Le rôle central de la voie mTOR (mammalian Target Of Rapamycin) dans l’homéostasie cellulaire suscite un réel intérêt dans de nombreux domaines thérapeutiques. Les inhibiteurs de mTOR (mTORi) sont utilisés en prévention du rejet de greffe par leur action d’inhibition de l’activation lymphocytaire T et l’induction de lymphocytes T régulateurs (Treg). En cancérologie, les mTORi sont utilisés pour leur action anti-proliférative et anti-angiogénique. Cependant, l’efficacité clinique de ces traitements reste modeste. Bien que des mécanismes de résistance aux mTORi intrinsèques à la cellule tumorale ont été identifiés, leur impact sur le système immunitaire émerge comme un facteur essentiel dans leur efficacité. Notre hypothèse est donc que l’efficacité anti-tumorale des mTORi serait également liée à la modulation de l’immunité anti-tumorale induite par ces traitements.Nous avons réalisé un immunomonitoring, du taux de Treg et de la réponse lymphocytaire Th1 anti-tumorale au sein d’une cohorte prospective de patients atteints de cancer rénal métastatique traités par éverolimus (mTORi).Nous avons observé une augmentation des Treg et de leur fonction suppressive à partir du 2ème mois de traitement dans la quasi-totalité des patients. Paradoxalement, une augmentation des réponses Th1 anti-tumorales a également été observée chez ces patients. Au moment de la progression tumorale, la majorité des patients ont présenté une forte augmentation des Treg associée à une perte de la réponse Th1. Ainsi, nous avons pu définir différents profils immunitaires basés sur la modulation de ces deux paramètres. Les patients présentant une diminution précoce des Treg associée à une augmentation de la réponse Th1 avaient une meilleure survie sans progression par rapport aux autres patients (13.2 vs 4 mois p= 0.02). De façon consistante, cette immunomodulation a été confirmée chez des patients atteints de tumeur neuroendocrine traités par éverolimus. De plus, l’impact du traitement sur les Treg et la réponse Th1 n’était pas influencé par la pharmacocinétique du médicament. Par la suite, nous avons étudié in vivo, chez des souris porteuses de différentes tumeurs, l’impact des mTORi sur la réponse immunitaire anti-tumorale. Comme chez les patients, nous avons observé une augmentation des Treg chez les souris traitées par mTORi. Ainsi, l’utilisation d’anticorps déplétants les Treg ou de souris transgéniques ont permis de démontrer le rôle délétère des Treg sur l’efficacité anti-tumorale des mTORi. Ces résultats suggèrent fortement que les mTORi ont un double effet sur le système immunitaire dans un contexte tumoral, un premier néfaste par l’induction d’un environnement immunosuppresseur et le second positif par l’augmentation d’une réponse Th1 anti-tumorale.Dans un deuxième travail, nous avons exploré l’effet positif des mTORi sur le système immunitaire afin d’optimiser les immunothérapies anti-tumorales. Nous avons montré que l’administration de temsirolimus (mTORi) améliore l’efficacité anti-tumorale d’un vaccin thérapeutique chez la souris. L’effet synergique de cette combinaison était lié à une augmentation de l’infiltration tumorale des LT CD8 anti-tumoraux de phénotype centro-mémoire. L’efficacité de cette combinaison a été nettement améliorée par l’addition d’un antagoniste de CCR4 permettant d’éliminer les Treg induits par le temsirolimus.En conclusion, les mTORi demeurent une stratégie prometteuse en cancérologie malgré la réduction de leur utilisation due à l’arrivée des nouvelles immunothérapies, comme les checkpoints inhibiteurs (notamment dans le cancer du rein) et d’autres thérapies ciblées (inhibiteurs de cycline dans le cancer du sein). En revanche, le futur développement des mTORi devra s’appuyer sur des biomarqueurs prenant en compte l’impact de ces traitements sur le système immunitaire et sur des combinaisons thérapeutiques notamment avec les immunothérapies / The key role of the mTOR (mammalian Target of Rapamycin) pathway in cellular homeostasis raises a real interest in many therapeutics areas. Inhibitors of mTOR (mTORi) are used for graft rejection prevention to inhibit the T lymphocyte (LT) activation and induce regulatory T cells (LTreg). In cancerology, they are used for their antiproliferative and antiangiogenic actions. However, the clinical efficacy of those treatments is low. Although several mTORi resistance mechanisms linked to the tumor cell have been identified, their impact on the immune system appears as a key factor in their efficiency. Thus, our hypothesis is that the clinical efficacy of mTORi could also be dependent of an anti-tumoral immunity modulation resulting from those treatments.We performed an immunomonitoring of the Treg rate and the tumor specific Th1 anti-tumor response among a prospective cohort of patients with a metastatic renal carcinoma treated by everolimus (mTORi). We observed that the Treg rate and the suppressive function increase after the second month of treatment for almost all the patients. Paradoxically, an increase of the Th1 anti-tumoral response has also been observed for these patients. At disease progression, a majority of the patients has shown an important increase of Treg with a decrease of the Th1 response. Thus, we have been able to define different immune profiles based on the modulation of these two parameters. Progression free survival of patients with an earlier decrease of Treg and an increase of Th1 response was significantly longer compared to other patients (13.2 vs 4 months p=0.02). This immunomodulation has been consistently confirmed with patients affected by neuroendocrine tumor and treated with everolimus. The treatment impact on Treg and the Th1 response was not related to the pharmacokinetic of the drug. Thereafter, we have studied in vivo the impact of mTORi on the anti-tumoral immune response with mice affected by different tumors. We have observed the same increase of Treg in mice treated with mTORi as the increase observed in the patients. Thus, using antibodies depleting the Treg or transgenic mice allowed us to confirm the deleterious role of Treg on the anti-tumor mTORi efficacy. Those results strongly suggest that mTORi have a double effect on the immune system, a harmful impact by the induction of an immunosuppressive environment and a positive impact by the increase of the anti-tumor Th1 response.In a second time, we have studied the positive effect of mTORi on the immune system in order to optimize the anti-tumoral immunotherapies. We have shown that the administration of temsirolimus (mTORi) improves the anti-tumoral efficiency of a therapeutic vaccination on mice. Indeed, the synergic effect of this combination is tied with an augmentation of tumor infiltrate anti-tumor T CD8 with a central memory phenotype. The efficiency of this combination has been greatly improved by the addition of an antagonist CCR4 allowing the elimination of Treg generated by temsirolimus.In conclusion, mTORi remain a promising strategy in cancerology even if their use is likely to be reduced as new immunotherapies such as checkpoint inhibitors (renal carcinoma) or other targeted therapies (cycline dependent kinase inhibitors in breast cancer) appeared. In the future, the development of mTORi must integrate biomarkers taking into account the impact of these treatments on the immune system, and therapeutic combination such as the immunotherapy

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 inibição de mTOR com rapamicina exacerba a intolerância glicose associada a obesidade, um efeito que pode estar associado ao desenvolvimento de processo pró-inflamatório no tecido adiposo. O tratamento de camundongos com rapamicina exacerbou a intolerância a glicose e inflamação do tecido adiposo induzida por dieta hiperlipídica. In vitro, a inibição dos complexos 1 e 2 da mTOR com rapamicina e torina induziu polarização espontânea de macrófagos para o fenótipo M1 e aumentou a atividade fagocítica de macrófagos M0. Camundongos com ativação constitutiva de mTORC1 exclusivamente em células mielóides foram protegidos do ganho de peso, adiposidade, intolerância a glicose e a insulina induzidos pela ingestão de dieta hiperlipídica e apresentaram um aumento da polarização de macrófagos para o fenótipo M2. Em conjunto, nossos dados indicam que a atividade do complexo 1 da mTOR atenua o desenvolvimento da inflamação do tecido adiposo associada a obesidade por um mecanismo que envolve a polarização de macrófagos para o fenótipo M2. / Pharmacological mTOR inhibition with rapamycin exacerbates the glucose intolerance associated with obesity, such an effect that may be associated to the development of inflammatory process in adipose tissue. Rapamycin treatment exacerbates the glucose intolerance and adipose tissue inflammation induced by high fat diet feeding. In vitro, inhibition of mTOR complexes 1 and 2 with rapamycin and torin induced spontaneous macrophage polarization into a pro-inflammatory M1 phenotype and increased M0 macrophage phagocytosis. Mice with constitutive activation of mTOR complex 1 in myeloid cells were protected from body weight gain, fat accretion, glucose and insulin tolerance induced by the intake of high fat diet and displayed a significant increase in macrophage polarization to a M2 phenotype. Altogether, our findings indicate that the activity of mTOR complex 1 attenuates the development of adipose tissue inflammation induced by high fat feeding, through a mechanism that involves a higher polarization of macrophages to anti-inflammatory M2 phenotype.

As células produtoras de anticorpos desempenham um papel chave na resposta efetora a microrganismos, sendo o foco principal da maioria das vacinas existentes. Entretanto, elas podem ter efeitos deletérios em doenças autoimunes e na rejeição a transplantes. Apesar de grandes avanços no controle da resposta humoral, alguns desafios permanecem e neste contexto, novos alvos terapêuticos têm sido explorados. Sabe-se que alterações metabólicas decorrentes da ativação de células B estão intimamente relacionadas com a função efetora destas células, o que anteriormente se imaginava ser apenas um reflexo de crescimento e proliferação celular. Estas alterações são controladas por sensores metabólicos ativados logo após a ativação de células B, como o mTOR, o qual é componente central de dois complexos: mTORC1 e mTORC2. Estudos anteriores já reportaram o papel positivo exercido por mTOR na via glicolítica em células T, bem como na função efetora destas células. Aqui, nós formulamos a hipótese de que a via do mTOR favorece a via glicolítica em detrimento de OXPHOS em células B, e que estas alterações metabólicas impactam as funções efetoras das mesmas. Desta maneira, para investigarmos alterações nestas vias em decorrência de mTOR, células B foram isoladas de animais controle (CT), ou de animais com células B deficientes de mTORC1 (RaptorΔB) ou mTORC2 (RictorΔB) e então estimuladas com LPS (lipopolissacarídeo) in vitro. Nossos dados indicam que a deficiência de mTORC2 beneficia OXPHOS em detrimento da via gicolítica, bem como a ativação de células B e a formação de plasmablastos. Na sequência, confirmamos que a redução nas taxas de glicólise, assim como a elevação da oxidação lipídica e de OXPHOS são cruciais para manter a elevada ativação de células B e formação de plasmablastos a partir de células B RaptorΔB e RictorΔB. Constatou-se ainda que a produção total de IgM é elevada em células RictorΔB após estímulo com LPS. Entretanto, identificamos que isso é decorrente do aumento de plasmablastos formados e não da capacidade individual de secreção dos mesmos. Diferentemente das células B deficientes, observamos que plasmablastos RaptorΔB e RictorΔB reduzem a atividade mitocondrial. Na sequência, confirmamos que a atividade mitocondrial via oxidação lipídica é fundamental para a produção de anticorpos. Além disso, demonstramos que a deficiência de mTORC2 eleva a troca de isotipo, enquanto a de mTORC1 a diminui após estimulo com LPS e IL4. Posteriormente, o impacto da deficiência de mTORC2 em células B foi avaliado in vivo em modelo de transplante de pele. Neste caso, não observamos diferenças significativas na sobrevida do enxerto entre CT e RictorΔB, mas foi constatado que apesar de ambos apresentarem formação de plasmócitos similares, animais deficientes apresentaram um número significativamente menor de células B na periferia. Assim, concluímos que a deficiência de mTORC1 ou mTORC2 em células B implica em uma maior diferenciação de plasmablastos e em uma maior produção total de anticorpos em RictorΔB in vitro, enquanto um papel funcional para essas moléculas no contexto das células B in vivo ainda precise ser determinado. / Antibodies are produced by Antibody Secreting Cells (ASCs), which are essential to fight infections. They are also the basis of most successful vaccines available, however they can present deleterious effects in autoimmune diseases and in graft rejection. Even though there have been great improvements in controlling the humoral response, its proper manipulation still remains a challenge, thus new targets need to be explored. It is known that metabolic shifts that occur upon B cell activation are not only essential for cell growth and proliferation, but are also interconnected with these cells effector function. Metabolic shifts are controlled by metabolic sensors, as the mTOR, which is a core component of two complexes, mTORC1 and mTORC2. Previous studies with T cells have already reported that mTOR exerts a positive role on glycolysis, which in turn impacts the effector function of T cells. We then hypothesized that mTOR favors glycolysis over Oxidative Phosphorylation (OXPHOS) in B cells, and that these metabolic changes impact the effector function of B cells. Thus, to investigate the impact of the mTOR pathway on B cells, we isolated B cells from mice with mTORC1 deficient B cells (RaptorΔB) or mTORC2 deficient B cells (RictorΔB) or Control mice (CT), and stimulated them in vitro with lipopolysaccharide (LPS). Our results indicate that the deficiency of mTORC2 favors OXPHOS over glycolysis, as well as B cell activation markers expression and plasmablast formation. Next, we confirmed that the reduced glycolysis levels, improved lipid oxidation and OXPHOS are in fact crucial for the enhanced activation and plasmablast formation observed in RaptorΔB and RictorΔB B cells. We also described that IgM secretion was elevated in B cells from RictorΔB after stimuli with LPS, however we found that this increase was due to the overall increase in plasmablasts in this group and not to their individual antibody secretion capacity. Interestingly, RaptorΔB and RictorΔB plasmablasts differently from B cells, reduce their mitochondrial activity. Subsequently, we confirmed that the mitochondria via lipid oxidation is actually essential for antibody secretion. In addition, we showed that mTORC2 deficiency increases isotype switching, while mTORC1 deficiency diminishes it when IL4 was added to LPS. We then sought to determine if mTORC2 deficiency in B cells would present an impact in vivo in a skin graft model. However, we did not observe a significant difference in graft survival between the CT and RictorΔB mice and in plasmacyte numbers, but we did observe a significant reduction in B cells in the periphery. Thus, we conclude that mTORC1 and mTORC2 deficiency leads to an improved plasmablast differentiation and an overall increase in antibody secretion in the last one in vitro, whereas the role of these sensors remains to be determined in vivo.

A pesquisa de substâncias bioativas apresenta diversas etapas de grande complexidade técnico-científica e envolve uma atividade multidisciplinar. No estudo de antineoplásicos, dentre outros candidatos a fármacos, a determinação do perfil de atividade biológica é um ponto essencial para a determinação da potência, toxidez e o índice de seletividade. Neste projeto, os ensaios in vitro de compostos inibidores de catepsinas serão realizados usando células de câncer de próstata resistentes à quimioterapia (DU 145 e PC-3). Os estudos envolverão análises fenotípicas da resposta celular para determinar a viabilidade e o tipo de perturbação do ciclo celular utilizando técnicas colorimétricas e citometria de fluxo, juntamente com o estudo de sinalização celular PI3K-Akt-mTOR. Os testes de atividade citotóxica serão realizados com as células de fibroblasto de camundongo (Balb-c 3T3). Os resultados levarão a caracterização do tipo de resposta biológica e também da citotoxidez das substâncias de interesse. / The search for bioactive substances leads to molecules that need to be qualified throughout many complex steps, involving multidisciplinary activities. For antineoplastic molecules, among other classes of candidate drugs, the determination of the biological activity is the core of the whole process with the aim of determining the potency, toxicity and the selectivity index. In this project, in vitro cell-based bioassays of cathepsin inhibitors are going to be performed using prostate cancer cells resistant to chemotherapy (DU 145 and PC-3). Phenotypic studies are going to be used as end-points to observe the cell viability and the perturbation of the cell cycle by means of colorimetric, flow cytometry, coupled with the study of PI3K-Akt-mTOR signaling pathway. Cytotoxic assays are going to be performed in mouse fibroblasts (Balb-c 3T3). As a result, the biological outcome and the cytotoxic profile of the compounds of interest will be addressed.

Orientador: Alexandrina Sartori / Resumo: A rapamicina, também conhecida como sirolimus, é um imunossupressor isolado da bactéria Streptomyces hygroscopicus. Este fármaco tem sido empregado clínicamente para o controle de rejeição de transplantes de órgãos sólidos, diabetes, esclerose tuberosa, doenças neurode-generativas e inclusive, no controle da esclerose múltipla (EM). O entendimento do mecanis-mo de ação da rapamicina requer o conhecimento da via de sinalização intracelular envolvendo os complexos mTOR (mammalian target of rapamycin). Evidências indicam que o mTOR está envolvido na ativação pró-inflamatória de leucócitos. Nesse contexto, o objetivo geral desta dissertação foi investigar o efeito in vitro da rapamicina em células envolvidas na imu-nopatogênese da encefalomielite autoimune experimental (EAE), um modelo murino de EM. Células do sisterma nervoso central (SNC) e do baço de camundongos com EAE foram trata-das com rapamicina e simultaneamente estimuladas com glicoproteína da mielina de oligo-dendrócitos. A rapamicina reduziu a produção de IL-17 em culturas de células do SNC e do baço. Adicionalmente, diminuiu a produção de IFN-γ, TNF-α e IL-10 e a expressão gênica de RORc em culturas de células do SNC. A microglia, que é uma célula fagocítica residente no SNC e envolvida na neuroinflamação, também foi testada. Em células da microglia (linhagem BV-2) estimuladas com LPS, a rapamicina diminuiu a produção de TNF-α e IL-6 e também reduziu a mediana da intensidade da fluorescência referente às moléculas MH... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rapamycin, also known as sirolimus, is an immunosuppressant produced by the bacterium Streptomyces hygroscopicus. Rapamycin has been clinically used for the control of rejection of solid organ transplants, diabetes, tuberous sclerosis, neurodegenerative diseases, including multiple sclerosis (MS). To better understand the mechanism of action of rapamycin, is necessary the knowledge of intracellular signaling of the mTOR (mammalian target of rapamycin) complex. Evidence indicates that mTOR is involved in the proinflammatory activation of leukocytes. In this scenario, the main objective of this investigation was to evaluate the in vitro effect of rapamycin in cells derived from mice with experimental autoimmune encephalomielits (EAE) and in a microglia cell line. CNS and spleen cells from EAE mice were treated with rapamycin and simultaneosly stimulated with a myelin oligodendrocyte glycoprotein peptide. Rapamycin reduced IL-17 production in both, CNS and spleen cell cultures. Additionally, rapamycin decreased IFN-γ, TNF-α and IL-10 production and RORc mRNA expression in CNS cell cultures. Microglia, that is a phagocytic immune cell in the CNS involved in neuroinflammation, was also tested. Microglia (BV-2 lineage) treated simultaneously with rapamycin and LPS showed a decreased production of TNF-α and IL-6 and also reduced expression of MHC II, CD40 and CD86. This downmodulatory effect was associated to upregulated TREM2 mRNA and downregulated iNOS mRNA expression. These resul... (Complete abstract click electronic access below) / Mestre

La production d’anion superoxyde (O2-) par le complexe NADPH oxydase 2 (NOX2) du polynucléaire neutrophile (explosion oxydative, EO) contribue à l’élimination d’agents pathogènes. Cette fonction de défense est stimulée par divers agents pro-inflammatoires, notamment par des peptides bactériens (fMLP), qui déclenchent une cascade de signalisation impliquant différentes protéines kinases (PKC, AKT, MAP-Kinases) et aboutissant à l’activation de la NOX2 (également nommée gp91phox), le coeur catalytique du complexe. Dans cette thèse, j’ai identifié la protéine kinase mTOR comme un nouvel effecteur majeur de l’EO dans les neutrophiles sains et j’ai comparé son mode d’action transductionnel dans les neutrophiles de patients ayant une cirrhose alcoolique décompensée en vue de comprendre leur grande susceptibilité aux infections bactériennes.Une contribution majeure de mTOR dans la production d’O2- induite par le fMLP a pu être démontrée à l’aide de son antagoniste spécifique, le médicament Rapamycine et par une approche antisense. mTOR agit en amont de la p38-MAPK qui phosphoryle la p47phox, un composant majeur du complexe NADPH oxydase. Dans les neutrophiles de patients cirrhotiques, l’EO est fortement défaillante, associée à un défaut d’activation de la voie p38-MAPK/p47phox(S345). Ce déficit d’EO est aggravé par la Rapamycine. Les neutrophiles de patients cirrhotiques présentent également un déficit d’expression de la gp91phox (NOX2), p22phox, p47phox et mTOR. Un déficit d’expression de la NOX2 a pu être reproduit en traitant les neutrophiles sains par le fMLP ou du plasma des patients. De plus, ce phénomène met en jeu une dégradation protéolytique insoupçonnée de la gp91phox impliquant l’élastase. Enfin, la déficience fonctionnelle des neutrophiles de patients a pu être corrigée dans des neutrophiles isolés et dans du sang total des patients cirrhotiques à l’aide d’un agoniste de récepteurs « Toll-like receptor (TLR) » qui agit en favorisant la transcription du gène de la gp91phox et sa synthèse protéique.En conclusion, mTOR émerge comme un nouvel effecteur transductionnel majeur de l’EO des neutrophiles, favorisant l’activation de la voie p47phox/gp91phox via les MAPK. Cette nouvelle voie de signalisation est fortement impactée au cours de la cirrhose alcoolique, ce qui favorise la susceptibilité des patients aux infections bactériennes. Bien que notre étude soulève ainsi des inquiétudes quant à l’utilisation d’inhibiteurs de mTOR chez les patients immunodéprimés, elle suscite par ailleurs la perspective de pouvoir corriger les déficits fonctionnels des neutrophiles à l’aide d’agents capables de stimuler des TLR intracellulaires. / Superoxide anion (O2-) production by NADPH oxidase 2 (NOX2) complex of polymorphonuclear neutrophil (i.e respiratory burst, RB) contributes to efficient elimination of pathogens. This defense function is stimulated by various pro-inflammatory agents, especially by bacterial peptides (fMLP) which trigger a signaling cascade involving many protein kinases (PKC, AKT, MAP-Kinases) resulting in activation of NOX2, also called gp91phox, the catalytic core of the complex. In this thesis, I identified the protein kinase mTOR as a novel major RB effector of healthy neutrophils and I compared its transductional activity in neutrophils from patients suffering from alcoholic decompensated liver cirrhosis, aiming at understanding their high susceptibility to bacterial infections.A major contribution of mTOR to fMLP-induced neutrophil superoxide production was demonstrated using its specific drug antagonist Rapamycin, and by an antisens strategy. mTOR is activated upstream of p38-MAPK which phosphorylates p47phox, a major component of the NADPH oxidase complex. In neutrophils from cirrhotic patients, the RB is dramatically impaired and this was associated with a deficient activation of the p38-MAPK/p47phox(S345) signaling pathway. This RB deficiency was aggravated by Rapamycin. Neutrophils from cirrhotic patients also exhibited a deficient expression of gp91phox (NOX2), p22phox, p47phox and mTOR. A deficient NOX2 expression can be reproduced by treating healthy neutrophils with fMLP or plasma from cirrhotic patients. Furthermore, this phenomenon involved an unexpected proteolytic degradation of gp91phox mediated by elastase. Finally, this deficient superoxide production by neutrophil from cirrhotic patients can be corrected ex vivo in isolated neutrophils and in patients’ whole blood, using a Toll-like receptor agonist that acts by promoting the transcription and traduction of gp91phox .In conclusion, mTOR emerges as a novel and major signaling effector of neutrophil RB, promoting the activation of p47phox/gp91phox through MAPKs. This novel signaling pathway is strongly impaired during alcoholic liver cirrhosis, which increases patients’ susceptibility to bacterial infections. Although our study raises concerns about the use of mTOR inhibitors in immunocompromised patients, it also provides therapeutic propects for correcting neutrophil functional deficiencies using agents capable of stimulating intracellular TLR .

O propósito do presente estudo foi verificar os efeitos do overtraining (OT) nas proteínas relacionadas com a via de sinalização da mammalian target of the rapamycin complex 1 (mTORC1), no conteúdo proteico de sterol regulatory element binding protein-1 (SREBP-1) e nas características morfológicas do fígado de camundongos C57BL/6. Os animais foram divididos em grupo controle (CT), overtraining em declive (OTR/down), overtraining em aclive (OTR/up) e overtraining sem inclinação (OTR). Teste do rotarod, incremental, exaustivo e força de preensão foram utilizados para avaliação de performance. Após 36 horas o teste de força de preensão, os fígados foram removidos e utilizados para immunoblotting ou análises histológicas. A fosforilação da proteína kinase B (pAkt; Ser473), mammalian target of the rapamycin (pmTOR; Ser2448), 70-kDa ribosomal protein S6 kinase 1 (pS6K1; Thr389) e da AMP-activated protein kinase (pAMPK; Thr172) foram significativamente maiores no grupo OTR/down quando comparado com os grupos CT e OTR. A fosforilação da 4E-binding protein-1 (p4E-BP1; Thr37/46) foi significativamente maior no grupo OTR/down quando comparado com o grupo CT. Os níveis proteicos de sterol regulatory element binding protein- 1 (SREBP-1; p125 precursor) foram significativamente maiores nos grupos OTR/down e OTR/up quando comparados com o grupo CT. Enquanto o grupo OTR/down apresentou sinais de esteatose com inchaço celular acompanhado de inflamação aguda, os grupos OTR/up e OTR demonstraram evidências de injúria hepática, com a presença de núcleos picnóticos, hepatócitos em balão e vacúolos citoplasmáticos. Conclui-se que o protocolo de OTR/down aumenta a modulação da via de sinalização da mTOR e induz a sinais de esteatose hepática. / The purpose of this study was to verify the effects of overtraining (OT) on the proteins related to the mammalian target of the rapamycin complex 1 (mTORC1) signaling pathway, the protein content of the sterol regulatory element binding protein-1 (SREBP-1) and the morphological characteristics in the livers of C57BL/6 mice. Rodents were divided into control (CT), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR) groups. Rotarod, incremental load, exhaustive and grip force tests were used to evaluate performance. Thirty-six hours after the grip force test, the livers were removed and used for immunoblotting or histological analysis. The phosphorylation of the protein kinase B (pAkt; Ser473), mammalian target of the rapamycin (pmTOR; Ser2448), 70-kDa ribosomal protein S6 kinase 1 (pS6K1; Thr389) and AMP-activated protein kinase (pAMPK; Thr172) were significantly higher in the OTR/down group when compared to the CT and OTR groups. The phosphorylation of the 4Ebinding protein-1 (p4E-BP1; Thr37/46) was significantly higher in the OTR/down group when compared to the CT group. The protein levels of the sterol regulatory element binding protein-1 (SREBP-1; p125 precursor) were significantly higher in the OTR/down and OTR/up groups when compared to the CT group. While the OTR/down group presented signs of steatosis with cell swelling accompanied by acute inflammation, the OTR/up and OTR groups demonstrated evidences of injury in liver, with the presence of pyknotic nuclei, ballooned hepatocytes and cytoplasmic vacuoles. In conclusion, the OTR/down protocol up-modulated the mTOR signaling pathway and induced signs of hepatic steatosis.

Pas de résumé en français disponible. / Pas de résumé disponible.

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.