Le but du projet est l’étude des régulateurs de la voie de signalisation intracellulaire de la voie Pi(3)K-Akt au travers de l’analyse du suppresseur de tumeur PTEN (Phosphatase and tenson homolog) et de la sérine/thréonine kinase mTOR (Mamalian Target of Rapamycin). Cette étude à plusieurs objectifs :1. Modulation de la voie Akt par HCV sur des foies humains, foies de souris FL-N/35 au niveau basal dans un premier temps et lors d’un boost de la voie in vitro sur des cultures primaires d’hépatocytes murins.2. Etude de l’expression ainsi que des modifications post-traductionnelles des modulateurs de la voie PI(3)K dans un modèle murin exprimant (FL-N/35) ou non l’ORF complète du virus de l’hépatite C (VHC).3. Confirmer nos précédentes données avec l’invalidation de PTEN dans un modèle de souris KO pour PTEN.4. Etendre ses données au niveau moléculaire dans l’optique d’une étude mécanistique grâce à l’analyse in vivo, ex vivo et in vitro d’un modèle murin KO pour PTEN.5. Compléter cette étude par l’analyse des déterminants viraux impliqués dans la dérégulation de la signalisation intracellulaire grâce à l’étude de souris NS5A.6. Examiner l’impact de la dérégulation de la voie PI(3)K-Akt dans le développement des Carcinomes hépatocellulaires (CHCs) induit par le VHC. / The goal of myt thesis is to study regulators of intracellular signaling pathway of Pi route (3) K-Akt through the analysis of tumor suppressor PTEN (Phosphatase and tenson homolog) and serine / threonine kinase mTOR (Target of Rapamycin Mamalian). This study has several objectives:1. Modulation of the Akt pathway by HCV in human liver, mouse livers FL-N / 35 to the basal level in a first time and at a track boost in vitro on primary cultures of mouse hepatocytes.2. Study of the expression and post-translational modifications of modulators of PI path (3) K in a murine model expressing (FL-N / 35) or not the complete ORF of hepatitis C ( HCV).3. Confirming our previous data with the invalidation of PTEN in a knockout mouse model for PTEN.4. Extending its data at the molecular level with a view to a mechanistic study through analysis in vivo, ex vivo and in vitro of a knockout mouse model for PTEN.5. Complete this study by analyzing the viral determinants involved in the dysregulation of intracellular signaling through the NS5A mouse study.6. Examine the impact of deregulation of IP route (3) K-Akt in the development of hepatocellular carcinoma (CHCs) induced by HCV.

Les cellules NK sont des lymphocytes de l'immunité innée impliqués dans la reconnaissance et l'élimination de cellules tumorales ou infectées par des pathogènes intracellulaires. La biologie des cellules NK est régulée par des facteurs intrinsèques comme les facteurs de transcription ainsi que par des facteurs environnementaux comme les cytokines, produites en condition homéostatique ou inflammatoire. Certaines cytokines, comme l'IL-15, l'IL-12 ou l'IL-18 sont connues pour potentialiser les fonctions effectrices des cellules NK. L'IL-15, en activant la voie STAT5 permet, d'une part, d'assurer la survie des cellules NK et, d'autre part via la kinase mTOR, d'induire leur prolifération, d'augmenter leur métabolisme ainsi que leurs fonctions effectrices. D'autres cytokines comme le TGF-ß sont connues pour inhiber les fonctions des cellules NK. Le TGF-ß1 est une des cytokines les plus immunosuppressives du système immunitaire et, en étant secrété´ par différents types de cancers, il participe a` l'échappement tumoral. Depuis longtemps, les effets du TGF-ß in vitro sont connus pour contrer ceux de l'IL-15. L'objectif de ce travail a été´ d'étudier les effets du TGF-ß sur la biologie des cellules NK. Nous avons observé´ que l'ajout de TGF-ß, in vitro, induit un blocage rapide de l'activation de la voie mTOR par l'IL-15, que le TGF-ß a des effets très proches de ceux de la rapamycine, un inhibiteur spécifique de mTOR et que, in vivo chez la souris, l'activation constitutive des voies de signalisation activées par le TGF-ß induit un phénotype proche de celui de la délétion de mTOR dans les cellules NK / NK cells are innate lymphocytes involved in the recognition and elimination of tumor or infected cells. The biology of NK cells is regulated by intrinsic factors such as transcription factors but also by cytokines produced at steady state or under inflammatory conditions. Some of these cytokines like IL-15, IL-12 or IL-18 are known to increase NK cells functions. IL-15 allows NK cell survival via STAT5 and, via mTOR, increase NK cell proliferation, metabolism and acquisition of functions. In the other hand, cytokines like TGF-ß are known to inhibit NK cell function. TGF-ß1 is a major immunosuppressive cytokine, often secreted by tumor cells and participates to tumor escape. The inhibitory effects of TGF-ß in vitro on IL-2/15 mediated NK cell activation have long been shown, but the mechanism remains unknown. The objective of this work was to characterize the effects of TGF-ß at a molecular level. We have observed that TGF-ß induces a rapid blockade of IL-15 induced mTOR activation, in vitro. TGF-ß and the mTOR inhibitor rapamycin have similar effects. Finally, using genetic mouse models in vivo, constitutive TGF-ß signaling or mTOR deletion results in similar developmental arrests in NK cells

O desenvolvimento de novas terapias para o câncer envolve um processo longo e custoso no qual apenas 5% dos candidatos a fármacos em ensaios clínicos são aprovados. Os ensaios celulares são um importante pilar neste processo, no entanto, eles são geralmente feitos em células cultivadas em monocamada, o que apresenta algumas limitações. Assim, o desenvolvimento e aplicação de modelos tridimensionais (3D) para ensaios pré-clínicos tem sido cada vez mais investigado, devido a suas maiores similaridades com tumores in vivo em relação a características físicas, espaciais e bioquímicas. A via PI3K-AKT-mTOR, que está frequentemente desregulada em diversos cânceres, é uma rota metabólica essencial por integrar fatores de crescimento e sinais internos com a expressão de proteínas relacionadas ao crescimento celular. Sendo assim, novos compostos inibitórios dessa via têm sido estudados pelo grupo NEQUIMED como alternativa terapêutica para essas doenças. Dessa forma, este trabalho teve o objetivo de avaliar o potencial citostático e citotóxico de novos inibidores da via da mTOR em culturas celulares bi e tridimensionais de câncer de próstata e hepatocarcinoma. Para isso, o modelo de cultura 3D foi padronizado para ambas linhagens usando duas técnicas diferentes, além da padronização da técnica de redução da resazurina para a determinação da viabilidade celular em 2D e 3D. No ensaio citotóxico, células em monocamada e esferoides foram tratadas com diferentes concentrações dos fármacos de referência e dos novos inibidores e tiveram sua viabilidade determinada pelo ensaio de redução da resazurina. Já para o ensaio citostático, baixas concentrações de células foram plaqueadas em monocamada e tratadas por até 6 dias, tendo sua viabilidade medida a cada 48 h pelo ensaio de MTT. Os esferoides foram tratados por 9 dias com as mesmas substâncias, tendo seu volume medido a cada 3 dias. No geral, os novos compostos não apresentaram efeitos citotóxicos relevantes, contudo, os compostos Neq0438 e seu análogo, Neq0679, tiveram maior efeito citostático que a Rapamicina em culturas 2D e 3D de HepG2. Além disso, os compostos tiveram maior efeito citostático em esferoides do que em células cultivadas em monocamada para as duas linhagens. Isso pode ser justificado pela alteração na expressão de proteínas da via da mTOR em relação ao modelo de cultura, já que foi demonstrado sua maior ativação nos modelos tridimensionais. Futuramente esses compostos poderão ser testados sozinhos, ou em terapia combinada em modelos animais para melhor avaliação de sua segurança e sua evolução no estudo pré-clínico. / The development of new cancer therapies involves a long and expensive process in which only 5% of the clinical trial candidates for new drugs get approval. Cell assays are an essential pillar in this process; however, they are usually carried out in cells grown as a monolayer, which have some limitations. Thus, the development and application of new tridimensional (3D) models for preclinical trials has been deeply investigated, due to their higher similarities with in vivo tumors, regarding physical, spatial and biochemical features. The PI3K-AKT-mTOR pathway, which integrates growth factors and the expressions of proteins for the cellular growth, is often upregulated in several types of cancer. Hence, the NEQUIMED group is studying new pathway inhibitors as a therapeutic alternative for cancer. Here, the cytotoxic and cytostatic effects of the new PI3K-AKT-mTOR inhibitors were assessed in two and three-dimensional cells models of prostate cancer and hepatocarcinoma. To that end, the 3D culture model was standardized for both cell lines using two different techniques. The resazurin reduction assay was also standardized to determine cell viability in 2D and 3D models. For the cytotoxic assay, cells grown in a monolayer and 3D spheroids were treated with a range of concentrations of the reference drugs and new mTOR inhibitors and had their viability assessed by the resazurin assay. For the cytostatic assay, low cell density was plated on 2D and treated for 6 days, having their viability determined every 48 h using the MTT assay. Spheroids were treated for 9 days with the same substances and had their volume measured every 3 days. Overall, the new mTOR inhibitors did not show relevant cytotoxic effects, however, Neq0438 and its analog Neq0679, had a higher cytostatic effect than Rapamycin for both 2D and 3D cultures of HepG2. Besides, all mTOR inhibitors had a higher cytostatic effect on 3D cultures when compared to monolayers, which can be related to the overexpression of the mTOR pathway in this culture system, already reported on the literature. Based on these results, Neq0438 and Neq0679 should now be used alone or in combination using in vivo models to investigate their antitumoral effects.

Os transtornos do espectro autista (TEA) são distúrbios neuropsiquiátricos bastante comuns, graves, e que propiciam grande impacto social e financeiro. A identificação de vias moleculares e processos celulares alterados que são compartilhados pelos pacientes, mesmo que estes apresentem causas etiológicas distintas, pode contribuir de forma significativa para o entendimento de sua patofisiologia desses transtornos. Ainda, a identificação destas vias pode propiciar o desenvolvimento de abordagens terapêuticas mais eficientes, uma vez que o uso de medicamentos nos TEA ainda é inadequado, envolvendo baixa melhora funcional e diversos efeitos colaterais, como o ganho excessivo de peso e anormalidades metabólicas associadas. Neste trabalho, selecionamos como uma primeira abordagem o estudo da via de sinalização PI3K-mTOR em pacientes com TEA não-sindrômico, via esta envolvida com diversos aspectos do desenvolvimento e funcionamento neuronal, assim como com a patofisiologia de síndromes monogênicas que apresentam alta prevalência de TEA em seu quadro clínico. Foram utilizadas como modelo experimental in vitro células-tronco mesenquimais provenientes de polpa de dente decíduo (SHEDs) de pacientes e indivíduos controles. Os resultados aqui obtidos sugerem a importância da desregulação da via PI3K/mTOR na patofisiologia de uma parcela importante dos casos de TEA não-sindrômico. Ainda, observamos que as células dos pacientes com alterações nessa via de sinalização apresentam maior capacidade proliferativa, e que a modulação deste fenótipo alterado por meio do uso concomitante de inibidores de PI3K e mTOR nas células de um destes pacientes sugere esta via como um alvo promissor para o desenvolvimento de novas abordagens terapêuticas para estes pacientes. Em seguida, na tentativa de desvendar os mecanismos subjacentes aos efeitos metabólicos adversos associados com o uso de antipsicóticos prescritos para o tratamento de pacientes com TEA, investigamos os efeitos destes psicofármacos sobre a biologia do tecido adiposo humano. Foram utilizadas como modelos in vitro células-tronco (ADSCs) e adipócitos maduros derivados de tecido adiposo humano de indivíduos controles. Os resultados obtidos sugerem que a ação direta dos antipsicóticos com alta propensão ao ganho de peso (como a olanzapina e a clozapina) sobre a proliferação, diferenciação, e o metabolismo do tecido adiposo humano parece não constituir um mecanismo importante associado ao ganho de peso apresentado pelos pacientes, e que a ação desses medicamentos sobre os sistemas centrais que regulam o peso e o metabolismo deve ser o mecanismo principal levando aos efeitos metabólicos adversos. Juntos, os resultados gerados neste trabalho podem, de certa forma, contribuir para da farmacoterapia dos TEA / Autism spectrum disorders (ASD) are common neuropsypchiatric disorders, which has serious social and economic impacts. Identification of common molecular and cellular processes altered in patients, despite the underlying genetic heterogeneity, can contribute significantly to our understanding of the disease pathophysiology and can help to develop more effective treatments, since available pharmacotherapy for ASD is inefficient and frequently associated with adverse side effects, such as weight gain and metabolic disturbances. Here, we used patient-derived Stem cells from Human Exfoliated Deciduous teeth (SHEDs) as an intro model system to investigate whether non-syndromic ASD patients show altered regulation of PI3K/mTOR signaling pathway, which is involved in multiple aspects of neuronal development and physiology, and in the pathogenesis of monogenic syndromes that share features with ASD. Our results suggest that dysregulation of PI3K/mTOR-linked networks play an important role in the pathogenesis of a subgroup of non-syndromic ASD. In addition, we found enhanced proliferative capacity in cells with altered PI3K/mTOR activity, which was rescued in one of these patients through combined pharmacological inhibition of both PI3K and mTOR kinase activity, suggesting that PI3K-mTOR signaling is a promising target for the development of new therapeutic approaches for these individuals. Next, in an attempt to better understand the mechanisms underlying the metabolic side effects of the antipsychotics prescribed for ASD treatment, we investigated the effects of some of these drugs on the biology of human adipose tissue using as in vitro model systems human adipose-derived stem cells (ADSCs) and mature adipocytes. Our results suggest that a direct and potent effect of antipsychotics with high weight gain liability (such as clozapine and olanzapine) on cell proliferation, differentiation, and metabolism of human adipose tissue is not an important mechanism by which these drugs induce metabolic disturbances. Consequently, our results suggest that these side effects may mainly reflect the action of these drugs on central pathways involved in weight control and metabolism. Together, our results can, to some extent, contribute to improving pharmacotherapy of ASD

A infecção por HPV de alto risco é considerada um dos principais fatores de risco para o desenvolvimento do carcinoma do colo uterino, um das neoplasias mais freqüentes em mulheres de todo o mundo. As oncoproteínas E6 e E7 de HPV-16 são capazes de induzir a degradação dos genes supressores de tumor p53 e pRb, respectivamente. Mais do que isso, a expressão dessas oncoproteínas está relacionada a alterações na via de PI3K/AKT/mTOR. A proteína quinase mTOR apresenta importante papel no controle da tradução de proteínas e é considerada o principal mediador entre crescimento celular e proliferação. A ativação de mTOR é correlacionada à fosforilação das proteínas eIF4G1 e 4EBP1, aumentando assim a taxa de síntese de proteínas. A Rapamicina é um inibidor específico de mTOR e seus análogos apresentam potente atividade antiproliferativa em um grande número de células tumorais e tumores gerados em animais. Uma vez que as proteínas E6 e E7 são capazes de interagir com diversas proteínas da via que controla a atividade de mTOR optamos por investigar o efeito da rapamicina na proliferação de culturas organotípicas de queratinócitos expressando esses genes. Também avaliamos o efeito dos genes E6 e E7 na atividade de mTOR após o tratamento com essa droga. Para geração de culturas organotípicas de queratinócitos infectamos essas células com vetores retrovirais recombinantes contendo os genes E6 e E7 de HPV-16 em conjunto ou separadamente. Nós também avaliamos o papel da degradação de p53 e pRb na resposta à rapamicina através da utilização de mutantes de E6 e E7 incapazes de induzir a degradação dessas proteínas celulares. Após a infecção dos queratinócitos, os mesmos foram semeados em uma matriz de colágeno. Após 6 dias as culturas foram tratadas com 100ng/ml de rapamicina e permaneceram 60h em contato com a droga. Para análise por imunohistoquímica os tecidos foram fixados em formalina tamponada e emblocados em parafina. A reação de imunohistoquímica foi realizada utilizando os anticorpos contra BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) e pAKT (ser 473). Os resultados obtidos ilustram que a rapamicina apresenta efeito antiproliferativo em culturas de queratinócitos contendo o vetor vazio. Por outro lado, culturas contendo o gene E7 são resistentes ao efeito antiproliferativo dessa droga. Essa resistência parece estar relacionada à capacidade de E7 induzir a degradação da proteína pRb, uma vez que em queratinócitos expressando o mutante de E7, incapaz de induzir a degradação dessa proteína, não foi observada resistência. Além disso, a fosforilação de eIF4G e 4EBP1 indica que a expressão de E7 impede que a rapamicina seja capaz de inibir a atividade de mTOR. Esses resultados mostram, pela primeira vez, que o efeito antiproliferativo da rapamicina pode ser superado pela expressão de uma proteína viral, no caso a proteína E7 de HPV-16. / High-risk HPV infection has a major etiologic role in development and progression of cervical cancer, one of the most frequent forms of cancer among women worldwide. HPV-16 E6 and E7 oncoproteins are able to induce degradation of p53 and pRb tumor suppressor proteins respectively. Moreover, the expression of these oncoproteins is related to alterations in the PI3K/AKT/mTOR pathway. The cellular kinase mammalian target of Rapamycin (mTOR) is an important regulator of the cellular protein synthesis machinery and has emerged as a principal mediator of cell growth and proliferation. mTOR activation has been shown to stimulates eIF4G1 and 4EBP1 phosphorylation, thus increasing the rate of protein synthesis. Rapamycin is a specific inhibitor of mTOR signaling pathway and its analogues have demonstrated impressive activity against a broad range of human cancer derived cell lines in culture and in human tumor xenograft models. Since E6 and E7 target several proteins controlling the mTOR pathway we aimed to investigate the effect of Rapamycin in the proliferation of organotypic raft cultures expressing these genes. We also evaluated the effect of E6 and E7 genes in mTOR activity after rapamycin treatment. To generate organotypic culture of keratinocytes we infect these cells with recombinant retroviruses containing HPV-16 E6 and E7 together or separately. We also analyzed the role of p53 and pRb degradation in rapamycin responsiveness by using E6 and E7 mutants lacking the hability to inactivate these cellular proteins. After infection, keratinocytes were seeded on to a collagen matrix. After 6 days, these cultures were treated with 100ng/ml of Rapamycin for 60 hours. BrdU was added in the last 12 hours to evaluate proliferation. For immunohistochemistry analysis tissues were fixed in buffered formalin and embedded in paraffin. Immunohistochemistry reactions against BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) and p-AKT (ser 473) were performed The results show that proliferation of organotypic cultures of keratinocytes transduced with empty vector is inhibited by Rapamycin. On the other hand, cultures generated with keratinocytes transduced with E7 gene were completely resistance to the antiproliferative effect of Rapamycin. Moreover, we found that this antiproliferative effect was dependent of Rb degradation since the cells transduced with E7 mutant unable do induce Rb degradation were sensitive. In addition, eIF4G and 4EBP1 phosphorylation indicates that E7 expression impairs mTOR inhibition by rapamycin. AKT phosphorilation indicates that rapamycin resistance could be dependent of Rb inactivation induced by E7 expression. These results show for the first time that the Rapamycin antiproliferative effect is bypassed by the expression of a viral oncogene, in this case the HPV-16 E7. Moreover, E7 expression impairs rapamycin to inactivate mTOR.

Les lymphocytes T (LT) jouent un rôle clé dans la mise en place d’une réponse immunitaire efficace. De part des besoins énergétiques et biosynthétiques distincts, leur activation, leur prolifération et leur différenciation fonctionnelle requièrent un contrôle métabolique très fin. La kinase mTOR est apparue comme un régulateur important de la biologie des LT. En effet, cette kinase contrôle le métabolisme et permet une augmentation de la synthèse d’énergie notamment par une augmentation de la glycolyse, indispensable à l’activation des LT. mTOR détecte la disponibilité des nutriments (ex. les acides aminés et le glucose) ainsi que les facteurs de croissance, puis intègre de tels signaux pour réguler le métabolisme des LT. Un rôle clé des acides aminés dans la réponse des LT a été mis en évidence. La petite protéine-G RagA joue un rôle clé dans la détection du glucose et des acides aminés ramifiés, requise pour l’activation de mTOR dans le complexe mTORC1, impliqué dans le contrôle du métabolisme. Afin d’appréhender l’influence du microenvironnement métabolique sur l’activation, la prolifération et la polarisation des LT auxiliaires, nous avons généré et analysé des souris avec des mutations de RagA dans les LT. Les souris mutantes ne présentent aucun signe macroscopique de perturbations du système immunitaire tel que des pathologies autoimmunes ou le développement de tumeurs. Le développement des LT dans le thymus est globalement normal, même si l’on peut observer une légère diminution du développement des LT régulateurs. En périphérie, l’homéostasie immunitaire ne semble pas altérée mis à part une légère diminution du pourcentage de LT mémoires. Nous avons constaté que la perte de RagA entraîne une diminution substantielle de l’activité de mTORC1 observée après activation des LT mais, de façon inattendue, pas une abolition totale. A l’inverse, nous avons observé une augmentation de l’activité de mTORC2 dans les cellules KO. De façon plus surprenante, nous avons mis en évidence que, très rapidement après la délétion de RagA dans le thymus, une faible activité basale de mTORC1 se met en place. Précocement après activation, les LT RagA KO ne présentent pas de problème de survie, cependant ils prolifèrent moins rapidement, ce qui est vraisemblablement dû à un apport d’énergie plus faible par glycolyse. Nous avons constaté que des LT RagA-KO activés in vitro dans des conditions « neutres » expriment spontanément des niveaux plus élevés de T-bet, facteur de transcription « maître » des lymphocytes T auxiliaires de type I (Th1). Aussi suite à une activation des LT en condition polarisante Th1, nous avons observé davantage de cellules RagA-KO que WT produisant de l’interféron-γ. Ces résultats montrent que l’activité de RagA, et par conséquent vraisemblablement de mTORC1, inhibe la différenciation Th1. Nous avons pu constater que les LT RagA-KO favorisent la différenciation Th1 au moins en partie par des mécanismes intrinsèques et extrinsèques. De plus, nous observons une activité tardive de mTORC1 dans les LT RagA-KO. Nous émettons l’hypothèse que RagA inhibe l’activité tardive de mTORC1 et que cette activité tardive permet une meilleure différenciation en Th1. En conclusion, nos résultats montrent que l’absence de la GTPase RagA dans les LT diminue l’activité de mTORC1 sans l’abolir totalement. De façon importante et surprenante, nous démontrons que malgré la baisse d’activité de mTORC1 en absence de RagA, la différenciation en lymphocytes Th1 est augmentée. Ainsi, la GTPase RagA semble avoir un rôle inhibiteur de la différenciation en Th1 potentiellement en inhibant une activité à long terme de mTORC1. / T lymphocytes play a key role in the development of an effective immune response. Because of their distinct energy and biosynthetic needs, their activation, proliferation and functional differentiation require very fine metabolic control. The mTOR kinase has emerged as an important regulator of the biology of helper T cells. Indeed, this kinase controls the metabolism and allows an increase in the synthesis of energy in particular by an increase in glycolysis, essential for the activation of T cells. mTOR detects the availability of nutrients, such as amino acids, glucose and growth factors, and then integrates such signals to regulate T cell metabolism. Studies have shown a key role of amino acids in the response of T cells. The small RagA-G protein plays a key role in the detection of glucose and branched amino acids required for the activation of mTOR in the mTORC1 complex involved in metabolic control. In order to understand the influence of the metabolic microenvironment on the activation, proliferation and polarization of helper T cells we generated and analyzed mice with mutations of RagA in T cells. Mutant mice show no signs of immune system disturbances such as autoimmune pathologies or tumor development. T cell development in the thymus is g normal even though a slight decrease in the development of regulatory T cells can be observed. In the periphery, immune homeostasis does not seem to be altered except for a slight decrease in the percentage of memory T cells.We found that the loss of RagA results in a substantial decrease in mTORC1 activity after T cell activation but unexpectedly not complete abolition. Conversely, we observed an increase in mTORC2 activity in KO cells. More surprisingly, we have shown that, very soon after the deletion of RagA in the thymus, a low basal activity of mTORC1 takes place. Early after activation, RagA KO T cells did not present a survival problem, however they proliferated less rapidly, which is probably due to a lower energy intake by glycolysis. We have found that RagA KO T cells activated in vitro under "neutral" conditions spontaneously express higher levels of T-bet, the "master regulator" transcription factor of type I (Th1) helper T cells. Therefore, following activation of T cells in polarizing condition Th1, we observed more RagA KO cells than wt producing interferon-γ. These results show that the activity of RagA, and therefore presumably of mTORC1, inhibits Th1 differentiation. We have seen that RagA KO cells favor Th1 differentiation by intrinsic and extrinsic mechanisms. We hypothesize that IFN-γ, more produced by RagA-KO cells, is involved. In addition, we observed a late activity of mTORC1 in RagA-KO LT. We hypothesize that RagA inhibits the late activity of mTORC1 and that this late activity allows better Th1 differentiation. In conclusion, our results show that the absence of RagA GTPase in T cells decreases the activity of mTORC1 without completely abolishing it. Significantly and surprisingly, we demonstrate that despite the decrease in mTORC1 activity, Th1 cell differentiation is increased in the absence of RagA. Thus, RagA GTPase appears to have an inhibitory role in Th1 differentiation potentially by inhibiting a long-term activity of mTORC1.

Les cellules NK sont des lymphocytes de l’immunité innée qui ont un rôle majeur dans le contrôle précoce des infections virales et dans l’immunosurveillance des tumeurs. Cependant, en cas de stimulation chronique, un état d’anergie, où les cellules NK n’exercent plus leur fonction a été mis en évidence. Les mécanismes conduisant à cette perte de fonction demeurent mal caractérisés et s’il s’agit d’une cause ou d’une conséquence de la chronicité n’est pas non plus déterminé. Cibler les cellules NK constitue une perspective thérapeutique de choix mais nécessite une meilleure compréhension de ces aspects. L’objectif de ce travail de thèse s’articule autour de trois points. Premièrement, nous avons généré un modèle tumoral murin sensible aux cellules NK permettant l’étude de la réponse anti-tumorale et l’établissement d’une stimulation chronique. Deuxièmement, l’utilisation de ce modèle a permis d’investiguer les mécanismes conduisant à la perte de fonctionnalité des cellules NK et de tester des stratégies de restauration. Enfin, nous avons mené une étude parallèle, chez l’homme, par analyse d’une cohorte de patients chroniquement infectés par le virus de l’Hépatite B dans le but de déterminer le phénotype des cellules NK et d’identifier les causes conduisant à leur perte de fonction dans ce contexte / NK cells are innate lymphocytes which play a crucial role in the early control of viral infection and in tumor immunosurveillance. However, a state of tolerance, where NK cells are poorly functional, occurs in the context of chronic stimulation. The mechanisms leading to this process remain poorly understood and whether this is a cause, or a consequence of chronicity is unknown. Targeting NK cells appears to be a potent therapeutic strategy but requires further investigation. With the lack of clarity in the field this work had three main objectives. First, we engineered a tumoral mouse model that was strongly immunogenic for NK cells and thus allowed us to study the anti-tumoral response of NK cells and to trigger chronic stimulation. Then, we used this model to investigate the mechanisms driving NK cell loss of function and to test potential therapeutic strategies to reverse this state. Finally, in the context of human chronic infection we analyzed samples from HBV infected patients in order to determine the phenotype, function and signaling capacity of NK cells to identify the drivers of NK cell dysfunction

As solid tumor types, breast and prostate cancer are rivalled only by lung cancer in their propensity to metastasize to bone in the later stages of disease. At advanced stages of disease, approximately 80% of breast and 90% of prostate cancer patients will present with bone metastases. Bone metastases are often a painful conclusion to the lives of these patients, resulting in bone pain, hypercalcemia, pathological fractures and spinal cord compression. The culmination of these comorbidities considerably reduces a patient’s quality of life and prolonged survival. Hormone depletion is used as a first line of treatment in the majority of cases, negatively regulating bone health due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts. Not only is bone integrity undermined, but this action of increased bone turnover is beneficial for the colonization of metastasizing cells which co-opt and enhance the same mechanisms to establish and maintain their own growth. This is termed ‘the vicious cycle’ of osteolytic bone metastasis. Current research approaches aim to identify bone-targeted therapies which not only inhibit tumor growth but concurrently protect bone. In this study, Dickkopf-1 (DKK-1), mechanistic target of rapamycin (mTOR) and p38 mitogen-activated kinases (p38 MAPK) are presented as novel targets. Pro-tumor roles have been described for all and clinical trials are currently investigating their efficacy in different cancer types. In normal bone biology DKK-1 is an inhibitor of the canonical Wnt signaling pathway which promotes osteoblastogenesis while mTOR signaling is a promoter of osteoclastogenesis. P38 MAPK inhibitors have been shown to regulate DKK-1 expression and bone destruction in preclinical models of multiple myeloma. The aims of this current study were to 1) investigate the role of DKK-1 in the biology of osteotropic breast cancer, 2) to assess the potential bone protective effects of mTOR inhibition by everolimus in the context of osteotropic cancers and 3) to test the hypothesis that p38 MAPK is a regulator of DKK-1 expression in prostate cancer, potentially supporting an osteolytic phenotype by impairing osteoblastogenesis. In aim 1, analysis of a breast cancer tissue microarray demonstrated that DKK-1 expression was elevated in advanced and invasive tumor stages. Strikingly, positive DKK-1 expression correlated with a significantly reduced survival rate only in estrogen receptor-negative (ER-) breast cancer patients compared to patients with tumors which were negative for DKK-1 expression. In MDA-MB-231 breast cancer xenograft models, neutralization of secreted DKK-1 by treating mice with the monoclonal DKK-1 antibody BHQ880 or knocking out the expression of DKK-1 in MDA-MB-231 cells using CRISPR-Cas9 mediated gene editing, resulted in reduced tumor growth and burden by ≥ 50% (p < 0.05). In aim 2, the mTOR inhibitor everolimus is presented as an anti-tumor and bone-protective agent. The anti-tumor effects of everolimus were confirmed in two subcutaneous tumor models and a model of breast cancer bone metastasis, were tumor burden in the bone was reduced by 45.4% (p < 0.01). Bone loss induced by a hormone-deprived environment in ovariectomized mice was prevented with everolimus treatment as was bone destruction in the metastasis model. In more detail, it could be shown that everolimus maintained osteoblast function while specifically inhibiting osteoclast function. In aim 3, p38 MAPK is presented as a regulator of DKK-1 in prostate cancer. While the activation of p38 MAPK upregulated DKK-1, inhibition of p38 MAPK using small molecule inhibitors and siRNAs inhibited DKK-1 expression. Furthermore, assessment of different p38 MAPK isoforms revealed MAPK11 as the most effective regulator of DKK-1 and inhibition of DKK-1 by interfering with p38 MAPK signaling was sufficient to prevent the inhibitory effects of prostate cancer-derived DKK-1 on osteoblastogenesis in vitro. This study has assessed multiple targets and their concurrent roles in cancer and bone cell biology. Specifically, DKK-1 has been proven to be a tumor promoter in ER- breast cancer and can be targeted therapeutically to inhibit tumor growth. MTOR inhibition by everolimus has been shown to be an effective mono-therapy in ER- breast cancer, inhibiting the growth of subcutaneous tumor and bone metastases and preventing bone loss induced by estrogen ablation. This further supports its use in postmenopausal women with breast cancer who are predisposed to developing osteoporosis and bone metastases. It also supports the use of everolimus in hormone receptor-negative or triple receptor-negative breast cancer, for which it has not yet been approved. A clear link has been made between p38 MAPK signaling and DKK-1 expression in prostate cancer and its consequent regulation of osteoblastogenesis. A future focus on the inhibition of a specific MAPK isoform, MAPK11 in particular, may help in translating these encouraging in vitro results into promising pre-clinical trials in vivo. As a whole, these investigations provide a foundation for further research and could be valuable for the design of future clinical trials, leading to improvements in the treatment and prognosis of osteolytic bone metastases.

La voie de signalisation mTOR intègre une variété de signaux environnementaux pour réguler la croissance et le métabolisme cellulaire. Cette voie est altérée dans 70% des cancers. Les inhibiteurs allostériques de mTOR, comme la rapamycine et ses dérivés (évérolimus et temsirolimus), sont administrés aux patients atteints de tumeurs métastatiques du sein, du rein et neuroendocrines. Cependant, leur efficacité reste modeste et une majorité de patients rechutent. L'utilisation de rapalogues fait donc face à deux problèmes cliniques majeurs : 1/l’absence de biomarqueur qui permette de stratifier les patients qui bénéficieraient le plus d'un traitement par rapalogues ; 2/ l’existence de plusieurs mécanismes de résistance décrits ou non. L’objectif de mon travail de thèse est d’identifier des nouveaux gènes cible des rapalogues utilisables comme biomarqueurs prédicteurs de l’efficacité du traitement ou comme cibles thérapeutiques pour vaincre la résistance.Nous avons identifié le gène TRIB3 comme cible des rapalogues. Sous traitement, son expression est diminuée dans un panel de lignées tumorales et des échantillons tumoraux. Nous avons démontré que cette régulation est indépendante de l’inhibition de la voie mTOR, mais médiée par le répresseur transcriptionnel GCF2. Des analyses protéomiques à haut débit ont identifié TRIB3 en tant que composant du spliceosome. De plus, nous avons démontré que la régulation négative de TRIB3 est nécessaire aux rapalogues pour modifier l'épissage des pré-ARNm. A l’inverse, la surexpression de TRIB3 supprime ces effets des rapalogues. En conclusion, ce travail de thèse ouvre plusieurs perspectives: 1 / l'utilisation potentielle de TRIB3 comme biomarqueur pour prédire ou évaluer l'efficacité du traitement par les rapalogues; 2 / de nouvelles opportunités thérapeutiques ciblant ces mécanismes indépendants de mTor ; 3/ la combinaison possible des rapalogues avec des composés ciblant l’épissage afin de surmonter la résistance. / The mTOR signaling pathway senses variety of environmental cues and integrates them to regulate cellular growth and metabolism. This pathway is altered in 70% of cancers. Allosteric inhibitors of mTOR like rapamycin and its derivatives (everolimus and temsirolimus) have become standard of care in patients with metastatic breast, kidney and neuroendocrine tumors. Unfortunately, their role is modest and most of patients will relapse. Thus, in clinic there are two major concerns related to the use of rapalogs: 1/ the absence of accurate biomarker to stratify patients who would benefit from rapalogs treatment; 2/ the existence of known and unknown mechanisms of resistance. Accordingly, the aim of my PhD project is to identify new target genes of rapalogs that could be used as biomarkers to predict treatment efficacy, or as therapeutic targets, to overcome resistance.We identified TRIB3 gene as a novel target of rapalogs. Upon treatment, its expression is down-regulated both in a panel of cancer cell lines and in cancer patient samples. We showed that this regulation is independent of the mTOR signaling inhibition, but relies on a transcriptional regulation via the co-repressor GCF2. High-throughput proteomic analyses identified TRIB3 as a component of the spliceosome. Additionally, we demonstrated that the down-regulation of TRIB3 is necessary for rapalogs to alter pre-mRNA splicing. In contrast, the, overexpression of TRIB3 abolishes these effects of rapalogs. In conclusion, this PhD work leads to the following important perspectives: 1/ the potential use of TRIB3 as a biomarker to predict or asses the efficacy of rapalogs treatment; 2/ new window of therapeutic possibilities by targeting this mTOR - independent mechanism of action; 3/ the potential combination of rapalogs with splicing targeting agents to overcome resistance.

Skeletal muscle protein synthesis is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. The first objective was to optimize the methodological procedures for assessing mTOR signaling in horses. The response of mTOR signaling (P-Akt Ser473, P-S6K1 Thr389, P-rpS6 Ser235/26 & 240/244, and P-4EBP1 Thr37/46 by Western blotting techniques) to meal consumption was determined at three gluteal muscle biopsy depths (6, 8, and 10 cm), and the repeatability of the contralateral side at 8 cm during 5 days of repeated biopsies. There was no effect (P > 0.05) of sampling side or biopsy depth on mTOR signaling in mature horses. During repeated biopsies there was an increase (P < 0.05) in downstream (P-S6K1 Thr389, P-rpS6 Ser235/236 & 240/244 and P-4EBP1 Thr389) mTOR signaling in response to feeding. The second objective was to characterize alterations in mTOR signaling throughout the equid lifespan. Adolescent horses (yearlings and two year olds) studied in the postprandial had a lowered (P < 0.05) activation of downstream mTOR signaling with aging. There was a lower (P < 0.05) abundance of P-S6K1 Thr389 in aged horses (23.5 years old) than in mature horses (11 years old) during the post-absorptive state. The final objective was to assess mTOR signaling during acute and chronic inflammation. Acute inflammation occurred during 5 days of repeated biopsies, and chronic inflammation is characteristic of the aged. During acute inflammation, characterized by increased muscle mRNA expression of inflammatory cytokines, there was an increase (P < 0.05) in downstream mTOR signaling. Chronic inflammation resulted in a decrease (P < 0.05) in the abundance of P-S6K1 Thr389. Phenylbutazone was administered to reduce (P < 0.05) acute and chronic inflammation in muscle. Phenylbutazone administration during acute inflammation reduced (P < 0.05) the activation of downstream mTOR signaling and trended to increase (P = 0.09) P-S6K1 Thr389 abundance during chronic inflammation. Whole-body protein synthesis determined using isotope infusion techniques increased (P < 0.05) when chronic inflammation was reduced due to phenylbutazone administration. This research provides new standards for muscle biopsy collection when examining mTOR signaling, and insight into management and feeding practices for adolescent and aging horses.