Les Sémaphorines constituent une grande famille de protéines sécrétées et membranaires, qui ont été initialement impliquées dans le guidage axonal au cours du développement du système nerveux, mais qui jouent aussi un rôle important dans la cancérogenèse. Ainsi, les Sémaphorines agissent via leurs récepteurs, qui font partie de la famille des Neuropilines et des Plexines, pour réguler des fonctions multiples qui contribuent au développement des cancers et à la progression métastatique, telles que l'adhésion cellulaire, la motilité, la survie, l'angiogenèse et la réponse immunitaire. Mon travail de thèse a visé à mieux comprendre le rôle de la signalisation induite par la Sémaphorine-3E (Sema3E) et son récepteur Plexine-D1 dans le cancer du sein. Il a été proposé récemment, que la signalisation autocrine induite par le couple Sema3E/Pleixne-D1 dans les cellules tumorales, favorise l’apparition de métastases via l’augmentation de leurs capacités de migration et d’intravasation/extravasation.Dans ce travail de recherche, nous avons trouvé qu’une nouvelle signalisation autocrine induite par Sema3E/Plexine-D1 régit à la fois la tumorigenèse et la formation de métastases. Ainsi, la sécrétion de Sema3E par les cellules tumorales favorise leur survie grâce à l’inhibition d’une voie de mort induite par le récepteur à dépendance Plexine-D1. Bloquer l’interaction entre Sema3E et son récepteur Plexine-D1, via l’utilisation d’un ligand TRAP dans des modèles murins de cancer du sein, permet de tuer spécifiquement les cellules tumorales et exerce une action anti-tumorale et anti-métastatique. Des données préliminaires sur la voie de mort induite par le récepteur Plexine-D1, suggèrent un rôle important du clivage de son domaine intracellulaire par les Caspases et/ou une translocation nucléaire. Ensemble, ces résultats indiquent que l’expression de Sema3E dans les cancers du sein chez la femme, permet un avantage sélectif pour la survie des cellules tumorales. Bloquer l’interaction entre Sema3E/Plexine-D1 apparaît donc comme une stratégie thérapeutique prometteuse pour traiter ces cancers. De plus, ces données permettent une nouvelle approche dans l’étude des fonctions du couple Sema3E/Plexine-D1 dans d’autres contextes physiologiques et pathologiques. / The Semaphorins constitute a large family of secreted and membrane-bound proteins, which have initially been implicated in axon guidance during development of the nervous system, but also have an important role in cancer. Semaphorins signal through Neuropilin and Plexin receptors to regulate multiple functions contributing to cancer development and progression, including cell adhesion and motility, cell survival, angiogenesis and immune response. My work is aimed at better understanding the role of the secreted glycoprotein Semaphorin 3E (Sema3E) and its binding receptor PlexinD1 in breast cancer. It has been recently proposed that Sema3E/PlexinD1 autocrine signaling in tumor cells promotes metastasis by enhancing cancer cell migration and intravasation/extravasion steps. Here we found another mechanism by which Sema3E/PlexinD1 autocrine signaling regulates both tumorigenesis and metastasis. We show that release of Sema3E by tumor cells promoted their survival through inhibition of an endogenous cell death pathway triggered by the unbound PlexinD1 “dependence receptor”. Interrupting Sema3E/PlexinD1 binding using a ligand-TRAP for PlexinD1 receptor specifically increased tumor cell death and exerted anti-tumoral and anti-metastatic activities in animal models of breast cancers. Preliminary investigations of the underlying cell death pathway suggested a crucial role of PlexinD1 intracellular domain via its cleavage by caspases or its possible translocation to the nucleus. Together, the results indicate that up-regulation of Sema3E in human breast cancers provides a selective advantage for tumor cell survival and that antagonizing Sema3E binding to PlexinD1 may represent a promising therapeutic strategy. Moreover, these data may provide new insight into the functions of Sema3E/PlexinD1 in other physiological and pathological contexts.

Les maladies allergiques sont en constante augmentation tant en prévalence qu’en gravité. Les éosinophiles sont fortement impliqués dans le dommage et le dysfonctionnement tissulaire et participent à l’entretien de l’inflammation allergique. Différentes cellules de l’immunité innée sont impliquées dans le contrôle de la réaction allergique. Parmi elles, les cellules NK, connues essentiellement pour leurs fonctions anti-tumorales et anti-microbiennes, pourraient réguler différents aspects de la réaction. Dans le sang périphérique de patients asthmatiques, les cellules NK présentent des capacités cytotoxiques accrues, ainsi qu’une prédominance de cellules NK2 comparativement à la prédominance de cellules NK1 chez les sujets non allergiques. Chez des patients atteints de dermatite atopique, le nombre et la cytotoxicité des cellules NK périphériques sont diminués, ainsi que leur capacité à produire de l’IFN-g. De plus, le dialogue entre les cellules NK et les cellules dendritiques est moins efficace chez le sujet asthmatique, menant ainsi à une capacité réduite de production d’IFN-g par les cellules NK. Dans des modèles murins d’inflammation pulmonaire, la déplétion en cellules NK par l’anti-NK1.1 ou l’anti-ASGM1 avant l’immunisation inhibe l’éosinophilie pulmonaire, l’infiltrat des LT CD3+ et l’augmentation des taux d’IL-4, IL-5 et IL-12 dans le LBA. Néanmoins, la déplétion avec l’anti-ASGM1 après l’établissement de l’inflammation éosinophilique retarde sa résolution, suggérant un rôle double des cellules NK dans l’inflammation allergique. Le recrutement et la fonction des cellules NK humaines dans l’allergie par le biais de l’analyse in vitro du rôle de CCL18 sur les cellules NK a été analysé. Cette chimiokine est préférentiellement produite au niveau du poumon et possède une double fonction dans la pathologie allergique puisqu’elle recrute les LTh2, mais également les LT reg et génère des DCs tolérogènes capables d’induire des LT reg, uniquement chez des donneurs non allergiques. Nous avons évalué la réponse des cellules NK de sujets allergiques vis-à-vis de CCL18 et l’avons comparée à celle de cellules NK provenant de sujets non allergiques. Nos travaux ont montré que CCL18 attire in vitro les cellules NK de sujets non allergiques et induit leur cytotoxicité, de façon dépendante des protéines G. Par contre, les cellules NK de sujets allergiques ne répondent pas au CCL18. La deuxième partie du travail s’est basée sur l’hypothèse d’un dialogue entre les cellules NK et les éosinophiles qui modifierait leurs fonctions respectives. Des cellules NK et des éosinophiles autologues ont été cocultivés pendant 3 et 12h, à différents ratios. Nous avons montré que les cellules NK activent directement les éosinophiles comme en témoignent l’augmentation de la libération de l’ECP, l’EDN, et de l’expression du CD63, du CD69 et la diminution de l’expression du CD62L sur les éosinophiles. De plus, les cellules NK induisent l’apoptose et la mortalité des éosinophiles dès la première heure de coculture. Cependant l’apoptose et la mortalité des cellules NK ne sont pas modifiées. La fixation des cellules NK empêche presque totalement l’activation et l’apoptose des éosinophiles, suggérant l’implication de molécules de surface et peut être de facteurs solubles. Les interactions entre molécules de surface restent à déterminer, et l’IFN-g et le TGF-β ne sont pas impliqués. Cependant, les voies de signalisation p38MAPkinase, ERK, JNK et PI3kinase interviennent dans l’activation des éosinophiles. La voie mitochondriale et ROS sont impliquées dans l’apoptose des éosinophiles induite par les cellules NK.En résumé, ces travaux ont permis de montrer que les cellules NK de sujets allergiques présentent un dysfonctionnement dans la réponse vis-à-vis de CCL18 comparativement aux sujets non-allergiques. De plus, nos résultats suggèrent que les cellules NK pourraient réguler l’inflammation à éosinophiles en induisant leur activation et/ou leur apoptose. / Allergic diseases are steadily increasing both in prevalence and severity. Known physiopathological mechanisms involve the induction of a Th2 response by dendritic cells, leading to IgE production and inflammation, in particular linked to the recruitment of eosinophils. Eosinophils are heavily involved in injury and tissue dysfunction and contribute to the maintenance of inflammation. Different cells of innate immunity were shown to be involved in the control of allergic reaction. Among them, (NK) cells, primarily known for their anti-tumor and anti-microbial functions, may regulate different aspects of allergic reaction as suggested by studies in humans or mice. In the peripheral blood of patients with asthma, NK cells exhibit increased cytotoxic capacity, and a predominance of NK2 cells compared to the prevalence of NK1 cells in non-allergic subjects. In patients with atopic dermatitis, the number and cytotoxicity of peripheral NK cells are reduced, as well as their ability to produce IFN-g. Moreover, the dialogue between NK cells and dendritic cells is less effective in asthmatic patients, leading to a reduced capacity of IFN-g production by NK cells. In murine models of pulmonary inflammation, depletion of NK cells by anti-NK1.1 or anti-ASGM1 before immunization inhibits pulmonary eosinophilia, the infiltration of CD3+ T cells and increased levels of IL-4, IL-5 and IL-12 in the bronchoalveolar lavage. However, depletion with anti-ASGM1 after the establishment of eosinophilic inflammation delays its resolution, suggesting a dual role of NK cells in allergic inflammation.We studied the recruitment and function of human NK cells in allergy through in vitro analysis of the role of CCL18 on NK cells. This chemokine is preferentially produced in the lungs and has a dual role in allergic diseases since it recruits Th2 cells but also regulatory T cells and generates tolerogenic DCs capable of inducing regulatory T cells only from non-allergic donors. We evaluated the response of NK cells in allergic subjects towards CCL18 and compared it to that of NK cells from non-allergic donors. We showed that CCL18 attracts NK cells from non-allergic subjects and induces their cytotoxicity in a G protein dependent pathway. However, NK cells from allergic subjects did not respond to CCL18. This chemokine has no effect on the proliferation of NK cells, but may negatively regulate IFN-g production.The second part of the thesis is based on the hypothesis of a dialogue between NK cells and eosinophils which would modify their respective functions. NK cells and autologous eosinophils were cocultured during 3 and 12 hours, at different ratios. We showed that NK cells directly activate eosinophils as evidenced by the increased release of ECP, eosinophil derived neurotoxin EDN, and the expression of CD63, CD69, and reduced expression of CD62L on living eosinophils. In addition, coculture with NK cells induced apoptosis and mortality of eosinophils in the first hours of coculture. However, apoptosis and death of NK cells were not changed. Fixation of NK cells prevented almost completely the activation and apoptosis of eosinophils, suggesting the involvement of surface molecules, however soluble factors cannot be excluded. These interactions require cell contact, but the molecules involved remain to be determined. Concerning soluble factors, IFN-g and TGF-β are not involved in these mechanisms. However, the signaling pathways p38MAPkinase, ERK, JNK and PI3-kinase are involved in eosinophils activation. Concerning eosinophil apoptosis induced by NK cells, the mitochondrial pathway is more involved than the caspase pathway.In summary, our studies show that NK cells from allergic patients exhibit a defect in their response towards CCL18 compared to non-allergic subjects. In addition, these results suggest that NK cells may regulate eosinophilic inflammation by inducing their activation and / or apoptosis.

L’infarctus du myocarde (IDM) est une cause majeure de mortalité dans le monde. La stratégie thérapeutique actuelle repose sur la reperfusion précoce du myocarde qui contribue largement à l’amélioration du pronostic des malades. Les investigations menées chez des modèles d’ischémie/reperfusion (I/R) cardiaque ont montré que l’apoptose des cardiomyocytes était contrôlée en partie par les protéines de type Bcl-2. La production de radicaux libres (RL), en particulier par la nicotinamide adénine dinucléotide phosphate (NADPH) oxydase, contribue aussi à l’altération de la fonction cardiaque. Récemment, les effets observés de l’érythropoïétine (Epo) sur l’I/R, sont principalement liés à ses effets anti-apoptotiques, anti-inflammatoires et à son rôle dans l’angiogénèse et le remodelage vasculaire. Ces propriétés suggèrent le potentiel de l’Epo dans la cardioprotection suite à un IDM. Dès lors, les objectifs de cette thèse ont été : 1) de confirmer les effets cardioprotecteurs d’un analogue de l’Epo, la darbepoétine-a (DA), chez un modèle d’I/R chez le rat; 2) d’étudier les voies de signalisation impliquées dans ses effets anti-apoptotiques et ; 3) de caractériser les effets antioxydants de la DA médiée par l’hème-oxygénase-1 (HO-1), et ; d’étudier le rôle de la NADPH oxydase. Dans la 1ère étude, le traitement par DA a permis de diminuer significativement la taille de l’infarctus, la production de RL et l’apoptose. La DA a activé la voie des protéines Jak2/Akt, augmenté l’expression des protéines P-Bad et P-GSK3ß et anti-apoptotiques, Bcl-2 et Bcl-xL. Ces mêmes effets bénéfiques ont été confirmés à plus long terme avec la réduction des lésions fibrotiques et l’augmentation du nombre da capillaires sanguins, suggérant une meilleure perfusion du ventricule gauche. La seconde étude, a confirmé les effets cardioprotecteurs de la DA. Parallèlement, la DA a induit l’expression et l’activité de l’HO-1 et régulé l’expression des sous-unités p47phox et Rac1 nécessaires à l’activation de la NADPH oxydase. Ces résultats sont concordants avec la baisse de la production des RL observés dans le groupe DA. Les effets bénéfiques de la DA ont été annihilés en présence de ZnPP, inhibiteur de l’HO-1. Des essais cliniques sont actuellement en cours pour démontrer que les bénéfices observés chez l’animal, peuvent être retrouvés chez l’homme. / Cardiovascular disease remains a leading cause of mortality in industrialized countries. Loss of cardiomyocytes via apoptosis is believed to contribute to the continuous decline of the ventricular function in heart failure. Several investigations revealed that following ischemia-reperfusion (I/R), cardiomyocytes apoptosis is controlled, at least, by the Bcl-2 proteins family members. The excessive reactive oxygen species (ROS) production, through NADPH oxidase, contributes also to cellular damages and death. Recently, erythropoietin (EPO), a hematopoietic cytokine, has been shown to protect heart exposed to ischemia or ischemia-reperfusion, limiting infarct size and cardiac remodeling. However, to date the precise cellular mechanism of DA-induced cardioprotection remains incompletely understood. Thus, the aims of this work were 1) to assess the short and long term cardioprotective effects of darbepoetin-a (DA), an Epo analog, in an in vivo rat model of I/R ; 2) to investigate the signaling pathway through which DA potentially limits apoptosis and ; 3) to elucidate whether its cardioprotective effect, and more particularly its antioxidative effect, is linked to an HO-1-dependent inhibition of the NADPH activity. In the first study, left ventricle infarct size (LV) was smaller than that in the control rats, in agreement with echocardiographics parameters. DA-treatment activated the JAK2/Akt signaling pathway, lowered cleaved caspase-3 and increased both P-Bad and P-GSK-3ß proteins. This was consistent with the decrease of ROS production and the lowered binding of Bad to Bcl-xL and Bcl-. Similarly, in long term study, histology alterations implicated lower LV cardiac fibrosis and greater capillary density; furthermore both Bcl-xL and Bcl-2 were upregulated. In the second study, both LVSF and LVEF were higher versus control and DA+ZnPP, a heme-oxygenase-1 (HO-1) inhibitor, matching with the decreased LV infarct size in DA rats. DA induced HO-1 and down regulated the expression of p47phox and the activation of Rac1, both regulatory subunits of the NADPH-oxidase. This was consistent with the decrease of ROS production and these DA effects were inhibited by ZnPP. Further experiments in humans are now required to prove benefits effects of DA and to promote the use of EPO as therapeutics in heart infarction.

Aujourd'hui, le cancer est devenu un problème majeur de santé publique avec 12,7 millions de nouveaux cas de cancer et 7,6 millions de décès enregistrés en 2008. Même si le nombre des personnes qui guérissent augmente, les décès sont toujours importants. Les raisons, malgré un diagnostic précoce et correct, sont l'absence de traitements efficaces et l'émergence des résistances à la thérapie anticancéreuse. C'est pourquoi les chercheurs s'intéressent aux nouvelles approches pour développer des traitements puissants et sélectifs pour vaincre le cancer. Dans notre première approche, nous avons développé une série de dérivés de composés naturels appelés aurones. Les aurones jouent un rôle important dans la pigmentation jaune lumineuse de certaines fleurs et certains fruits et montrent des nombreuses activités biologiques. Nous avons combiné le motif benzofuranone des aurones avec d'autres motifs de la coumarine et chromone inspirées par la nature. Ces nouveaux composés montrent une activité anticancéreuse prometteuse, car ils sont capables de bloquer le cycle cellulaire dans les cellules cancéreuses K562 et peuvent y induire l'apoptose. Ils sont donc un motif intéressant pour un développement ultérieur de recherches. Ensuite nous avons concentré notre attention sur un objectif épigénétique. Les méthyltransférases d'ADN sont considérées comme une cible intéressante en oncologie. L'usage d'inhibiteurs spécifiques de la méthyltransferase d'ADN (DNMTi) pourrait réactiver les gènes suppresseurs de tumeurs et induire la reprogrammation des cellules cancéreuses, conduisant à l'arrêt de leur prolifération et finalement à leur mort. Nous avons amélioré le composé connu SGI1027 par modification de la structure. Nous avons obtenu des nouveaux inhibiteurs de la méthyltransferase d'ADN non- nucléosidiques, plus puissants et plus sélectifs que le composé principal. L'activité anti-cancéreuse de nos composés quinoléiniques et pyrimidiniques est testée sur différentes lignées cellulaires de cancer indiquant leur future utilisation possible dans un traitement anti-cancéreux puissant et sélectif. Une troisième série d'analogues de curcuminoïdes à base de coumarine a été préparée et testée pour sa capacité potentielle à moduler la voie TNF-alpha induit par NF-kB dans les cellules cancéreuses K562. Cependant, nous n'avons pas été capable de montrer l'implication de la voie ciblée jusqu'à maintenant. Des études complémentaires et plus approfondies doivent être menées afin d'estimer les propriétés biologiques de ces composés dans la participation éventuelle à différentes voies / Today, cancer is becoming a major public health problem with 12.7 million new cancer cases and 7.6 million cancer deaths registered in 2008. Although the number of people cured of cancer is increasing, people still die because of cancer. The reasons, besides an early and correct diagnosis, are the lack of effective treatments and the emergence of drug-resistant cancers. Therefore, researchers are interested in new approaches to develop potent and selective therapies to fight cancer. To start with, we developed a series of natural compound derivatives related to aurones. Aurones play an important role in the bright yellow pigmentation of some flowers and fruits exhibiting a strong and broad variety of biological activities. We combined the benzofuranone motif of the aurone with other coumarin and chromone motifs inspired by nature. These new compounds displayed spromising anticancer activity because they are able to block the cell cycle in K562 cancer cells and are able to induce apoptosis being an interesting scaffold for further development. Secondly, we focused on an epigenetic target. DNA methyltransferases are considered as an interesting target in Oncology. The use of specific inhibitors of DNMT (DNMTi) might reactivate tumor suppressor genes and induce the reprogramming of cancer cells, leading to their proliferation arrest and ultimately to their death. We improved the known compound SGI1027 through structure modification leading to novel non-nucleoside DNMT inhibitors, more potent and more selective than the lead compound. The anticancer activity of our quinoline and pyrimidine based compounds - tested in different cancer cell lines - suggests their use as possible potent and selective future cancer therapy. A third series of coumarin-based curcuminoid analogues were prepared and tested for their potential ability to modulate the TNF-alpha induced NF-kB pathway in K562 cancer cells. However we were not able to demonstrate the involvement of the targeted pathway so far. Complementary and deeper investigations need to be conducted in order to elicit deeper biological properties of these compounds with the possible involvement of different pathways

Les protéines receptor-interacting protein kinase 1 (RIP1) et RIP3 ont été identifiées comme intervenant dans la régulation de la mort cellulaire apoptotique ou nécroptotique mais également dans la survie cellulaire. Ces deux protéines possèdent un domaine sérine/thréonine kinase, un domaine d’interaction spécifique RHIM (RIP homotypic interacting motif) et diffèrent dans leur domaine C-terminal car seule RIP1 possède un domaine de mort. Ces protéines font partie d’un ensemble de protéines régulatrices nommé ripoptosome. Des études ont montré une altération du ripoptosome dans les leucémies lymphoïdes chroniques (LLC) et les leucémies aigües lymphoïdes (LAL). Nous nous sommes intéressés aux leucémies aigües myéloïdes (LAM). L’analyse de l’expression des protéines RIP1 et RIP3 a été réalisée dans des blastes triées CD34+ de patients atteints de LAM ou dans des cellules CD34+ de donneurs sains en Q-RT-PCR. Les premières analyses montrent que RIP3 est significativement sous-exprimée chez les patients atteints de LAM en comparaison avec les cellules CD34+ issues de donneurs sains. Aucune différence n’a été mise en évidence pour l’expression de RIP1 dans les deux types de cellules CD34+. Afin de comprendre l’implication de l'extinction de RIP3 dans les LAM, nous avons étudié sa réexpression dans une lignée cellulaire leucémique murine (DA1-3b) où RIP3 n’est pas exprimée par métylation de son promoteur, au moyen d'un système d’expression conditionnelle (LacSwith II, IPTG). Après 10h d’induction de l’expression, on constate que la protéine RIP3 sauvage (RIP3-WT) induit une apoptose dans les cellules DA1-3b. Afin de déterminer l’implication des domaines de RIP3, nous avons utilisé une protéine mutante kinase Dead (RIP3-KD, activité kinase abolie) et une protéine mutante dans la séquence d’interaction spécifique avec RIP1 (RIP3-RHIM). L’analyse de la mortalité cellulaire en cytométrie en flux et en microscopie électronique montre que les protéines RIP3-WT et -KD induisent toutes les deux la mort apoptotique des cellules DA1-3b respectivement de 15% et de 50% après 10h d’expression. On constate donc que la protéine RIP3-KD induit une mort plus importante et plus précoce que la protéine sauvage. La protéine RIP3 mutée dans son domaine RHIM ne peut plus induire de mort cellulaire. Il semble donc que le domaine kinase de RIP3 jouerait un rôle régulateur dans la mort cellulaire induite par RIP3. L’utilisation du modèle de leucémie murine DA1-3b a permis de réaliser un étude in vivo de l’expression conditionnelle de RIP3-WT et -KD. Seule l'expression de RIP3-KD est capable de prolonger significativement la survie des souris.De plus, il a été démontré que RIP3 pouvait également induire la nécroptose dans les cellules lorsque l’apoptose ne peut aboutir, notament lorsque les caspases sont inhibées à l’aide d’un inhibiteur de pan-caspases le Z-VAD-FMK. Le traitement des cellules exprimant RIP3-WT par 50µM de Z-VAD-FMK induit une plus forte mortalité (45%) des cellules tandis que dans les cellules exprimant RIP3-KD, l’inhibiteur des caspases inhibe complètement le processus apoptotique et permet la survie des cellules (10%). Une étude en microscopie électronique a permis de déterminer que la présence de Z-VAD-FMK induit un switch de l’apoptose vers la nécroptose. Il semble donc que le domaine de kinase possède un rôle important dans la signalisation de la nécroptose car la protéine RIP3-KD n’est plus capable d’initier le switch entre l’apoptose et la nécroptose. Quelques données préliminaires semblent indiquer que les calpaïnes ainsi que la caspase 12 pourraient également être impliquées dans la balance apoptose/nécroptose. [...] / The receptor-interacting protein kinase 1 (RIP1) and 3 (RIP3) are key signaling molecules in the regulation of apoptotic cell death or in the execution of a specific instance of regulated necrosis, named necroptosis, as well as in cell survival processes. These proteins have in common a serine/threonine kinase domain and a specific interacting motif RHIM (RIP homotypic interacting motif), while they differ in their C-terminal domain, as only RIP1 is characterized by a death domain. They belong to a family of regulatory proteins forming a cell death-inducing platform, referred to as Ripoptosome. Previous studies showed that the ripoptosome was altered in chronic lymphoid leukemia (CLL) and acute lymphoid leukemia (ALL). We decided to focus our studies on acute myeloid leukemia (AML).Expression profile of RIP1 and RIP3 was established by Q-RT PCR on CD34+ sorted cells of AML patients or healthy donors. Our first results show that if RIP3 is significantly under expressed in CD34+ cells of AML patients compared to healthy donors, there was no difference in RIP1 expression pattern in both cell types.To further understand the functional relevance of RIP3 down-regulation in the leukemia, we used a murine leukemic cell line (DA1-3b) in which RIP3 promoter is methylated, inhibiting its expression.A conditional expression system in DA1-3b cells has been realized (LacSwith II). IPTG (Isopropyl-beta-D-thiogalactoside) treatment (1mM) allows expression of the proteins of interest in these cell lines. We noticed that, 10 hours after expression’s induction, the wild-type RIP3 protein (RIP3-WT) induces apotptosis in DA1-3b cells. In order to decipher the role of each RIP3 domains in this cell-death-induced phenomenon, several mutants were employed. We used a mutant protein with a non functional kinase domain, RIP3 Kinase Dead (KD) and a mutant unable to interact with RIP1, RIP1-RHIM. Cell death analysis, performed by flow cytometry and by electron microscopy, shows that both RIP3-WT and RIP3-KD induce apoptosis in DA1-3b cells (respectively 15% and 50%) after 10 hrs of expression. However, these results show that RIP3-KD induces a stronger and more rapid cell-death than the wild-type protein. In agrement with previous findings, we found that the protein mutated in the RHIM domain cannot engage a cell-death process. Our results thereby suggest that the RIP3 kinase domain palys a major regulatory role in cell-death.Taking advantage of a mouse model of leukemia, we also performed an in vivo study of conditional expression RIP3-WT and –KD. As a matter of fact, DA1-3b cells, obtained from C3H mice are able to induce a leukemia in these mice after transplantation. We have shown that the inducible expression of RIP3-KD in DA1-3b cells can increase significatively mice survival. Moreover, we have shown that, in certain conditions, RIP3 could also induce necroptosis when apoptosis is prevented by a pan-caspase inhibitor, Z-VAD-FMK. Treating RIP3-WT expressing cells with 50µM of Z-VAD-FMK induces a strong mortality (45%) while it inhibits totally the apoptotic process in RIP3-KD expressing cells, allowing cell survival (10%).Using electronic microscopy, we were able to demonstrate that the use of Z-VAD-FMK leads to a switch from apoptosis to necroptosis. Thus, as the kinase dead protein is not able to induce this switch, the kinase domain could play an important role in necroptotic signaling. Preliminary results suggest that calpains, as well as caspase 12, could also be involved in apoptosis/necroptosis balance. It has been shown that RIP1 and RIP3 could play a role in Nf-kB pathway regulation. Studying the effects of RIP3-KD expression on Nf-kB pathway, we were able to demonstrate that it led to a specific cleavage of Nf-kB p65 protein, forming two fragments of about 25 kDa and 40 kDa. [...]

Le récepteur à neurotrophine TrkC a été identifié comme étant un récepteur à dépendance : en l'absence de son ligand NT-3, il déclenche l'apoptose. En effet, la survie des cellules qui expriment ces récepteurs dépend de la disponibilité en ligand, un mécanisme qui inhibe la prolifération incontrôlée et la migration des cellules tumorales. TrkC, en tant que récepteur à tyrosine kinase, est généralement considéré comme un proto-oncogène. Or nous montrons que l'expression TrkC est diminuée dans une grande fraction des cancers colorectaux humains, principalement par méthylation du promoteur de TrkC. En outre, ce mécanisme confère un avantage sélectif aux lignées cellulaires colorectales pour inhiber la mort des cellules tumorales. De plus, la réexpression de TrkC dans les lignées tumorales colorectales est associée à la mort des cellules tumorales et à l'inhibition in vitro des caractéristiques de transformation cellulaire, et in vivo de la croissance tumorale. Ensemble, ces données permettent de conclure que TrkC est un gène suppresseur de tumeur dans le cancer colorectal. Le mécanisme moléculaire par lequel TrkC déclenche l'apoptose implique le clivage de son domaine intracellulaire, ce qui libère un fragment pro-apoptotique (TrkC KF). Nous montrons que TrkC KF interagit avec Cobra1, un cofacteur de BRCA1, et que Cobra1 est nécessaire à l'apoptose induite par TrkC. Cobra1 conduit TrkC KF à la mitochondrie, où il favorise l'apoptose apoptosome-dépendante. Ainsi, nous proposons qu'en l'absence de NT-3, le clivage protéolytique de TrkC conduit à la libération d'un fragment tueur qui déclenche l'apoptose mitochondriale, via le recrutement de Cobra1 / The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. Indeed cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC, as a classic tyrosine kinase receptor, is generally considered to be a proto-oncogene. We show that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor. TrkC triggers apoptosis in the absence of its ligand NT-3 : the molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic fragment (TrkC KF). We show that TrkC KF interfacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1

Non-canonical bioenergetics concerns with those physiological and pathophysiological situations under which ATP synthesis is suppressed. This thesis brings an outcome of three types of studies within the field of the non-canonical bioenergetics, investigating specific bioenergetic phenotypes of cancer cells, on one hand; and a role of mitochondrial uncoupling proteins as deduced from their transcript distribution in various tissues and organs; plus a role of a novel and likely pro-apoptotic factor CIDEa in mitochondria. Cancer cells generally present abnormal bioenergetic properties including an elevated glucose uptake, a high glycolysis and a poorly efficient oxidative phosphorylation system. However, the determinants of cancer cells metabolic reprogramming remain unknown. The main question in this project was how environmental conditions in vivo can influence functioning of mitochondrial OXPHOS, because details of mitochondrial bioenergetics of cancer cells is poorly documented. We have combined two conditions, namely glucose and oxygen deprivation, to measure their potential interaction. We examined the impact of glucose deprivation and oxygen deprivation on cell survival, overall bioenergetics and OXPHOS protein expression. As a model, we have chosen a human breast carcinoma (HTB-126) and appropriate control (HTB-125) cultured cells, as large fraction of breast malignancies exhibit hypoxic tumor regions with low oxygen concentrations and poor glucose delivery. The results demonstrate that glucose presence or absence largely influence functioning of mitochochondrial oxidative phosphorylation. The level of mitochondrial respiration capacity is regulated by glucose; by Crabtree effect, by energy substrate channeling towards anabolic pathways that support cell growth and by mitochondrial biogenesis pathways. Both oxygen deprivation and glucose deprivation can remodel the OXPHOS system, albeit in opposite directions. As an adaptative response to hypoxia, glucose inhibits mitochondrial oxidative phosphorylation to the larger extent than in normoxia. We concluded that the energy profile of cancer cells can be determined by specific balance between two main environmental stresses, glucose and oxygen deprivation. Thus, variability of intratumoral environment might explain the variability of cancer cells´ bioenergetic profile. Mitochondrial uncoupling proteins are proteins of inner mitochondrial membrane that uncouple respiration from ATP synthesis by their protonophoric activity. Originally determined tissue distribution seems to be invalid, since novel findings show that UCP1 is not restricted exclusively to brown fat and that originally considered brain-specific isoforms UCP4 and UCP5 might have wider tissue distribution. Hence, in second part of this thesis, I discuss consequences of findings of UCPn transcripts in the studied mouse and rat tissues. We have shown that mRNA of UCPn varies up to four orders of magnitude in rat and mouse tissues with highest expression in rat spleen, rat and mouse lung, and rat heart. Levels of the same order of magnitude were found for UCP3 mRNA in rat 100 and mouse skeletal muscle, for UCP4 and UCP5 mRNA in mouse brain, and for UCP2 and UCP5 mRNA in mouse white adipose tissue. Further, we have shown that expression pattern of UCPn varies between animal species, rat versus mouse, such as the dominance of UCP3/UCP5 vs. UCP2 transcript in mouse heart and vice versa in rat heart; or UCP2 (UCP5) dominance in rat brain contrary to 10-fold higher UCP4 and UCP5 dominance in mouse brain. spontaneous apoptosis due to CIDEa overexpression in HeLa cells, adapted for a tetracycline-inducible CIDEa expression, a portion of mitochondria-localized CIDEa molecules migrates to cytosol or nucleus. / Résumé non disponible

Orientador: Márcia Marinho / Coorientador: Flávia Lombardi Lopes / Banca: Helio Langoni / Banca: Simone Baldini Lucheis / Banca: José Fernando Garcia / Banca: Flávia Resende Eugênio / Resumo: O objetivo do presente trabalho foi verificar a dinâmica da resposta imune celular in vitro utilizando-se cultivos de macrófagos de camundongos (J774A.1) quando expostos às amostras virulenta, atenuada e saprófita de Leptospira, observados nos períodos de 1h, 3h, 6h, 12h, 24h, 36, 48h e 72h após a exposição. Foram realizados ensaios que determinaram a produção de intermediários reativos do nitrogênio (NO), expressão de genes para citocinas e interleucinas, pela técnica de reação em cadeia de polimerase (PCR) e a presença e quantificação das mesmas no sobrenadante do cultivo celular pelo teste ensaio imunoenzimático (ELISA). Avaliam-se as diferenças na modulação da expressão gênica do hospedeiro por cepas de Leptospira com variados graus de virulência, pelo método do microarranjo. Os resultados demonstraram que as amostras virulenta e atenuada proporcionaram um estímulo maior aos macrófagos em comparação à amostra saprófita, principalmente na fase tardia da infecção, considerando-se os valores expressos por óxido nítrico (NO), óxido nítrico sintase endotelial (eNOS) e induzido (iNOS). Os valores, embora estatisticamente não significativos, apresentaram uma dinâmica maior à resposta de citocinas (TNF-α, IL-1β) pela amostra virulenta em comparação à atenuada e saprófita. Outro fator relevante foi o encontro de Caspase-3 e -8 na fase inicial da infecção, sugerindo que a apoptose dos macrófagos ocorreu no começo do processo, logo após a internalização das Leptospiras, atingindo ní... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the present work was verify the dynamic of the cellular immune response in vitro using macrophages cultures of mice (J774A.1) when exposed to virulent, attenuated and saprophytic Leptospira samples, observed in eight time intervals after exposure: 1h, 3h, 6h, 12h, 24h, 36, 48h and 72h. The assays that determined the production of reactive nitrogen intermediates (NO), the expression of genes for cytokines and interleukins by polymerase chain reaction (PCR) and the presence and quantification of them in the supernatant of the cell culture were performed by the assay ELISA. In this context, we aimed to evaluate the differences in modulation of host gene expression by Leptospira strains with varied virulence degrees, using the microarray method. The results demonstrated that the virulent and attenuated samples provided a greater stimulus to the macrophages compared to the saprophyte sample, especially in the late phase of the infection, considering the values expressed by nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and induced (iNOS). The values, although statistically insignificant, presented a greater dynamics to the cytokine response (TNF-α, IL-1β) by the virulent sample compared to the attenuated and saprophyte. Another important factor was the finding of Caspase-3 and -8 in the initial phase of the infection, suggesting that macrophage apoptosis occurred early in the process, soon after Leptospiras internalization, reaching high levels at 24 ... (Complete abstract click electronic access below) / Doutor

Orientador: Tereza Cristina Cardoso da Silva / Resumo: Coronavirus são RNA vírus sentido positivo, envelopados, comumente associados a infecções brandas em aves e mamíferos. A infecção por CCoV é comum em cães jovens, principalmente em animais que vivem em canis e abrigos, associada à ocorrência de diarreia branda e autolimitante, causada pela infecção das células das vilosidades do intestino delgado. São conhecidos dois genótipos: CCoV-I e CCoV-II, o qual é subdividido em CCoV-IIa e CCoV-IIb. O CCoV-IIa, é uma variante altamente patogênica associada à doença sistêmica e acentuada linfopenia. Diferentemente de outros CCoV realiza viremia e, assim, determina a disseminação do vírus para diversos órgãos, incluindo tecidos linfóides. Nesse sentido, o envolvimento da infecção de macrófagos correlacionada à gravidade da doença e linfopenia, vem sendo sugerido. Este trabalho teve por objetivo promover a infecção de macrófagos caninos derivados de monócitos sanguíneos e avaliar a replicação viral, despolarização da membrana mitocondrial e os complexos da cadeia respiratória mitocondrial às 6, 12, 18 e 24 horas pós-infecção. A estatística descritiva incluiu média ± desvio padrão (s.d.). As médias foram comparadas através da análise de variância, ANOVA. Foi possível observar que a infecção por CCoV induziu a liberação de novas partículas virais entre 18 e 24 horas pós-infecção. Ainda, a infecção viral esteve associada à despolarização e disfunção da membrana mitocondrial, afetando o complexo III da cadeia respiratória. Desse modo, acredit... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coronaviruses are enveloped positive-sense RNA viruses commonly associated with mild infections in birds and mammals. CCoV infection is common in young dogs, especially kennel and shelter animals, associated with the occurrence of mild, self-limiting diarrhea caused by infection of small intestinal villus cells. Two genotypes are known: CCoV-I and CCoV-II, which is subdivided into CCoV-IIa and CCoV-IIb. CCoV-IIa is a highly pathogenic variant associated with systemic disease and marked lymphopenia. Unlike other CCoV it carries viremia and thus determines the spread of the virus to various organs including lymphoid tissues. In this sense, the involvement of macrophage infection correlated with disease severity and lymphopenia has been suggested. This study aimed to promote the infection of canine macrophages derived from blood monocytes and to evaluate viral replication, mitochondrial membrane depolarization and mitochondrial respiratory chain complexes at 6, 12, 18 and 24 hours post-infection. Descriptive statistics included mean ± standard deviation (s.d.). Means were compared by analysis of variance, ANOVA. It was observed that CCoV infection induced the release of new viral particles between 18 and 24 hours after infection. Moreover, viral infection was associated with depolarization and mitochondrial membrane dysfunction, affecting respiratory chain complex III. Thus, CCoV is believed to induce mitochondrial bioenergetic failure, acting as a decoupler of the respiratory c... (Complete abstract click electronic access below) / Doutor

Introdução: A isquemia cerebral é uma desordem da função cerebral ocasionado pela supressão sanguínea no tecido cerebral sem nenhuma outra causa aparente do que a vascular. Estudos revelam os danos causados pela isquemia cerebral focal repercutem não apenas na região da lesão isquêmica, mas também em outras regiões do encéfalo, dentre elas o cerebelo. O etanol atua diminuindo o tempo de reação do corpo e a resposta reflexa, produzindo até mesmo perda de coordenação motora. Por tempos, estudos tem verificado a ação do etanol no cerebelo. Objetivos: Este trabalho tem por objetivo analisar o córtex cerebelar de ratos submetidos a um modelo experimental de isquemia cerebral focal transitória por oclusão da ACM durante 90 minutos, seguida por reperfusão de 48 horas, associado ou não a modelo de alcoolismo. Material e métodos: Foram utilizados 50 ratos Wistar adultos, subdivididos em 5 grupos experimentais: grupo controle (C): animais submetidos apenas à anestesia; grupo sham (S): animais submetidos à simulação completa do procedimento cirúrgico; grupo isquêmico (I): animais submetidos à isquemia cerebral focal por 90 minutos seguido por reperfusão de 48 horas; grupo alcoolizado (A): animais que receberam diariamente álcool etílico absoluto diluído a 20% em água durante quatro semanas; e, grupo isquêmico e alcoolizado (IA): animais submetidos ao mesmo tratamento do grupo A e que, após quatro semanas foram submetidos à isquemia cerebral focal durante 90 minutos, seguido por reperfusão de 48 horas. As amostras do cerebelo coletadas e realizado a análise de imunohistoquímica da proteína CASPASE-9 e a análise sérica por meio de PCR - RT dos miRNAS miR-21, miR-126 e o miR155. Resultados: A expressão de CASPASE 9 teve maior expressão no grupos I, A e I+A. A análise dos miRNAS, o miR-126 foi maior nos grupos A e I+A, o miR-155 foi maior nos grupos I e I+A. Conclusões: Podemos concluir que a ocorrência de apoptose no córtex cerebelar, mesmo distante do foco isquêmico e ques miRNAs 126 e 155 apresentam correlação com a apoptose celular em ratos isquêmicos e submetidos ao modelo de alcoolismo crônico / INTRODUCTION: Cerebral ischemia is a disorder of brain function caused by blood suppression in brain tissue with no apparent cause other than vascular. Studies reveal the damage caused by focal cerebral ischemia to affect not only the region of the ischemic lesion but also other regions of the brain, including the cerebellum. Ethanol acts by decreasing the reaction time of the body and the reflex response, producing even loss of motor coordination. For some time, studies have verified the action of ethanol in the cerebellum. AIMS: This study aims to analyze the cerebellar cortex of rats submitted to an experimental model of transient focal cerebral ischemia by ACM occlusion for 90 minutes, followed by reperfusion of 48 hours, associated or not with the model of alcoholism. METHODS: Fifty adult Wistar rats were used, subdivided into 5 experimental groups: control group (C): animals submitted to anesthesia only; sham group (S): animals submitted to complete simulation of the surgical procedure; ischemic group (I): animals submitted to focal cerebral ischemia for 90 minutes followed by reperfusion of 48 hours; alcoholic group (A): animals that received daily absolute ethanol diluted 20% in water for four weeks; and ischemic and alcoholized group (AI): animals submitted to the same treatment as group A and after four weeks were submitted to focal cerebral ischemia for 90 minutes, followed by reperfusion of 48 hours. The cerebellum samples were collected and the immunohistochemical analysis of the CASPASE-9 protein and the serum analysis by means of RT-PCR of miRNAS miR-21, miR-126 and miR155 were performed. RESULTS: The expression of CASPASE 9 had higher expression in groups I, A and I + A. The miRNAS analysis, miR-126 was higher in groups A and I + A, miR-155 was higher in groups I and I + A. CONCLUSIONS: We can conclude that apoptosis occurs in the cerebellar cortex, even if it is distant from the ischemic focus, and that miRNAs 126 and 155 present a correlation with cellular apoptosis in ischemic rats and submitted to the chronic alcohol model