Base teórica: Nos Estados Unidos da América (EUA) e no Norte da Europa o carcinoma de ovário é a principal causa de morte por câncer ginecológico. A maioria dos casos é diagnosticada em estágios avançados – III ou IV FIGO . A sobrevida em 5 anos neste grupo é de 30% das pacientes. O tratamento envolve cirurgia oncológica para citorredução tumoral e quimioterapia adjuvante, na maioria dos casos. A via do apoptose está envolvida no desenvolvimento tumoral e resistência ao tratamento. As proteínas da família Bcl2 são ativas na via apoptótica e sua atividade se divide em anti e próapoptose. Estudos em pacientes com câncer de ovário foram realizados nos EUA, Europa e Canadá e os resultados observados foram controversos com relação ao papel dessas proteínas no câncer de ovário. Objetivos: Determinar a prevalência de Bcl2, Bcl-xL, Bax, Bad e p53 em uma amostra de pacientes do HCPA. Estudar a possível correlação entre a expressão dessas proteínas e aspectos de desfecho clínico (resposta ao tratamento, sobrevida total e livre de doença). Pacientes e métodos: Foram avaliadas, retrospectivamente, 45 pacientes com diagnóstico de carcinoma epitelial de ovário, tratadas de forma padronizada no HCPA no período de 1996 a 2004. Tecido tumoral foi avaliado através da técnica de imunohistoquímica com relação à positividade para as proteínas Bcl2, Bcl-xL, Bax, Bad e p53. Resultados: A prevalência das proteínas encontrada nessa amostra de pacientes foi: Bcl2 49%, Bcl-xL 80%, Bax 98%, Bad 87% e p53 58%; apresentando similaridade, em geral , com a literatura mundial revisada. A expressão positiva da proteína p53 correlacionou-se com pior sobrevida livre de doença (16 versus 58 meses – p=0.04) na análise univariada. As demais proteínas não apresentaram correlações com resposta ao tratamento ou sobrevida, nesta amostra. Conclusão: O padrão de expressão das proteínas da família Bcl-2 e da proteína p53 encontrado nesta coorte foi similar ao descrito na literatura mundial disponível. A expressão positiva de p53 demonstrou correlação com menor sobrevida livre de doença na análise univariada, o que aponta para as investigações, já em andamento, acerca desta proteína, suas mutações e potencial alvo terapêutico. As proteínas da família Bcl-2 não se correlacionaram com resposta ao tratamento ou sobrevida nesta amostra e requerem investigações em estudos prospectivos, utilizando novas tecnologias de detecção. / Background: In the USA, Europe and Canada epithelial ovarian cancer is the principal cause of death from gynecological cancer and most patients are diagnosed in advanced stages of the disease – Stages III or IV FIGO. The 5 years survival for these patients is around 30%. Treatment is based in laparoscopy to stage and debulk the tumor volume and most cases will need chemotherapic treatment, based on platinum compounds. Apoptosis route is involved in the tumor development and, probably in treatment resistance also. Bcl2 family proteins are active in the apoptosis route and its activity is divided in pro and anti-apoptosis pathways. Studies in ovarian cancer and Bcl2 proteins family activity were done in USA, Europe and Canada. However, the results remain controversial concerning the role of these proteins in ovarian cancer. Objective: Determine Bcl2, Bcl-xL, Bax, Bad and p53 prevalence in a sample of patients from HCPA. Evaluate a possible correlation between these proteins expression and the clinical response, as well as the survival. Methods and patients: Forty-five patients were retrospectively analyzed. They had epithelial ovarian carcinoma diagnosed and treated in a standardized way on HCPA, between the years 1996 to 2004. Tumor tissue samples were evaluated through immunohistochemical analysis to detect positive expression of the proteins Bcl2, Bcl-xL, Bax, Bad and p53. Results: The prevalence found for the studied proteins in this sample was: Bcl2 49%, Bcl-xL 80%, Bax 98%, Bad 87% and p53 58%; similar to the levels described in the literature. Positive expression of p53 correlated with worst disease free survival (16 versus 58 months – p=0.04) in the univariate analysis. The other proteins did not show correlation with treatment response or with survival in this sample. Conclusion: The expression pattern of Bcl-2 protein family and of the p53 protein in this cohort was similar to the one usually described in the literature. The positive expression of p53 was correlated with a smaller time of disease free survival in the univariate analysis. In fact, there are currently investigations about this protein and its mutations as a potential therapeutic goal. Bcl-2 family proteins did not correlate with response to treatment or survival in this sample. Prospective studies are required, as well as the use of new technologies, to know better the role of these proteins in ovarian cancer.

Base teórica: Nos Estados Unidos da América (EUA) e no Norte da Europa o carcinoma de ovário é a principal causa de morte por câncer ginecológico. A maioria dos casos é diagnosticada em estágios avançados – III ou IV FIGO . A sobrevida em 5 anos neste grupo é de 30% das pacientes. O tratamento envolve cirurgia oncológica para citorredução tumoral e quimioterapia adjuvante, na maioria dos casos. A via do apoptose está envolvida no desenvolvimento tumoral e resistência ao tratamento. As proteínas da família Bcl2 são ativas na via apoptótica e sua atividade se divide em anti e próapoptose. Estudos em pacientes com câncer de ovário foram realizados nos EUA, Europa e Canadá e os resultados observados foram controversos com relação ao papel dessas proteínas no câncer de ovário. Objetivos: Determinar a prevalência de Bcl2, Bcl-xL, Bax, Bad e p53 em uma amostra de pacientes do HCPA. Estudar a possível correlação entre a expressão dessas proteínas e aspectos de desfecho clínico (resposta ao tratamento, sobrevida total e livre de doença). Pacientes e métodos: Foram avaliadas, retrospectivamente, 45 pacientes com diagnóstico de carcinoma epitelial de ovário, tratadas de forma padronizada no HCPA no período de 1996 a 2004. Tecido tumoral foi avaliado através da técnica de imunohistoquímica com relação à positividade para as proteínas Bcl2, Bcl-xL, Bax, Bad e p53. Resultados: A prevalência das proteínas encontrada nessa amostra de pacientes foi: Bcl2 49%, Bcl-xL 80%, Bax 98%, Bad 87% e p53 58%; apresentando similaridade, em geral , com a literatura mundial revisada. A expressão positiva da proteína p53 correlacionou-se com pior sobrevida livre de doença (16 versus 58 meses – p=0.04) na análise univariada. As demais proteínas não apresentaram correlações com resposta ao tratamento ou sobrevida, nesta amostra. Conclusão: O padrão de expressão das proteínas da família Bcl-2 e da proteína p53 encontrado nesta coorte foi similar ao descrito na literatura mundial disponível. A expressão positiva de p53 demonstrou correlação com menor sobrevida livre de doença na análise univariada, o que aponta para as investigações, já em andamento, acerca desta proteína, suas mutações e potencial alvo terapêutico. As proteínas da família Bcl-2 não se correlacionaram com resposta ao tratamento ou sobrevida nesta amostra e requerem investigações em estudos prospectivos, utilizando novas tecnologias de detecção. / Background: In the USA, Europe and Canada epithelial ovarian cancer is the principal cause of death from gynecological cancer and most patients are diagnosed in advanced stages of the disease – Stages III or IV FIGO. The 5 years survival for these patients is around 30%. Treatment is based in laparoscopy to stage and debulk the tumor volume and most cases will need chemotherapic treatment, based on platinum compounds. Apoptosis route is involved in the tumor development and, probably in treatment resistance also. Bcl2 family proteins are active in the apoptosis route and its activity is divided in pro and anti-apoptosis pathways. Studies in ovarian cancer and Bcl2 proteins family activity were done in USA, Europe and Canada. However, the results remain controversial concerning the role of these proteins in ovarian cancer. Objective: Determine Bcl2, Bcl-xL, Bax, Bad and p53 prevalence in a sample of patients from HCPA. Evaluate a possible correlation between these proteins expression and the clinical response, as well as the survival. Methods and patients: Forty-five patients were retrospectively analyzed. They had epithelial ovarian carcinoma diagnosed and treated in a standardized way on HCPA, between the years 1996 to 2004. Tumor tissue samples were evaluated through immunohistochemical analysis to detect positive expression of the proteins Bcl2, Bcl-xL, Bax, Bad and p53. Results: The prevalence found for the studied proteins in this sample was: Bcl2 49%, Bcl-xL 80%, Bax 98%, Bad 87% and p53 58%; similar to the levels described in the literature. Positive expression of p53 correlated with worst disease free survival (16 versus 58 months – p=0.04) in the univariate analysis. The other proteins did not show correlation with treatment response or with survival in this sample. Conclusion: The expression pattern of Bcl-2 protein family and of the p53 protein in this cohort was similar to the one usually described in the literature. The positive expression of p53 was correlated with a smaller time of disease free survival in the univariate analysis. In fact, there are currently investigations about this protein and its mutations as a potential therapeutic goal. Bcl-2 family proteins did not correlate with response to treatment or survival in this sample. Prospective studies are required, as well as the use of new technologies, to know better the role of these proteins in ovarian cancer.

Le traitement du mélanome métastatique a été révolutionné par le développement de thérapies ciblées, qui ont montré un bénéfice significatif sur la survie globale. En particulier, l'inhibition de la sérine-thréonine kinase BRAF, mutée dans 60% des mélanomes, par le Vémurafénib (PLX4032), a montré un gain de survie de 6 à 8 mois comparée à la chimiothérapie de référence, la Dacarbazine. Cependant, une très faible proportion de patients répond sur le long terme. En effet, la majorité des patients développent un échappement thérapeutique dans un délai médian de 6 mois. Des mécanismes cellulaires ont été mis en évidence dans l'apparition de cette résistance acquise, notamment l'implication de MITF, un facteur de transcription majeur des mélanocytes, ainsi que des modifications de l'expression de plusieurs membres de la famille de Bcl-2. Cependant, une meilleure compréhension des mécanismes de résistance aux thérapies anti-BRAF semble essentielle, tout comme l'utilisation de nouvelles approches thérapeutiques combinées afin d'optimiser l'efficacité des traitements et la durée du bénéfice clinique. Notre groupe a récemment identifié des altérations du métabolisme du céramide et de l'un de ses dérivés, la Sphingosine 1-phosphate (S1P), dans les cellules de mélanome humain comparé à des mélanocytes sains. En effet, nous avons montré que la S1P lyase (SPL), qui dégrade irréversiblement la S1P est sous exprimée. Au contraire, l'expression de la sphingosine kinase 1 (SK1), qui produit la S1P, est augmentée dans les cellules de mélanome, conséquence directe de la mutation BRAF. Ces perturbations ont pour effet d'augmenter les niveaux de S1P. Ce lysophospholipide favorise la survie cellulaire ainsi que la résistance vis-à-vis d'agents thérapeutiques dans diverses cellules tumorales. L'objectif de cette thèse a été d'évaluer si le métabolisme de la S1P peut moduler la résistance acquise des cellules de mélanome humain aux inhibiteurs de BRAF. Nous avons montré que la surexpression de la SPL ou l'inhibition pharmacologique de la SK1 (SKI-I) sensibilise les mélanomes métastatiques à l'apoptose induite par la thérapie ciblée. Ce phénomène est associé à une diminution de MITF et de l'une de ses cibles directes, la protéine anti-apoptotique Bcl-2. La diminution d'expression protéique de MITF peut être réversée par un traitement de S1P exogène. De plus, nous avons montré pour la première fois une augmentation de l'expression des récepteurs 1 et 3 à la S1P (S1PR1 et S1PR3), dans les cellules de mélanome présentant une résistance acquise au PLX4032. Ces modifications sont associées à l'expression accrue de MITF. La surexpression de la SPL, le traitement par le SKI-I ou par des inhibiteurs ciblant les S1PR1 et S1PR3, surmonte la résistance acquise de ces cellules au PLX4032 via la diminution d'expression des S1PRs, de MITF, et de Bcl-2. Par conséquent, en contrôlant l'expression de protéines clés de la survie et de la résistance, le métabolisme de la S1P représente une nouvelle approche thérapeutique pour améliorer l'efficacité des thérapies ciblées. / The treatment of metastatic melanoma has changed considerably in recent years with the development of targeted therapies, which have shown a significant benefit in overall survival. In particular, the inhibition of the frequently mutated serine-threonine kinase BRAF, by Vemurafenib (PLX4032) showed that survival rates increase by 6 to 8 months compared to standard chemotherapy, Dacarbazine. However, a very small proportion of patients will respond to the long term, and the majority of patients relapses in a median of 6 months. Cellular mechanisms have been identified in the appearance of this acquired resistance, including the involvement of MITF, a major transcription factor of melanocytes, as well as changes in the expression of several members of Bcl-2 family. However, a better understanding of these mechanisms seems essential, as is the use of new therapeutic strategies to optimize treatment efficacy and duration of clinical benefit. Our group recently showed some alterations of ceramide metabolism and its derivative sphingosine 1-phosphate (S1P) in human melanoma cells compared to healthy melanocytes. For instance, S1P lyase (SPL), which degrades S1P, is under-expressed. Conversely, sphingosine kinase 1 (SK1), which produces S1P, is over-expressed in tumor cells, as a direct result of BRAF mutation. These alterations increases the levels of S1P. This lysophospholipid promotes cell survival and the resistance to therapeutic agents in a variety of tumor cells. This PhD project aimed at defining whether S1P metabolism could modulate the resistance of human melanoma cells to PLX4032. Here, we show that SPL overexpression or pharmacological inhibition of SK1 by SKI-I sensitizes metastatic melanoma cells to PLX4032-induced apoptosis. This phenomenon is associated with a decreased expression of the master regulator of melanocyte differentiation MITF as well as its direct cellular target Bcl-2. The decrease in MITF protein can be reversed by treating cells with exogenous S1P. Interestingly, we also report for the first time an increased expression of SK1 as well as the S1P receptors, S1PR1 and S1PR3, in melanoma cells with acquired resistance to PLX4032 as compared to sensitive counterparts. These modifications are associated with high expression of MITF. Overexpression of SPL, treatment with SKI-I or antagonists of S1PR1 ans S1PR3, strongly overcomes acquired resistance to PLX4032 through a decrease in the expression of S1PR, MITF as well as Bcl-2. Thus, by controlling the expression of key proteins in melanoma cell survival and resistance, S1P metabolism could represent a new therapeutic approach to enhance the effectiveness of targeted therapies.

Le cancer de l’ovaire représente la quatrième cause de décès par cancer chez la femme. En France, près de 4 500 nouveaux cas par an sont diagnostiqués, avec plus de 3000 décès chaque année. Ce cancer reste longtemps silencieux : un diagnostic souvent tardif, et le développent d’une chimiorésistance qui touche jusqu'à 75 % des patientes, expliquent le fort taux de mortalité et une survie à 5 ans estimée à moins de 45 %. Les mécanismes responsables du développement de la chimiorésistance sont nombreux. Aujourd’hui, un fort intérêt est porté au développement de nouvelles stratégies de traitement en ciblant les protéines cellulaires inhibitrices de l’apoptose (mort cellulaire programmée). Deux familles de protéines jouent un rôle cruciale dans la régulation de l’apoptose : la famille Bcl-2 et la famille IAP. Nous nous sommes intéressés à la synthèse de petites molécules abiotiques, appelées foldamères. Ces composés sont capables de perturber les interactions entre les protéines anti-apoptotiques des familles Bcl-2 ou IAP, surexprimées dans le cancer de l’ovaire, et leurs partenaires biologiques (pro-apoptotiques). Ceci permet de libérer les protéines pro-apoptotiques et rétablir l’apoptose dans les cellules cancéreuses. Ce travail de thèse a permis la conception, la synthèse, la caractérisation et l’évaluation biologique de molécules inhibitrices des protéines anti-apoptotiques Mcl-1 et Bcl-xL de la famille Bcl-2, et de possibles inhibiteurs de la protéine XIAP de la famille IAP. / Ovarian cancer is the 4th leading cause of cancer death in women. In France, nearly 4,500 new cases per year are diagnosed, with more than 3,000 deaths each year. This cancer remains silent for a long time: late diagnosis and chemoresistance development for up to 75% of patients, explain the high mortality rate and the 5-year survival estimated at less than 45%. Mechanisms responsible for the development of drug resistance are numerous. Today, there is a strong interest in the development of new treatment strategies by targeting cellular proteins that inhibit apoptosis (programmed cell death). Two families of proteins play a crucial role in the regulation of apoptosis: the Bcl-2 family and the IAP family. We are interested in the synthesis of small abiotic molecules, called foldamers. These compounds are able to disrupting the interactions between the anti-apoptotic proteins of the Bcl-2 or IAP families, overexpressed in ovarian cancer, and their biological (pro-apoptotic) partners. This releases the pro-apoptotic proteins and restores apoptosis in the ovarian cancer cells. This PhD work allowed the design, synthesis, characterization and biological evaluation of inhibitors of Mcl-1 and Bcl-xL anti-apoptotic proteins of Bcl-2 family, and the development of possible inhibitors of the XIAP protein of IAP family.

No description available.

Nur77 is a member of the orphan nuclear receptor family, where it is known to play an important role in apoptosis in both negative selection in T cells and in cancer cell lines. In the development of T cells, it is critical for the immune system to discriminate self from non-self by eliminating auto-reactive cells. It was originally thought that Nur77 initiated apoptosis by activating downstream gene targets. However, it is now clear that Nur77 has its own distinct role outside of the nucleus and the precise mechanisms by which Nur77 induces apoptosis in T cells still needs to be clarified. Calcium plays an important role as a second messenger in various cellular responses, one of which includes apoptosis. The IP3 receptor controls efflux of calcium from the ER and can be activated through TCR activation. This signal induces a rise in cytoplasmic calcium levels ultimately causing cell death through mechanisms that remain unclear. Here, we use a double positive DO11.10 T cell line with tetracycline responsive Nur77, to examine the effects of cytosolic Nur77. Through co-immunoprecipitation experiments we suggest, that the presence of Nur77 disrupts the IP3R/Bcl-2 interaction. In this study, we also investigated the effect of Nur77 on intracellular calcium levels. We show that Nur77 increases baseline calcium levels and causes emptying of ER calcium stores. We suggest a model where cytosolic Nur77 disrupts the IP3R/Bcl-2 interaction by binding Bcl-2 at the mitochondria or ER, causing calcium release through the IP3R and apoptosis of the cell.

No description available.

No description available.

No description available.

During the course of an immune response, CD4+ T helper cells differentiate into a number of subsets including: T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. The functional diversity of CD4+ T effector cells results in a coordinated, pathogen-specific immune response. For example, the production of IFNγ by TH1 cells is vital for the clearance of intracellular pathogens, while TFH cell engagement with cognate B cells is required for germinal center (GC) formation and the generation of pathogen- and vaccine- induced antibody production. The development of CD4+ subsets is contingent on extracellular signals, in the form of cytokines, and downstream transcriptional networks responsible for promoting the unique gene expression profile for each subset while simultaneously suppressing alternative cell fates. However, the exact composition of, and stage-specific requirements for, these environmental cytokines and transcription factor networks in the governance of TFH cell differentiation remain incompletely understood. The work in this dissertation seeks to understand how cell-extrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Here, we demonstrate that in response to decreased IL-2 and constant IL-12 signaling, T helper 1 (TH1) cells upregulate a TFH-like phenotype, including expression of the TFH lineage defining transcription factor Bcl-6. Intriguingly, our work established that signals from IL-12 were required for both the differentiation and function of this TFH-like population. Mechanistically, IL-12 signals are propagated through both STAT3 and STAT4, leading to the upregulation of the TFH associated genes Bcl6, Il21, and Icos, correlating with increased B cell helper activity. Conversely, exposure of these TFH-like cells to IL-7 results in the STAT5-dependent repression of Bcl-6 and subsequent inhibition of the TFH phenotype. Finally, we describe a novel regulatory mechanism wherein STAT3 and the Ikaros zinc finger transcription factors Ikaros and Aiolos cooperate to regulate Bcl-6 expression in these TFH-like cells. Collectively, the work in this dissertation significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines. / Ph. D. / Specialized cells called T helper cells serve as a critical interface between the innate (first line of defense) and adaptive (specialized and long-term) immune systems. During the course of an infection, T helper cells are responsible for orchestrating the immune-mediated elimination of invading viruses, bacteria, and parasites. This wide breadth of functionality is achieved through the formation of distinct T helper subsets including T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. Individual subsets have distinct developmental requirements and have unique functions within the immune system. For example, TFH cells are required for the production of effective antibodies that recognize invading pathogens, leading to their subsequent elimination. This naturally occurring process is the basis for a number of modern medical therapies including vaccination. Conversely, aberrant generation of antibodies that recognize host tissues can result in the onset of various autoimmune diseases including lupus, multiple sclerosis, and crohn’s disease. Due to the importance of TFH cells to human health, there is intense interest in understanding how these cells are formed. It is recognized that the generation of these therapeutically important immune cells is mediated by numerous cell-extrinsic andintrinsic influences, including proteins in their cellular environment called cytokines, and important proteins inside of the cell called transcription factors. However, as this is a complicated and multi-step process, many questions remain regarding the identity of these cytokines and transcription factors. The work in this dissertation seeks to understand how cellextrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Collectively, this body of work significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines.