L’étude d’échantillons humains montre que les voies de signalisation RAS/MAPK et PI3K/AKT/mTOR sont fréquemment activées de manière aberrante dans les tumeurs de la prostate, d’autant plus dans les phases de résistance aux traitements. Ces deux voies de signalisation sont sensibles aux facteurs de croissances et impliquées dans la régulation de processus cellulaires fondamentaux tels que la prolifération, la croissance ou encore la différenciation cellulaire. Ces données suggèrent qu’elles ont un rôle essentiel dans la tumorigenèse prostatique. Cependant, le rôle respectif de chacune de ces voies dans la carcinogenèse prostatique, particulièrement dans les phases précoces, n’est pas clairement établit. L’objectif de ma thèse est donc de définir le rôle possible de ces deux voies dans l’initiation et la progression du cancer de la prostate, ainsi que les mécanismes impliqués dans leur co-dérégulation. Cette étude est réalisée dans un modèle in vivo alternatif, la drosophile, qui possèdent un équivalent fonctionnel de la prostate : les glandes accessoires. La première partie des travaux réalisés montre que seule la suractivation de la voie RAS/MAPK dans la glande accessoire conduit à un processus de tumorigenèse, avec la production de masses cellulaires récapitulant de nombreuses caractéristiques cancéreuses : croissance cellulaire et prolifération incontrôlée, expression de métalloprotéases, perte de l’expression de marqueurs épithéliaux et formation de nouvelles trachées. Cependant, les deux voies de signalisation sont nécessaires à la tumorigenèse, mais avec des rôles différents : la voie RAS/MAPK est activée précocement et est capable de recruter la voie PI3K/AKT/TOR grâce à la mise en place de deux boucles autocrines de régulation. La première dépend de spitz (dEGF) et du récepteur EGFR pour amplifier l’activation de la voie RAS/MAPK. La seconde dépend de l’activation d’ILP6 (dIGF1), produit suite à l’activation de la voie RAS/MAPK, et permet le recrutement de la voie PI3K/AKT/TOR par l’intermédiaire du récepteur à l’insuline InR. La deuxième partie des travaux réalisés montre que l’activation de la voie RAS/MAPK conduit à la production de MMP1 dans les cellules qui seront à l’origine des tumeurs avant leur extravasation hors de l’épithélium. Cette expression temporelle contrôlée correspond à une étape où une réorganisation du cytosquelette a lieu et où le microenvironnement est altéré. Ces données placent donc la dérégulation de la voie RAS/MAPK comme un évènement précoce de la tumorigenèse prostatique, capable de recruter la voie PI3K/AKT/TOR et d’entrainer la production de MMP1, pour in fine conduire à l’extravasation des cellules et à la formation de tumeurs. / Clinical studies have demonstrated that, in prostate cancer, RAS/MAPK and PI3K/AKT/TOR signaling pathways are often aberrantly co-activated in tumors, their activation levels increasing again in resistance phases. These pathways, that are regulated by growth factors, are implicated in fundamental cellular processes regulation such as proliferation, growth and cellular differentiation. These data suggest that they are likely implicated in prostate tumorigenesis. However, the relative implication of each of these two pathways during prostate tumorigenesis, especially during early phases, is not fully understood. Thus, the aim of my thesis is to define the possible implication of these pathways in prostate cancer initiation and progression and which molecular mechanisms are implicated in their co-deregulation. Therefore, we have developed an alternative in vivo model of prostate tumorigenesis in drosophila, where accessory glands are a functional equivalent of the human prostate. The first part of my work shows that only the hyperactivation of the RAS/MAPK pathway in accessory glands can promote tumorigenesis, with the formation of cell masses that recapitulate many cancer hallmarks including uncontrolled cell growth and proliferation, enhanced matrix metalloproteinases expression, loss of epithelial markers expression, neovascularization-like tracheogenesis. However, both pathways are necessary to tumorigenesis, even though they display different roles: the RAS/MAPK pathway is activated earlier and is able to recruit the PI3K/AKT/TOR pathway thanks to the formation of two feedback loops. The first depend on Spitz (dEGF) and EGFR receptor to amplify RAS/MAPK pathway activation. The second depends on ILP6 (dIGF1) activation, produced following RAS/MAPK pathway activation and allow PI3K/AKT/TOR pathway recruitment via insulin receptor InR. The second part of the work shows that RAS/MAPK pathway activation allows MMP1 production restricted to the cells that will be the origin of the tumors, before their actual extravasation. This temporally controlled step of MMP1 expression corresponds to a time window where the cells show strong cytoskeletal reorganization and where microenvironment is disturbed. These data place the RAS/MAPK pathway deregulation as an early event of prostate tumorigenesis, able to recruit the PI3K/AKT/TOR pathway and to induce MMP1 production to allow cell extravasation and tumor formation.

Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer pre- disposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient. Methods: The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide3-kinase (PI3K)/AKT/ mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro. Results: Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly. Conclusion: Sirolimus treatment led to an improvement of the patient’s clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.

No description available.

La résistance aux anti-aromatases (AAs) constitue un obstacle thérapeutique majeur dans le traitement des cancers du sein RE+. Les objectifs de ce travail étaient : (i) de caractériser les événements moléculaires associés à la résistance acquise aux AAs ; (ii) d’identifier de manière globale de nouveaux profils de miRNAs spécifiquement associés à la résistance aux AAs. Notre étude a mis en évidence le rôle central de la voie Akt/mTOR dans la résistance acquise et de novo aux AAs dans des modèles cellulaires, mais également dans des échantillons de patientes ayant récidivé sous anastrozole. La combinaison d’un AA avec le MK-2206, inhibiteur d’Akt ou avec la rapamycine, inhibiteur de mTOR, augmente la sensibilité à l’AA dans les cellules contrôles et est suffisante pour surmonter la résistance et restaurer la sensibilité à l'hormonothérapie dans les cellules résistantes. Notre travail propose également un modèle de résistance acquise aux AAs basé sur la sélection de cellules « cancer-initiating-like » dotées de propriétés d'auto-renouvellement, d’une résistance intrinsèque aux AAs et d’une sensibilité au MK-2206. Notre étude à grande échelle des miRNAs a identifié la voie Akt/mTOR comme une des cibles privilégiées de ces miRNAs. Nous avons identifié et validé trois miRNAs dérégulés capables de moduler le statut d’activation de la voie Akt/mTOR, qui représentent des cibles potentielles. En conclusion, notre projet a mis en évidence de nouvelles voies de signalisations ciblées par ces miRNAs et de nouveaux évènements moléculaires, qui représentent des candidats potentiels dans la résistance aux AAs / Resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of ER+ breast cancers. Our objectives were (i) to characterize molecular events associated with acquired AI resistance (ii) to capture a global view of the miRNA expression profiles associated with AI resistance. Our results showed the major role of the Akt/mTOR pathway in both de novo and acquired resistance to AI in cellular models and also in breast tumors of patients who relapsed under anastrozole. Combining AI with the Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings propose a model of AI-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to AI and sensitivity to MK-2206. Our large-scale study identified the Akt/mTOR pathway as one of the main targets of the deregulated miRNAs. We identified and validated three miRNAs able to modulate the Akt/mTOR activation status, suggesting these miRNAs as potential targets. To conclude, our project identified new miRNA-targeted signaling pathways and new molecular events, representing strong candidates in the mediation of AI resistance

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis. Como resultado desse primeiro tratamento com 5-ALA-PDT, observou-se que as células sobreviventes ao tratamento apresentaram intensa marcação para pmTOR e exibiram potencial de crescimento durante o período analisado. Após esses ensaios, as células que sobreviveram a essa primeira sessão foram coletadas, replaqueadas e novamente cultivadas, sendo então submetidas a novo ciclo de 5-ALA-PDT. Esse processo foi realizado 5 vezes, variando-se a intensidade de irradiação à medida que se observava aumento na viabilidade celular. As populações celulares que exibiram viabilidade 1,5 vezes maior do que a detectada no primeiro ciclo PDT foram consideradas resistentes ao tratamento. Os mesmos marcadores analisados no primeiro ciclo de PDT foram novamente avaliados nas populações resistentes. Foram obtidas quatro populações celulares resistentes, com viabilidade de até 4,6 vezes maior do que a do primeiro ciclo de PDT e irradiação com LED que variou de 5,86 a 9,38J/cm2. A população mais resistente apresentou ainda menor intensidade de protoporfirina IX, maior capacidade de migração e modificação na morfologia nuclear. As populações resistentes testadas exibiram aumento na expressão de pNF?B, iNOS, pmTOR e pAkt, mas não da proteína anti-apoptótica Bcl- 2. Ensaio in vivo foi também conduzido em ratos, nos quais CEC bucal foi quimicamente induzido e tratado ou não com 5-ALA-PDT. Houve intensa expressão imuno-histoquímica das proteínas pNF?B, Bcl-2, iNOS, pmTOR e pAkt em relação ao controle não tratado, nas células adjacentes à área de necrose provocada pela PDT. Concluiu-se que as células de CEC bucal tratadas com 5-ALA-PDT a uma dose de 90% de letalidade desenvolveram viabilidade crescente após ciclos repetidos do tratamento, bem como exibiram superexpressão de proteínas relacionadas à sobrevivência celular, tanto in vitro quanto in vivo. Esses fatos, aliados à maior capacidade de migração, sugerem a aquisição de fenótipo de resistência à 5-ALAPDT. Esse aspecto deve ser cuidadosamente considerado no momento da instituição dessa terapia para os CECs bucais. / Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period. After these assays, the cells that survived the first cycle were collected, plated, and cultured and were subjected to another cycle of 5- ALA-PDT. This process was repeated five times at various irradiation intensities, and cell viability gradually increased. The cell populations that exhibited 1.5-times higher viability than that detected after the first PDT cycle were considered to be resistant to treatment. The markers analyzed after the first PDT cycle were again assessed in the resistant populations. Four resistant cell populations were obtained with a viability of up to 4.6-times higher than that of the first PDT cycle and LED light treatment, which varied between 5.86 and 9.38J/cm2. The most resistant population exhibited lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. The resistant populations tested showed increased expression of pNF?B, iNOS, pmTOR, and pAkt, but not of the anti-apoptotic Bcl-2 protein. Moreover, an in vivo assay was conducted in rats; oral SCCs were chemically induced and treated with 5-ALA-PDT. The intensity of the immunohistochemical expression of proteins pNF?B, Bcl-2, iNOS, pmTOR, and pAkt in cells adjacent to the area with necrosis caused by PDT was higher than that observed in the untreated control. In conclusion, oral SCCs treated with 5-ALA-PDT at a lethal dose of 90% exhibited increasing viability after several treatment cycles and overexpression of proteins associated with cell survival both in vitro and in vivo. These results, together with the higher migration capacity, suggest the acquisition of the phenotype of resistance to 5-ALA-PDT. This aspect should be carefully considered when initiating this therapy for oral SCCs.

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis. Como resultado desse primeiro tratamento com 5-ALA-PDT, observou-se que as células sobreviventes ao tratamento apresentaram intensa marcação para pmTOR e exibiram potencial de crescimento durante o período analisado. Após esses ensaios, as células que sobreviveram a essa primeira sessão foram coletadas, replaqueadas e novamente cultivadas, sendo então submetidas a novo ciclo de 5-ALA-PDT. Esse processo foi realizado 5 vezes, variando-se a intensidade de irradiação à medida que se observava aumento na viabilidade celular. As populações celulares que exibiram viabilidade 1,5 vezes maior do que a detectada no primeiro ciclo PDT foram consideradas resistentes ao tratamento. Os mesmos marcadores analisados no primeiro ciclo de PDT foram novamente avaliados nas populações resistentes. Foram obtidas quatro populações celulares resistentes, com viabilidade de até 4,6 vezes maior do que a do primeiro ciclo de PDT e irradiação com LED que variou de 5,86 a 9,38J/cm2. A população mais resistente apresentou ainda menor intensidade de protoporfirina IX, maior capacidade de migração e modificação na morfologia nuclear. As populações resistentes testadas exibiram aumento na expressão de pNF?B, iNOS, pmTOR e pAkt, mas não da proteína anti-apoptótica Bcl- 2. Ensaio in vivo foi também conduzido em ratos, nos quais CEC bucal foi quimicamente induzido e tratado ou não com 5-ALA-PDT. Houve intensa expressão imuno-histoquímica das proteínas pNF?B, Bcl-2, iNOS, pmTOR e pAkt em relação ao controle não tratado, nas células adjacentes à área de necrose provocada pela PDT. Concluiu-se que as células de CEC bucal tratadas com 5-ALA-PDT a uma dose de 90% de letalidade desenvolveram viabilidade crescente após ciclos repetidos do tratamento, bem como exibiram superexpressão de proteínas relacionadas à sobrevivência celular, tanto in vitro quanto in vivo. Esses fatos, aliados à maior capacidade de migração, sugerem a aquisição de fenótipo de resistência à 5-ALAPDT. Esse aspecto deve ser cuidadosamente considerado no momento da instituição dessa terapia para os CECs bucais. / Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period. After these assays, the cells that survived the first cycle were collected, plated, and cultured and were subjected to another cycle of 5- ALA-PDT. This process was repeated five times at various irradiation intensities, and cell viability gradually increased. The cell populations that exhibited 1.5-times higher viability than that detected after the first PDT cycle were considered to be resistant to treatment. The markers analyzed after the first PDT cycle were again assessed in the resistant populations. Four resistant cell populations were obtained with a viability of up to 4.6-times higher than that of the first PDT cycle and LED light treatment, which varied between 5.86 and 9.38J/cm2. The most resistant population exhibited lower intensity of protoporphyrin IX, higher migration capacity, and changes in nuclear morphology. The resistant populations tested showed increased expression of pNF?B, iNOS, pmTOR, and pAkt, but not of the anti-apoptotic Bcl-2 protein. Moreover, an in vivo assay was conducted in rats; oral SCCs were chemically induced and treated with 5-ALA-PDT. The intensity of the immunohistochemical expression of proteins pNF?B, Bcl-2, iNOS, pmTOR, and pAkt in cells adjacent to the area with necrosis caused by PDT was higher than that observed in the untreated control. In conclusion, oral SCCs treated with 5-ALA-PDT at a lethal dose of 90% exhibited increasing viability after several treatment cycles and overexpression of proteins associated with cell survival both in vitro and in vivo. These results, together with the higher migration capacity, suggest the acquisition of the phenotype of resistance to 5-ALA-PDT. This aspect should be carefully considered when initiating this therapy for oral SCCs.

Les β2-agonistes sont couramment utilisés pour prévenir et réduire les symptômes de l'asthme et de la bronchoconstriction induite par l'exercice. Mais, pris en quantités supérieures aux doses thérapeutiques, les β2-agonistes ont un effet anabolisant qui a été clairement démontré in vivo. Un certain nombre d’acteurs sont mis en jeu dans la réponse biologique du tissu musculaire aux β2-agonistes. L’un de ces acteurs est la voie de signalisation PI3K/Akt/mTOR, voie d’initiation de la traduction, ayant un rôle majeur dans la synthèse protéique. Dans ce contexte, notre première étude avait pour objectif de déterminer la cinétique des événements moléculaires responsables de l’hypertrophie du muscle squelettique de rat après administration de formotérol pendant 1 jour (J1), 3 jours (J3) et 10 jours (J10). Nous avons montré que l’administration de formotérol induisait une hypertrophie musculaire à J3 et J10 associée à l’activation transitoire de la voie de signalisation PI3K/Akt/mTOR (J1 et J3), et à une diminution de l’expression de l’E3 ubiquitine ligase MAFbx/Atrogin-1 (J3). La voie autophagie lysosome ne semblait pas être affectée. Ainsi, l’ensemble de ces résultats suggère que l’activation de la voie PI3K/Akt/mTOR est associée à la voie ubiquitine-protéasome mais pas à la voie autophagie-lysosome. La régulation transitoire de la voie PI3K/Akt/mTOR suggère que d’autres voies de signalisation sont impliquées dans l’hypertrophie musculaire induite par le formotérol. Le 007-AM, analogue de l’AMPc, a été décrit comme pouvant stimuler la voie de signalisation PI3K/Akt/mTOR via l’activation de la protéine Epac, suggérant que le 007-AM puisse constituer une molécule de substitution à l’utilisation des β2-agonistes. Notre seconde étude avait pour but de déterminer si le 007-AM avait une action anabolisante sur le tissu musculaire, mais également de déterminer si la 007-AM était une molécule stable permettant d’envisager son usage dans un cadre pharmacologique. L’administration de 007-AM pendant 7 jours chez des souris n’engendrait pas d’hypertrophie musculaire. En revanche, in vitro sur cellules C2C12, le 007-AM activait la voie de signalisation PI3K/Akt/mTOR comme en témoignait l’augmentation de la phosphorylation des protéines rpS6 et 4E-BP1. Nos résultats montraient également que le 007-AM était instable dans le plasma alors que son produit de dégradation, le 007 était plus stable. Pris ensembles, ces résultats suggèrent qu’un traitement de 7 jours au 007-AM n’est pas suffisant pour induire une hypertrophie musculaire et que l’absence d’hypertrophie musculaire pourrait provenir de l’instabilité du 007-AM dans le plasma. Toutefois, des études supplémentaires seront nécessaires pour confirmer ces résultats / Β2-agonists are traditionally used to prevent and reduce asthma symptoms and bronchoconstriction induced by exercise. Nevertheless, when administrated in vivo, at relatively high, far away from therapeutic doses, β2-agonists induce anabolic effects. Numerous actors are involved in biological response of the skeletal muscle, induced by β2-agonists. PI3K/Akt/mTOR signaling pathway, which initiates translation, is one of these actors. In this context, our first study aimed at determined the kinetic of molecular events responsible for skeletal muscle hypertrophy after 1 day (D1), 3 days (D3) and 10 days (D10) of formoterol administration. We have shown that formoterol administration induced skeletal muscle hypertrophy at D3 and D10 associated with a transient activation of PI3K/Akt/mTOR signaling pathway (D1 and D3), and, with a decrease in E3 ubiquitin ligase MAFbx/atrogin-1 expression (D3). The autophagy-lysosome pathway seems not to be regulated by formoterol administration. Taken together, these results suggest that PI3K/Akt/mTOR activation is temporally associated with the regulation of ubiquitin-proteasome but not the autophagy-lysosome pathway. The transient nature of the regulation of PI3K/Akt/mTOR signaling pathway also indicates that other unidentified pathways are probably activated to sustain the increase in skeletal muscle mass. Recently, 007-AM synthetic molecule has been described to stimulate PI3K/Akt/mTOR signaling pathway through Epac protein activation, suggesting that 007-AM could be an alternative to the use of β2-agonists. The purpose of our second study was to determine whether 007-AM had an anabolic action on skeletal muscle and if 007-AM was stable allowing considering its use in pharmacology. 007-AM administration for 7 days to mice does not lead to muscle hypertrophy. Nonetheless, in vitro on C2C12 cells, 007-AM activated PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of rpS6 and 4E-BP1. Our results showed that contrary to 007, 007-AM was instable in plasma. Altogether, these results suggest that a 7-day 007-AM treatment is not sufficient to induce skeletal muscle hypertrophy. This lack of hypertrophy could be due to 007-AM instability in plasma. However, supplemental studies are needed to confirm these results

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Abstract : Long-term survival of T lymphocytes in a quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. Interaction of the T cell antigen receptor (TCR) with self-peptide/MHC synergizes with IL-7-induced anti-apoptotic signals to promote T cell survival. These extrinsic stimuli are also implicated in T cell metabolism and survival by regulating several signaling pathways including the phosphatidyl-inositol-3 kinase (PI3K)/Akt pathway. In mice and in rats, loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanisms responsible for the pro-survival function of GIMAP5 in T lymphocytes remain largely unknown. Previous work from my laboratory has shown that T cells from GIMAP5-deficient rats show reduced influx of calcium (Ca[superscript 2+]) from the extracellular milieu following stimulation of the TCR complex. In this thesis, I characterized the mechanism by which GIMAP5 regulates Ca[superscript 2+] homeostasis, and elucidated the signaling pathways modulated by GIMAP5 to facilitate the survival of T cells. Firstly, I investigated if GIMAP5 prevents apoptotic death of T lymphocytes by affecting the Ca[superscript 2+] buffering capacity of mitochondria, which is required for sustained Ca[superscript 2+] influx via the plasma membrane channels. I observed that mitochondrial Ca[superscript 2+] accumulation following capacitative Ca[superscript 2+] entry is defective in T cells from Gimap5 deficient rats. Disruption of microtubules, but not the actin cytoskeleton, abrogated Ca[superscript 2+] sequestration by mitochondria in T cells from control but not Gimap5 deficient mice. Similarly, mice lacking functional GIMAP5 displayed defective T cell development and Ca[superscript 2+] influx. Furthermore, I observed that the proximal signaling events following TCR stimulation was reduced and was accompanied by defective proliferation in T cells from Gimap5 deficient mice. Additionally, IL-7-induced STAT5 phosphorylation was decreased in CD4[superscript +] T cells from Gimap5 deficient mice. I also showed that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Instead, the constitutive activation the PI3K pathway contributed to the spontaneous high mTOR activation. Collectively, my observations suggest that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to the regulation of diverse signaling pathways in a context dependent manner. GIMAP5 also facilitates microtubule-dependent mitochondrial buffering of Ca[superscript 2+] following capacitative entry. GIMAP5 is required to integrate the survival signals generated following activation through TCR and IL-7R. / Résumé : La survie à long terme des lymphocytes T en état de repos est essentielle pour maintenir leurs nombres dans les organes lymphoïdes secondaires. Le récepteur antigénique des cellules T (TCR) en contact avec les peptides du soi / CMH et en synergie avec l'IL-7 induit des signaux anti-apoptotiques pour favoriser la survie des cellules T. Ces stimuli extrinsèques sont également impliqués dans le métabolisme et la survie des cellules T grâce à la régulation de plusieurs voies de signalisation dont la voie phosphatidyl-inositol-3 kinase (PI3K) /AKT. Chez la souris et chez le rat, la perte de l’activité de GIMAP5 (GTPase of Immune Associated Nucleotide Binding Protein 5), provoque une lymphopénie T périphérique en raison de la mort spontanée des cellules T. Le mécanisme sous-jacent responsable de la fonction de survie de GIMAP5 dans les lymphocytes T reste largement inconnu. Nous avons observé que les cellules de rats déficients en GIMAP5, après stimulation par complexe TCR, montrent un afflux de calcium (Ca[indice supérieur 2+]) réduit provenant du milieu extracellulaire. Dans cette thèse, J’ai caractérisé le mécanisme d’action de GIMAP5 dans la régulation de l'homéostasie du Ca[indice supérieur 2+], ainsi que les voies de signalisation modulées par GIMAP5 pour faciliter la survie des cellules T. Tout d'abord, j’ai étudié si GIMAP5 empêche l’apoptose des lymphocytes T en affectant la capacité des mitochondries à réguler la concentration du Ca[indice supérieur 2+], ce qui est nécessaire pour soutenir l’influx de Ca[indice supérieur 2+]. J’ai trouvé que l’accumulation du Ca[indice supérieur 2+] mitochondrial après l’entrée capacitive de Ca[indice supérieur 2+] est défectueuse dans les cellules T de rat déficientes en Gimap5. La disruption des microtubules, mais pas du cytosquelette d'actine, abroge la séquestration du Ca[indice supérieur 2+] mitochondrial dans les cellules T primaires de rat, mais pas dans les cellules T déficientes en Gimap5. J’ai observé que les cellules T provenant de souris déficientes en Gimap5 démontrent une diminution de l’entrée de Ca[indice supérieur 2+]. De plus, la prolifération des cellules T déficientes en Gimap5 est diminuée suite à la stimulation du TCR. En outre, la phosphorylation de STAT5 induit par l'IL-7 est diminuée dans les cellules T CD4[indice supérieur +] de souris déficientes en Gimap5. Également, la perte de Gimap5 aboutit à une activation accrue de la cible mammalienne de la rapamycine (mTOR), indépendamment de la protéine phosphatase 2A (PP2A) ou de la protéine kinase activée par l'AMP (AMPK). Au lieu de cela, l'activation constitutive de la voie PI3K contribue à une forte activation spontanée de mTOR. Collectivement, la fonction de survie de GIMAP5 dans les lymphocytes T peut être liée à la régulation de différentes voies de signalisation. GIMAP5 facilite la fonction, microtubule dépendant, des mitochondries dans leurs actions de régulation du Ca[indice supérieur 2+] après l’entrée capacitive de Ca[indice supérieur 2+]. GIMAP5 est nécessaire pour intégrer les signaux de survie produits suite à l'activation du TCR et de l’IL-7R, qui pourrait être associée à la régulation de l'activité PI3K / AKT / mTOR.

Le muscle squelettique s'adapte en réponse à diverses influences en modulant sa masse et ses propriétés contractiles et métaboliques. Il est ainsi rapporté que l'hypoxie sévère a un effet délétère sur la masse et les capacités oxydatives du muscle, et pourrait ralentir la maturation du phénotype contractile au cours du développement post-natal. Cependant, les mécanismes de contrôle de cette plasticité musculaire ne sont pas clairement identifiés. Le but de ce travail était de déterminer le rôle de l'hypoxie environnementale sur le contrôle de la masse et l'adaptation du phénotype du muscle en croissance (hypertrophie de surcharge du plantaris après ablation de ses muscles agonistes et régénération du soléaire après lésions étendues induites par la notexine). L'exposition hypoxique limite transitoirement l'hypertrophie induite par la surcharge fonctionnelle, tandis qu'elle accentue la fonte musculaire en réprimant la formation et la croissance des néo-fibres au cours des étapes précoces de la régénération. Ces résultats seraient en partie expliqués par la désactivation partielle de la principale voie de protéosynthèse, la voie mTOR, par un mécanisme indépendant d'Akt. Parmi les inhibiteurs endogènes de mTOR étudiés (REDD1, BNIP-3 et l'AMPK), nous montrons que l'activation prononcée de l'AMPK en hypoxie pourrait réprimer l'activité de mTOR au cours de la régénération, alors que le mécanisme responsable de l'inhibition de mTOR n'a pas pu être identifié dans le modèle de surcharge. Le système protéolytique ubiquitine/protéasome-dépendant, évalué à partir de l'expression des atrogènes MURF1 et MAFbx, pourrait également expliquer en partie l'altération de l'hypertrophie de surcharge en hypoxie. Nos résultats soulignent par ailleurs que l'activité des cellules satellites serait réprimée au cours des premiers jours de régénération musculaire, conduisant à réduire la formation et la croissance des myotubes. Malgré cette perturbation précoce de la croissance musculaire, l'exposition prolongée en hypoxie ne limite pas l'hypertrophie de surcharge et la récupération de la masse du muscle lésé. Ceci démontre que les signaux anaboliques induits dans ces deux situations de croissance musculaire l'emportent très largement sur les signaux cataboliques de l'hypoxie. L'analyse des propriétés métaboliques et contractiles met en évidence que l'hypoxie altère les capacités oxydatives du muscle en croissance, mais les mécanismes impliqués dans cette réponse adaptative restent à identifier. Par ailleurs, l'hypoxie ne constitue pas un stimulus métabolique suffisant pour altérer la transition du phénotype contractile du muscle en surcharge et la récupération complète du phénotype contractile du muscle lésé. Elle contribue uniquement à ralentir très modérément et transitoirement l'adaptation phénotypique du muscle en surcharge, et à modifier le profil contractile du muscle durant la phase de dégénérescence musculaire.