Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Este trabalho avaliou o efeito da dieta hiperlipídica em camundongos para o estudo da inflamação cardiovascular. Camundongos C57Bl/6 machos com 8 semanas de vida foram utilizados nos ensaios, sendo divididos nos grupos dieta controle e dieta hiperlipídica. Após 8 semanas foram avaliados: o ganho de peso, a porcentagem de tecido adiposo, pressão arterial sistólica, frequência cardíaca, perfil lipídico e glicêmico séricos. A partir de cortes histológicos de aortas e corações corados com picrossirius foram feitas análises morfométricas. Em cortes histológicos de aorta foram realizadas a análise fibras elásticas e colágenas usando a coloração de Weigert-Van Gieson. Também foram realizadas a quantificação de fibras colágenas em aortas, usando a coloração de picrossirius. Nos tecidos aórticos e cardíacos foram feitos: 1) Ensaios de atividade enzimática para ECA e MPO. 2) Ensaios de Immunoblotting para a detecção proteíca para ECA e TGF-b. Também foram feitos ensaios de imuno-histoquímica para marcação e localização de ECA e TGF-b no tecido aórtico. / This work evaluated the effect of high-fat diet in mice for the study of cardiovascular inflammation. C57BL / 6 mice at 8 weeks of age were used in the tests were divided in groups control diet and high fat diet. After 8 weeks were evaluated: weight gain, percentage of fat, systolic blood pressure, heart rate, serum lipids and glucose levels. From histological aortas and hearts stained with picrosirius morphometric analyzes. Histological sections of the aorta were performed to analyze elastic and collagen fibers using Weigert-Van Gieson staining. Also the quantification of collagen fibers in aortas using picrosirius staining. In aortic and cardiac tissues were made: 1) Enzymatic activity assays for ACE and MPO. 2) Immunoblotting assays to detect proteinous for ACE and TGF-b. Also were peformed Immunohistochemistry assays for marking and localization of ACE and TGF- b in the aortic tissue.

In order to effectively treat disorders whose pathology is marked by neovascularization, a better understanding of the pathways that mediate the processes involved in angiogenesis is needed. To this end we have identified two important pathways that regulate endothelial cell capillary morphogenesis, a key process in angiogenesis. We have identified the small GTPase RhoB as being induced by vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Depletion of RhoB inhibited endothelial cell VEGF - mediated migration, sprouting, and cord formation. Cells depleted of RhoB showed a marked increase in RhoA activation in response to VEGF. Defects in cord formation in RhoB - depleted cells could be partially restored through treatment with the Rho inhibitor C3 transferase or ROCK I/II inhibitors, indicating increased RhoA activity and enhanced downstream signaling from RhoA contribute to the phenotype of decreased cord formation observed in cells depleted of RhoB. Interestingly, we did not observe a significant change in RhoC activity in RhoB - depleted cells suggesting differential regulation of RhoA and RhoC by RhoB in HUVECs. We have also identified microRNA - 30b (miR - 30b) as being negatively regulated by VEGF and as being a negative regulator of HUVEC capillary morphogenesis. Overexpression of miR - 30b significantly reduced HUVEC cord formation in vitro, while inhibition of miR - 30b enhanced cord formation. Neither overexpression nor inhibition of miR - 30b affected migration or viability of endothelial cells. Interestingly, miR - 30b regulated the expression of TGFβ2 but not TGFβ1, with overexpression of miR - 30b inducing expression of TGFβ2 mRNA and protein, and inducing phosphorylaton of Smad2 , suggesting TGFβ2 produced in response to miR - 30b overexpression functions in an iii autocrine manner to stimulate HUVECs . MiR - 30b effects on TGFβ2 expression were found to be regulated to an extent by ATF2, as miR - 30b overexpressing cells exhibited increased levels of phosphorylated ATF2 , with depletion of ATF2 via siRNA resulting in inhibition of miR - 30b - induced TGFβ2 expression. Treatment of HUVECs with TGFβ2 inhibited cord formation, while TGFβ1 had no effect, indicating a major difference in how endothelial cells respond to these two related growth factors. Inhibition of TGFβ2 with a neutralizing antibody restored cord formation in miR - 30b overexpressing cells to levels similar to control cells, thus identifying TGFβ2 expression as contributing to the inhibitory effects of miR - 30b overexpression on capillary morphogenesis. Thus, we have identified two signaling pathways regulated by VEGF in HUVECs that further our understanding of the process of angiogenesis and may provide novel targets for therapeutic intervention into diseases involving angiogenesis.

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Les tumeurs solides emploient diverses stratégies afin de se maintenir dans l’organisme et d’échapper à l’inhibition du système immunitaire. Un des mécanismes les plus puissants est la production de la cytokine Transforming Growth Factor Beta (TGF-bêta). Cependant, cette cytokine est sécrétée dans le micro-environnement tumoral sous une forme inactive, incapable de se lier à son récepteur et donc d’exercer ses fonctions hautement immunosuppressives. Ces travaux de thèse démontrent qu’une population de lymphocytes T (LT) CD4+ dite T régulateurs (Tregs), qui exprime le facteur de transcription Forkhead box P3 (Foxp3), est responsable de l’activation du TGF-bêta au sein de la tumeur. Nous avons montré que parmi les cellules du système immunitaire, les Tregs constituent la principale population exprimant l’intégrine avb8 (Itgb8), protéine responsable de l’activation du TGF-bêta. L’absence de l’Itgb8 spécifiquement à la surface des Tregs entraîne une forte diminution de la croissance tumorale. Par conséquent, l’activation de la signalisation du TGF-bêta est réduite dans les LT CD8+ qui infiltrent la tumeur, conduisant à une exacerbation de leurs fonctions cytotoxiques et donc à une élimination accrue des cellules tumorales. La relevance de ces données obtenues chez la souris a été confirmée chez l’Homme à la fois par des approches ex vivo sur des tumeurs fraîches ainsi que par des approches bio-informatiques et biostatistiques à partir d’étude de cohortes de patients. Nous proposons donc que les Tregs et les cellules tumorales travaillent de concert pour fournir une source bio-active de TGF-bêta capable de réprimer efficacement la réponse immunitaire anti-tumorale et donc de permettre à la tumeur d’échapper au système immunitaire / Solid tumors employ diverse strategies to be maintained in the organism and escape the suppression mediated by the immune system. One of the most powerful mechanisms they use is through the production of Transforming Growth Factor Beta (TGF-beta). However, this cytokine is secreted within the tumor microenvironment in its inactive form, unable to bind to its receptor and exert its highly immunosuppressive functions. The present thesis project demonstrates that a population of CD4+ T lymphocytes called regulatory T cells (Tregs), which express the transcription factor Forkhead box P3 (Foxp3), is responsible for TGF-beta activation in tumors. We show that among the cells of the immune system, Tregs constitute the main population expressing the integrin avb8 (Itgb8) which is responsible for TGF-beta activation. The absence of Itgb8 specifically on Tregs surface leads to strong decrease of tumor growth. As a result, TGF-beta signaling pathway is impaired in tumor infiltrating CD8+ T lymphocytes leading to exacerbation of their cytotoxic and efficient elimination of tumor cells. The relevance of these data obtained in mice was confirmed in the human pathology by ex vivo approaches using fresh tumors as well as by bioinformatics and biostatistics approaches from studies on patient cohorts. We propose that Tregs and tumor cells cooperate to provide a bioactive source of TGF-beta which is able to efficiently repress the anti-tumor response and thus allowing tumors to escape the immune system

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Thirty five million people are currently living with HIV-1 today with women accounting for half of infected individuals globally. Sexual transmission is the main route of HIV transmission with approximately 40% of HIV infections occurring when the mucosal lining of the female genital tract (FGT) is exposed to HIV in semen from an infected male partner. Seminal plasma (SP), the fluid portion of semen, is a complex fluid which plays an immunomodulatory role in the FGT for successful conception, largely due to its high concentrations of TGF-β1. Several factors in SP from HIV-uninfected men have been shown to either inhibit or enhance HIV infection in target cells, however it is not clear how SP from HIV infected men would modulate genital epithelial cells (GECs), the first cells that encounter HIV in the FGT. The overall goals of this thesis were to compare inflammatory and regulatory cytokine concentrations in SP from HIV-uninfected and infected men, and subsequently compare GEC cytokine responses following exposure to SP from HIV-uninfected and HIV-infected men. I also investigated how SP and TGF-β regulated cytokine production and barrier function in GECs in the presence of HIV. The results summarized in this thesis demonstrated that HIV infection leads to different cytokine profiles in SP, based on stage of HIV-1 infection. HIV-infected men in acute stage contained higher levels of proinflammatory cytokines in their SP compared to HIV-uninfected and chronically infected men (CI men) which subsequently lead to higher levels of proinflammatory cytokines from GECs compared to CI men. In the follow up to this study we found that active TGF-β, which was found in higher concentrations in SP from CI men and led to decreased inflammatory response from GECs, was compartmentalized between blood plasma and seminal plasma. Higher levels of active TGF-β in SP correlated with decreased semen viral load and the immune activation marker sCD14 leading us to believe that ART-naive CI men in our cohort were naturally controlling their immune activation status, as active TGF-β levels were lower in ART-treated men. Short-term exposure of GECs to SP from CI men or TGF-β at comparable concentrations to SP protected the GEC barrier against HIV by decreasing inflammatory cytokines and preventing tight junction breakage. However, long-term exposure to TGF-β in the presence of HIV further increased inflammation in GECs suggesting a biphasic role for TGF-β in the FGT. This body of work summarized in this thesis demonstrates for the first time how semen from HIV-infected men modulates FGT epithelial cell cytokine responses and barrier function, an important consideration in the design of local therapeutic strategies to protect the FGT against HIV infection. / Thesis / Doctor of Philosophy (Medical Science)

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