleiomorphic effects throughout the body. TNF is a key causative agent of chronic inflammatory diseases and is of an intense clinical interest as a therapeutic target. At the outset of this thesis, little was known about how macrophages secrete TNF. Notably, the pathways, carriers and molecules that regulate TNF secretion had not been characterised. A main goal of this work was to identify compartment and molecules involved in the intracellular trafficking of TNF. Live cell imaging of GFP-TNF was established and this provided novel and important new insights into trafficking. Both endogenous and GFP-tagged TNF were followed in macrophages using fluorescence microscopy. The trafficking of other molecules in macrophages was also studied. The major findings of this work include the identification of a new two-step secretory pathway for TNF and other proteins from the trans-Golgi Network (TGN) to the cell surface. This pathway goes via the recycling endosome as an intermediate station. Pleiotrophic tubular-vesicular carriers containing TNF bud off the TGN for the post-Golgi trafficking of TNF and their characterization both in live cell imaging and in biochemical analysis of isolated vesicles constituted the main parts of this work. Functional studies, including endosome inactivation and overexpression of Rab11 mutants (proteins functioning at the level of the recycling endosome) revealed that recycling endosomes have indeed an essential role in the exocytic trafficking of TNF in macrophages. This thesis also provides further insight into recycling endosomes as a possible intermediate step in the exocytic trafficking of several other proteins including the adhesion protein E-Cadherin, that function at the cell surface. Finally, the last chapter of this thesis examines endocytic pathways in activated macrophages. Assays for fluid phase endocytosis and receptor-mediated endocytosis were established and the regulation of both pathways was compared. The results show that LPS has opposite effects on fluid phase and receptor mediated endocytosis, decreasing and increasing their activity respectively. Recycling of transferrin through the recycling endosome was also measured, providing a link with studies on TNF exocytosis. Overall, the work in this thesis has made a major contribution to our understanding of TNF trafficking in macrophages, of macrophage pathways more generally and of trafficking at a fundamental level. The findings herein set the stage for more in depth analysis at a single molecular level to explore TNF regulation in normal and disease cells.

Small molecular Inhibitor of Apoptosis (IAP) antagonists, known as Smac mimetic compounds (SMCs), are a novel class of anti-cancer drugs currently undergoing clinical trials. SMCs were designed to mimic the function of the pro-apoptotic protein, Smac, which directly depletes cells of cIAP1 and cIAP2, and consequently renders tumour cells sensitive to death in the presence of proinflammatory ligands such as TNFα. The Korneluk lab recently reported that SMCs synergize with the attenuated oncolytic virus Vesicular stomatitis virus (VSVΔ51) by eliciting an enhanced immune response in mice, such that the combined therapy is vastly superior to stand-alone therapies. To improve on this SMC-mediated synergistic response, I generated variants of TNFα-armed VSVΔ51. Due to high ectopic expression of TNFα in infected cells, a five times lower viral dose of TNFα-armed VSVΔ51 combined with SMC treatment was sufficient to improve the survival rate as compared to SMC and VSVΔ51 co-therapy. This improved synergistic response is attributed to a bystander effect whereby the spread of TNFα from infected cells leads to the death of neighbouring, uninfected cells in the presence of a SMC. In addition, the double treatment induced vasculature collapse in solid tumours, revealing another mechanism by which cytokine-armed VSVΔ51 in combination with a SMC can induce cancer cell death. This approach demonstrates great potential for engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.