Abnormal thyroid cell proliferation causes human diseases, such as goiter, thyroid adenoma or carcinoma and primary hypothyroidism resulting from hypoplasia. Thyrotropin (TSH), mainly acting through cAMP and cAMP-dependent protein kinases (PKA), is considered the main regulator of thyrocyte proliferation and differentiation. The general aim of this thesis was to get new mechanistic insights in the regulatory action of the cAMP pathway on various functions of thyrocytes, including proliferation. During the first part of this thesis work, we have collaborated to a study of the effects of the TSH/cAMP pathway on small G proteins of the Rho family and their impact on the actin cytoskeleton and thyroid cell function. This study, performed in canine thyrocytes, showed for the first time, that the TSH/cAMP/PKA pathway inactivates the three small G proteins RhoA, Rac1 and Cdc42 and the RhoA/ROCK/LIMK/cofilin pathway. Inactivation of the latter appeared both necessary and sufficient to mediate the action of TSH and PKA on the reorganization of actin microfilaments and its morphological impact. Moreover, this inactivation by PKA of Rho-mediated actin polymerization also played an important role in the cAMP-dependent expression of thyroid differentiation genes. On the other hand, a residual RhoA activity appeared to be required for mitogenesis. This dependence of DNA synthesis on RhoA activity was not mediated by ROCK-dependent events nor by the integrity of the actin cytoskeleton. Indeed, DNA synthesis induction was unexpectedly resistant to actin depolymerisation in canine thyrocytes, which explains how it could be compatible with the cAMP-dependent microfilament disruption. This first study did not provide new insights on how the cAMP/PKA-dependent mitogenic stimulus can trigger cell cycle progression, which, in thyrocytes, depends on the phosphorylation of pRb by the cyclin D3-CDK4 complex. In the various in vitro thyroid models, the only convergent early signaling event found in response to TSH/cAMP, insulin and growth factors was the phosphorylation and activation of p70 S6K1 which largely depends on mTOR (mammalian Target Of Rapamycin). The main part of the work in this thesis has been devoted to investigation of the action of TSH/cAMP on the mTOR pathway. mTOR is a therapeutic target for a wide variety proliferative disorders and rapamycin derivates are now considered in anti-cancer treatments. We have shown for the first time in PC Cl3 rat thyroid cells that TSH, through cAMP, activates mTORC1, leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMPK and GSK3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate PKB, nor induce TSC2 phosphorylation at Thr1462 and Tyr1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity associated with augmented 4E-BP1 binding to raptor. This could be caused in part by Thr246-phosphorylation of PRAS40, which was found as an in vitro substrate of PKA, but other regulatory events likely remain to be uncovered. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and pRb phosphorylation induced by TSH and insulin. Rapamycin reduced cyclin D3 accumulation but not the abundance of cyclin D3-CDK4 complexes. However, rapamycin inhibited the activity of these complexes and the activating Thr172-phosphorylation of CDK4 stimulated by both TSH and insulin. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 Thr172-phosphorylation.

The mTOR inhibitor and rapalogue everolimus was approved use in 2012, in HR+, HER-2-, post-menopausal patients, who had previously failed aromatase inhibitor treatment. mTOR pathway activation has been associated with resistance to breast cancer therapies, but how cells may become resistant to mTOR therapies themselves in breast cancer is currently not well explored, due to the relative recentness of everolimus approval. Drug resistance across all areas of cancer research is a major clinical issue, often leading to the spread of a patient's cancer. This project set out to create in vitro breast cancer models that were resistant to everolimus, and thus explore any changes that had developed in these models, help determine the mechanisms behind resistance and discover drugs/drug combinations that could overcome resistance. Cell lines T47D and MDA-MB-361 were subsequently developed into everolimus resistant lines (EveR) over the course of 4-6 months using an on/off exposure routine. The exact mechanism behind the everolimus resistance was not fully determined but EveR cells did show multiple intriguing characteristics. An increase in dormancy and stem-cell like phenotype was noted, as revealed by a decrease in cell cycle progression and an increase in increase ALDH activity. mTORC2 components and signalling was up-regulated although siRNA down-regulation of PKCα did not decrease everolimus resistance, suggesting other mTORC2 targets may be involved. The rapalogue 'receptor', FKBP12, was up-regulated which was accompanied by an increased growth inhibition by the rapalogue, temsirolimus, possible due to temsirolimus lower binding affinity for FKBP12 compared to everolimus. No resistance to the dual mTOR/PI3K inhibitor BEZ-235 was observed, in line with similar published work. The combination of vitamin D/calcitriol and everolimus had no added effect compared to everolimus alone, in parental cells, but the addition of 1μM calcitriol did drastically lower EveR cell resistance to everolimus. Future work focusing on the exact nature of calcitriol's interaction with the mTOR pathway is required to advance calcitriols role as a breast cancer therapeutic. Research with everolimus resistant breast cancer patients has not yet been published on, but the work presented here aims to help guide such studies, when they are carried out in the future.

Polyphosphates (PolyP) are linear chains of inorganic phosphates joined together by phosphoanhydride bonds. This polyanionic molecule has been found in all organisms and has been implicated in diverse biological processes such as cell growth and blood coagulation. Nonetheless, the mechanism of polyP’s involvement in these processes and its impact at a molecular level is still unknown. In 2015, the addition of polyP chains to lysine residues was found to be a novel post-translational modification. However, a hurdle in studying the impact of polyP in the mammalian system is its low endogenous levels. In this study, I applied an ectopic expression system using E. coli-derived polyphosphate kinase 1 (PPK) in HEK293T cells to induce excess production of polyP. In mammalian cells, increased intracellular polyP levels lead to increased activation of both Erk1/2 and p70s6k. I also expanded our system by utilizing the yeast exopolyphosphatase, Ppx1, to deplete polyP. Overall, this work presents a novel role for polyP in mediating key signalling pathways and will help probe the function of polyP in mammalian cells.

Les travaux de la thèse portent sur l'implication de la voie de signalisation Akt (sérine/thréonine kinase Akt) et mTOR (mammalian target of rapamycin) dans la physiopathologie de l'hypertension artérielle pulmonaire (HTAP) expérimentale. L'HTAP résulte d'une prolifération exagérée des cellules constitutives des vaisseaux pulmonaires, principalement les cellules musculaires lisses artérielles pulmonaires (CML-AP). De nombreux effecteurs biologiques et physiques préalablement identifiés agissent sur les CML-AP et participent à l'hyperplasie de celles-ci. Nous montrons que ces nombreux effecteurs convergent vers une voie de signalisation intracellulaire commune, la voie Akt/mTOR, qui de fait représente une cible thérapeutique pour le traitement de l'HTAP, et pourrait conditionner l'hyperplasie des CML-AP. mTOR est présent dans la cellule sous forme de deux complexes, mTORC1 et mTORC2, qui phosphorylent des substrats variés contrôlant la prolifération cellulaire. Les effecteurs de mTORC1 incluent les S6 kinases (S6K1 and S6K2) et les "eIF4E-binding proteins" (4EBP) alors que mTORC2 active la sérine/thréonine kinase Akt et parmi les kinases sous-jacentes, la kinase GSK3. La première étude est consacrée à l'évaluation des effets des inhibiteurs de protéases du VIH (ritonavir, amprenavir, nelfinavir) sur la progression de HTAP expérimentale, induite par la monocrotaline ou l'hypoxie. Nous montrons que ces deux formes d'HTAP sont associées à une activation de la voie Akt/mTOR dans les artères pulmonaires. Les traitements respectifs par les trois inhibiteurs des protéases du VIH durant 3 semaines induisent une réversibilité de l'HTAP, de l'hypertrophie ventriculaire droite et du remodelage des vaisseaux pulmonaires, de même qu'une inhibition de la phosphorylation d'Akt, de S6K et de GSK3. La prolifération des CML-AP induite par le PDGF ou le SVF 5%, associée à une augmentation de p-Akt et p-GSK3, est également bloquée par les inhibiteurs des protéases, de façon similaire et non additive à celle d'inhibiteurs spécifiques de la PI3 kinase et de GSK3. La conclusion est que ces traitements antirétroviraux inihibent la progression de l'HTAP en inhibant la voir Akt/mTOR dans les CML-AP. Cette proposition permettrait d'expliquer l'effet suspecté en clinique des traitements antirétroviraux sur l'HTAP compliquant l'infection par le VIH.Dans la seconde étude, nous montrons que les CML-AP en culture prélevées à partir de rats ayant développé une HTAP induite par la monocrotaline proliférent de façon exagérée en comparaison avec les cellules de rats témoins. Ce phénotype prolifératif est observé en présence de nombreux facteurs mitogènes parmi lesquels le SVF 5%, le PDGF, la sérotonine ou 5-HT, l'IGF1 ou l'IL1-beta, et est associé à une activation des substrats de mTORC1 et mTORC2. Le traitement in vitro par la rapamycine des CML-AP de rats avec HTAP établie permet d'inhiber la prolifération de ces cellules et de bloquer à la fois mTORC1 et mTORC2. De même, le traitement par la rapamycine de rats porteurs de l'HTAP préétablie pendant une semaine permet de normaliser la prolifération des CML-AP in vitro et in vivo et d'inhiber mTORC1 et mTORC2, effets non observés par l'Imatinib ou la Fluoxetine. De plus, le traitement des rats par la rapamycine prévient ou corrige l'HTAP induite par la monocrotaline de façon plus importante que l'Imatinib ou la Fluoxétine.Ces résultats indiquent donc que l'activation de la voie Akt/mTOR, très étroitement associée au développement de l'HTAP expérimentale, pourrait expliquer le phénotype prolifératif anormal des CML-AP inhérent à la pathologie, et ainsi représenter une cible thérapeutique de choix pour le traitement de l'HTAP humaine. / The major objectives of research described in this thesis is focused on the cell signaling pathway of Akt (serine/threonine kinase Akt) and mTOR (mammalian target of rapamycin) in the patho-physiology of experimental pulmonary arterial hypertension (PAH). PAH occurs as a result ofhyperplasia of the components of pulmonary vessels, principally the pulmonary arterial smooth muscle cells (PA-SMCs). Numerous previously identified biological and physical effectors act on the PA-SMCs and participate in PA-SMC hyperplasia. Here we show studied that these different effectors converge into a common intracellular signaling pathway, Akt/mTOR signaling pathway, which represents actually a therapeutic target for PAH treatment, and could be involved in the hyperplasia of PA-SMCs. In cells mTOR, is presented in the form of two complexes, mTORC1 and mTORC2, which phosphorylate various substrates controlling the cellular proliferation. The effectors of mTORC1 include the S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4EBP), meanwhile mTORC2 activates the serine/threonine kinase Akt and the underlying kinases, e.g. GSK3 kinase.The first study is devoted to evaluate the effects of the protease inhibitors of HIV (ritonavir, amprenavir, nelfinavir) on experimental PAH development induced by monocrotaline or hypoxia. We studied that the two forms of PAH are associated with an activation of Akt/mTOR signaling pathway in pulmonary arteries. The treatment by the three protease inhibitors of HIV during 3 weeks causes reversibility in experimental PAH with decreased right ventricular hypertrophy and pulmonary vascular remodeling as well as inhibition of phosphorylation of Akt, S6K and GSK3. The proliferation of PA-SMCs stimulated by PDGF or FCS 5%, which is associated with an increased p-Akt and p-GSK3, is also blocked by the proteases inhibitors, in a similar and non additive way like the specific inhibitors of PI3 kinase and GSK3. We conclude that the antiretroviral treatments significantly inhibits PAH development by inhibiting Akt/mTOR signaling pathway in PA-SMCs. This proposition allows explaining the effect of antiretroviral treatments of PAH accompanied with HIV in patients.In the second study, we studied that the cultured PA-SMCs extracted from the rats with monocrotaline induced-PAH(MCT-PAH) proliferates faster as compared to control. This proliferative phenotype is observed in the presence of different mitogenic factors including FCS 5%, PDGF, 5-HT, IGF1 or IL-1β, and is associated with an activation of the substrates of mTORC1 and mTORC2. Treatment with rapamycin in the PA-SMCs extracted from the rats with PAH in vitro inhibits the proliferation and also blocks the activation of mTORC1 and mTORC2. The treatment by rapamycin in the rats with PAH during one week allows normalizing the proliferation of PA-SMCs in vitro and inhibiting the activation of mTORC1 and mTORC2 in vivo. These effects were not observed when treated with imatinib or fluoxetine. Moreover, treatment with rapamycin prevents or reverse MCT induced PAH more significantly than that by imatinib or fluoxetine.These results indicate that the activation of Akt/mTOR signaling pathway isclosely related to experimental PAH development, which can explain the abnormal proliferative phenotype of PA-SMCs involved in the patho-physiology of PAH, and represent a therapeutic target for the treatment of PAH in human.

Atualmente, alguns pesquisadores tem se dedicado ao estudo do efeito da manipulação do treinamento de força sobre a ativação das vias de sinalização intracelular para hipertrofia. Tem-se sugerido que o grau de tensão muscular desempenhe um papel importante nesta sinalização. Dentre os diferentes tipos de ações musculares, as ações excêntricas (AE) reconhecidamente proporcionam maior grau de tensão à estrutura do músculo esquelético. Em particular, AE de alta velocidade parecem exercer um efeito interessante sobre os ganhos de hipertrofia muscular. Porém, pouco se sabe sobre o efeito da manipulação da velocidade sobre as vias de sinalização da hipertrofia. Assim, o presente estudo teve como objetivo verificar o efeito agudo da AE de alta e baixa velocidade sobre a sinalização para hipertrofia muscular. Vinte sujeitos foram aleatoriamente divididos em dois grupos. Um realizou cinco séries de oito AE máximas à 20º/s (EXC20) e o outro à 210o/s (EXC210) do exercício extensão de joelhos. Amostras do músculo vasto lateral foram obtidas antes, imediatamente após e duas horas após o exercício. As análises de quantificação protéica de Akt e p70S6K totais não apresentaram diferenças significantes intra ou inter grupos. A avaliação da fosforilação das mesmas proteínas revelou um efeito principal de tempo, indicando um aumento da fosforilação nos tempos imediatamente após e duas horas após o término do exercício em relação à amostra controle (p<0,05), porém não foram observadas diferenças entre os grupos. Para os dados de expressão gênica de MGF e mTOR, não foram observadas diferenças intra ou inter grupos. Em conclusão, a manipulação aguda da velocidade das AE parece não influenciar a fosforilação ou expressão gênica das proteínas em questão / Recently, many studies have focused on the effects of strength training variables manipulation on the activation of intracellular signaling pathways for skeletal muscle hypertrophy. It has been suggested that the muscle tension plays a major role in such process. Eccentric muscle actions (EE) are notorious for imposing a greater amount of tension on the active muscle. In particular, EE performed at high velocities seems to exert an interesting effect on hypertrophy gains. However, little is known about the effect of EE velocity manipulation on hypertrophy pathways signaling. Thus, the present study aimed to investigate the acute effect of low and high velocity EE on muscle hypertrophy signaling. Twenty subjects were randomly assigned to either a slow velocity group 20o/s (ECC20) or fast group 210o/s (ECC210). Muscle biopsy samples were taken before, immediately after and two hours after the completion of five sets of eight maximal repetitions at the designated velocity, knee extension exercise. Akt and p70S6K analysis did not reveal any differences inter or intra groups. Akt and p70S6K phosphorylation results indicated a main effect for time (p<0,05), with increased phosphorylation values for immediately after and two hours after time points in comparison to control samples. MGF and mTOR mRNA analysis did not return any inter or intra groups differences. In conclusion, the acute manipulation of EE velocity does not seem to differently influence the phosphorylation or expression of the proteins studied

A proteína colibistina (CB), uma Rho GEF neuro-especifica, apresenta papel importante na formação e funcionamento das sinapses inibitórias do sistema nervoso central por interagir com a proteína scaffold gefirina e com receptores GABAA e promover o agrupamento e transporte dessas proteínas para a membrana pós-sináptica. Recentemente, nosso grupo de pesquisa identificou interação de CB com um complexo proteico que controla o início da tradução em eucariotos (complexo eIF3), o que sugeriu pela primeira vez que essa proteína pode estar envolvida também na regulação da tradução em células neurais. Ainda, já havia sido descrito que gefirina, parceira funcional de CB, interage com mTOR, uma quinase que desempenha papel fundamental no controle do início da tradução. Contudo, até o momento não havia estudos adicionais investigando o papel de CB neste cenário. Assim sendo, o presente trabalho teve como objetivo investigar o envolvimento da proteína CB no controle do início da síntese proteica mediada pela via de sinalização mTORC1. Foram utilizados dois modelos experimentais: i) um sistema de expressão heteróloga - superexpressão de CB em células HEK293T, e ii) um modelo endógeno de expressão - células neuroprogenitoras derivadas de células-tronco pluripotentes induzidas (iNPCs) provenientes de indivíduos controles e de um paciente com deleção no gene da CB. Por meio de experimentos de coimunoprecipação nós verificamos que CB interage fisicamente com mTOR nos dois modelos experimentais utilizados. Ainda, nossos resultados mostraram que CB parece modular a atividade da via mTORC1, e nas iNPCs derivadas do paciente a ausência de CB leva a um aumento na ativação desta via de sinalização. Em concordância com esses resultados, nós observamos aumento em neo-síntese proteica nas iNPCs provenientes do paciente, o que pode ser um mecanismo patofisiológico contribuindo para as alterações cognitivas e comportamentais observadas no paciente. Embora estudos adicionais sejam necessários para melhor entender os mecanismos moleculares deste controle de início de tradução mediado por CB, nós sugerimos um modelo no qual CB, por interagir fisicamente com mTOR e eIF3, sequestra estas proteínas e impede que mTOR ative seus alvos e desencadeie a formação do complexo de inicio de tradução. Em conclusão, nossos resultados oferecem novas evidências do envolvimento de CB no controle da síntese proteica / Collybistin (CB), a neural specific RhoGEF, plays key roles in inhibitory synapse formation and function that cluster and localize the scaffold protein gephyrin and GABAA receptors to the neural postsynaptic membrane. We have recently reported that CB interacts with a protein complex that controls translation initiation in eukaryotic cells (eIF3 complex), which suggested for the first time that this protein may also act as regulator of protein synthesis in neural cells. Moreover, it has been previously described that gephyrin, the CB functional partner, interacts with mTOR, a kinase that plays a pivotal role in the control of translation initiation. However, until now there were no further studies investigating the role of CB in this scenario. The purpose of this study was to investigate if CB is involved in the control of translational initiation mediated by the mTORC1 signaling pathway. Two experimental models were used: i) a heterologous expression system - overexpression of CB in HEK293T cells, and ii) an endogenous expression model - neural progenitor cells derived from induced pluripotent stem cells (iNPCs) from control individuals and a patient with a deletion of the entire CB gene. We performed coimmunoprecipitation experiments and verified that CB physically interacts with mTOR both in 293T cells and in control iNPCs. In addition, our results show that CB appears to modulate the activity of mTORC1 pathway, and the absence of CB leads to increased mTORC1 signaling activation in patient\' iNPCs. In agreement with these results, we observed increased de novo cap-dependent translation in patient cells, which could be a pathophysiological mechanism contributing to cognitive and behavioral abnormalities observed in the patient. Although further studies are needed to better understand the molecular details of CB-mediated translational control, we suggest a model whereby CB, by physically interacting with mTOR and eIF3, sequesters these proteins, thereby preventing both the ability of mTOR to activate its targets and the formation of the translational initiation complex. In conclusion, our results offer new insights into the role of CB in protein synthesis control

Atualmente, alguns pesquisadores tem se dedicado ao estudo do efeito da manipulação do treinamento de força sobre a ativação das vias de sinalização intracelular para hipertrofia. Tem-se sugerido que o grau de tensão muscular desempenhe um papel importante nesta sinalização. Dentre os diferentes tipos de ações musculares, as ações excêntricas (AE) reconhecidamente proporcionam maior grau de tensão à estrutura do músculo esquelético. Em particular, AE de alta velocidade parecem exercer um efeito interessante sobre os ganhos de hipertrofia muscular. Porém, pouco se sabe sobre o efeito da manipulação da velocidade sobre as vias de sinalização da hipertrofia. Assim, o presente estudo teve como objetivo verificar o efeito agudo da AE de alta e baixa velocidade sobre a sinalização para hipertrofia muscular. Vinte sujeitos foram aleatoriamente divididos em dois grupos. Um realizou cinco séries de oito AE máximas à 20º/s (EXC20) e o outro à 210o/s (EXC210) do exercício extensão de joelhos. Amostras do músculo vasto lateral foram obtidas antes, imediatamente após e duas horas após o exercício. As análises de quantificação protéica de Akt e p70S6K totais não apresentaram diferenças significantes intra ou inter grupos. A avaliação da fosforilação das mesmas proteínas revelou um efeito principal de tempo, indicando um aumento da fosforilação nos tempos imediatamente após e duas horas após o término do exercício em relação à amostra controle (p<0,05), porém não foram observadas diferenças entre os grupos. Para os dados de expressão gênica de MGF e mTOR, não foram observadas diferenças intra ou inter grupos. Em conclusão, a manipulação aguda da velocidade das AE parece não influenciar a fosforilação ou expressão gênica das proteínas em questão / Recently, many studies have focused on the effects of strength training variables manipulation on the activation of intracellular signaling pathways for skeletal muscle hypertrophy. It has been suggested that the muscle tension plays a major role in such process. Eccentric muscle actions (EE) are notorious for imposing a greater amount of tension on the active muscle. In particular, EE performed at high velocities seems to exert an interesting effect on hypertrophy gains. However, little is known about the effect of EE velocity manipulation on hypertrophy pathways signaling. Thus, the present study aimed to investigate the acute effect of low and high velocity EE on muscle hypertrophy signaling. Twenty subjects were randomly assigned to either a slow velocity group 20o/s (ECC20) or fast group 210o/s (ECC210). Muscle biopsy samples were taken before, immediately after and two hours after the completion of five sets of eight maximal repetitions at the designated velocity, knee extension exercise. Akt and p70S6K analysis did not reveal any differences inter or intra groups. Akt and p70S6K phosphorylation results indicated a main effect for time (p<0,05), with increased phosphorylation values for immediately after and two hours after time points in comparison to control samples. MGF and mTOR mRNA analysis did not return any inter or intra groups differences. In conclusion, the acute manipulation of EE velocity does not seem to differently influence the phosphorylation or expression of the proteins studied

O overreaching não funcional (NFOR) induzido pelo exercício excêntrico (EE) em modelo animal está associado com a diminuição de desempenho físico, dano no DNA (amostras de músculo esquelético e soro), estresse oxidativo (amostras de músculo esquelético e soro), inflamação crônica de baixo grau (amostras de músculo esquelético e soro) e prejuízo da via de sinalização da insulina (amostras de músculo esquelético). No entanto, as adaptações do músculo cardíaco em resposta ao estado de NFOR induzido ou não pela predominância do EE ainda não foram investigadas. Além disso, sabe-se que a mTOR (mammalian target of rapamycin) possui um efeito protetor no músculo cardíaco, suprimindo o aumento de citocinas pró-inflamatórias, que estão relacionadas à disfunções cardíacas. Assim, o presente estudo teve como objetivo comparar os efeitos do NFOR em declive com outros dois protocolos de mesmo volume e intensidade, mas realizados sem inclinação e em aclive, no conteúdo das proteínas relacionadas às vias moleculares inflamatória e hipertrófica, no conteúdo de fibrose intersticial e na expresão gênica em músculo cardíaco de camundongos. Os animais foram divididos em 6 grupos: Naíve (N; camundongos sedentários), Controle (C; camundongos sedentários submetidos aos testes físicos), Treinado (TR; camundongos submetidos ao protocolo de treinamento), Overtraining em declive (OTR/down; camundongos submetidos ao protocolo de OT com corrida na descida), Overtraining sem inclinação (OTR; camundongos submetidos ao protocolo de OT com corrida sem inclinação) e Overtraining em aclive (OTR/up; camundongos submetidos ao protocolo de OT com corrida na subida). Em relação aos parâmetros metabólicos, o grupo OTR/down apresentou menor variação de peso corporal na semana 8. Todos os grupos que passaram pelo protocolo de OT demonstraram queda de desempenho ao final da semana 8, aumento no conteúdo de tecido conjuntivo no ventrículo esquerdo e menor ativação da proteína AMPKalfa. O grupo OTR/down apresentou menor conteúdo da proteína mTOR e S6RP, e aumento na expressão do gene beta-MHC. Pode-se concluir que os protocolos de OT provocaram indícios de hipertrofia patológica no ventrículo esquerdo, sendo esse efeito mais pronunciado no grupo OTR/down. / Nonfunctional overreaching (NFOR) induced by eccentric exercise (EE) in animal model is associated with performance decrement, DNA damage (muscle and serum samples), oxidative stress (muscle and serum samples), low grade chronic inflammation (muscle and serum samples) and insulin signaling impairment (muscle and serum samples). However, the adaptations of cardiac muscle in response to NFOR induced or not induced by EE are unknown. In addition, the mammalian target of rapamycin (mTOR) has a protector effect in the cardiac muscle, suppressing the increase of the proinflammatory cytokines that is related to cardiac dysfunctions. Thus, the main aim of present study was to compare the effects of NFOR based on EE (downhill running) with other two protocols with similar intensity and volume, but performed in uphill and without inclination, on the protein contents related to hypertrophic and inflammatory signaling, on the content of interstitial fibrotic tissue and on genes expression in mice cardiac muscle. The animals were divided on six groups: Naïve (N; sedentary mice), Control (C; sedentary mice submitted to physical tests), Trained (TR; mice submitted to training protocol), Overtraining in downhill (OTR/down; mice submitted to the overtraining protocol in downhill), Overtraining without inclination (OTR; mice submitted to the overtraining protocol without inclination), and Overtraining in uphill (OTR/up; mice submitted to the overtraining protocol in uphill). Regarding metabolic parameters, OTR/down group presented reduced body weight variation at week 8. All OT groups presented performance drop at end of week 8, increased connective tissue content in left ventricle and reduced AMPKalpha activation. OTR/down group presented reduced mTOR and S6RP protein content, and increased betaMHC gene expression. The conclusion is that OT protocols provoked signs of pathological hypertrophy in left ventricle, being this effect more pronounced in OTR/down group.

La Nucléophosmine (NPM1/B23) est une petite chaperonne moléculaire impliquée dans de nombreux processus cellulaires, tels que la régulation de l’expression génique ou le contrôle du cycle cellulaire. De nombreuses études rapportent une surexpression de NPM1 dans divers types de tumeurs solides incluant les cancers de la prostate, et son rôle proH prolifératif dans des lignées cellulaires tumorales d’origines variées est bien établi. La première partie de notre travail s’est attaché à évaluer le potentiel oncogénique de NPM1 dans l’épithélium prostatique in vivo. Pour cela, nous avons généré un modèle de souris transgéniques qui surexpriment NPM1 spécifiquement dans l’épithélium de la prostate. Ces animaux présentent une hyperplasie prostatique associée à une augmentation de l’index prolifératif de l’épithélium. Nos expériences révèlent que NPM1 pourrait lever la quiescence des cellules épithéliales différenciées en dérégulant l’expression de gènes clés de la régulation du cycle cellulaire, comme la Cycline E ou p27kip1. Bien que ces souris ne développent pas de lésions néoplasiques, ces données suggèrent que NPM1 participe à la carcinogenèse prostatique en association avec d’autres lésions oncogéniques. La seconde partie du travail visait à comprendre la nature des mécanismes qui supportent la surexpression de NPM1 dans les tumeurs prostatiques. Des données récentes de la littérature indiquent un enrichissement de la protéine kinase mTOR au niveau du promoteur proximal de NPM1 dans des foies de souris. Pour déterminer s’il existe un lien fonctionnel entre mTOR et NPM1, nous avons tiré parti d’un modèle de fibroblastes embryonnaires de souris invalidés pour le suppresseur de tumeur PTEN dont l’inactivation mène à une hyperactivité de mTOR. Dans ce contexte, les taux d’ARNm et de protéines NPM1 sont augmentés par rapport aux cellules sauvages. Nos résultats montrent également que mTOR contrôle l’expression de NPM1 i) en se fixant sur son promoteur et en stimulant l’expression du gène et ii) en stabilisant l’ARNm de NPM1. Nous avons confirmé le lien entre NPM1 et mTOR in vivo grâce à notre modèle de souris invalidées pour PTEN dans l’épithélium prostatique. Enfin, nous avons montré que l’expression de NPM1 est nécessaire pour transduire les effets prolifératifs de la voie PI3K/AKT/mTOR. Ces données placent donc NPM1 comme nouvel effecteur en aval de cette voie de signalisation, faisant de cette protéine une potentielle cible thérapeutique dans les tumeurs présentant une perte de PTEN. / Nucleophosmin (NPM1/B23) is a small molecular chaperone involved in a large array of cellular processes, including the regulation of gene expression and the control of the cell cycle. Several studies have reported the overexpression of NPM1 in solid tumors from various histological origin, including prostate cancer, and its proliferative impact on several human cancer cell line is being well described. The first part of our work aimed at assessing the NPM1 oncogenic properties in the prostate gland in vivo. To do so, we generated a new transgenic mouse model that overexpresses NPM1 specifically in the prostatic epithelium. These mice harbor prostatic hyperplasia associated with an increase of the ki67 proliferative index. Our molecular investigations revealed that NPM1 could be an inhibitor of the quiescent state of epithelial cells through a dysregulation of key cell-cycle controlers such as Cyclin E or p27kip1. Although these mice do not develop neoplastic lesions, our data suggest that NPM1 overexpression accelerate prostate cancer progression when associated with other oncogenic alterations. The second part of the work aimed at understanding the mechanisms underlying NPM1 overexpression in prostate tumors. The serine/threonine Kinase mTOR was recently shown to bind to the proximal promoter of NPM1 in the mouse liver. In order to characterize a fonctionnal link between NPM1 and mTOR, we took advantage of murine embryonic fibroblast (MEF) deleted for PTEN, since these cells display a constitutive mTOR activity. In such cells, NPM1 protein and mRNA levels are increased compared to wild type MEF. We also demonstrated that mTOR controls NPM1 expression i) through its binding to NPM1 promoter, thus stimulating NPM1 gene expression and ii) by stabilizing NPM1 mRNA. We have confirmed the functional link between NPM1 and mTOR in vivo in a mouse model deleted for PTEN specifically in the prostatic epithelium. Finally, we have shown that NPM1 expression is necessary for the proliferation of PTEN knock-out MEF. These data set NPM1 as a new downstream effector of the PI3K/AKT/mTOR pathway, and suggest that it could be a new potential therapeutic target in PTEN negative human prostate cancer.

A proteína colibistina (CB), uma Rho GEF neuro-especifica, apresenta papel importante na formação e funcionamento das sinapses inibitórias do sistema nervoso central por interagir com a proteína scaffold gefirina e com receptores GABAA e promover o agrupamento e transporte dessas proteínas para a membrana pós-sináptica. Recentemente, nosso grupo de pesquisa identificou interação de CB com um complexo proteico que controla o início da tradução em eucariotos (complexo eIF3), o que sugeriu pela primeira vez que essa proteína pode estar envolvida também na regulação da tradução em células neurais. Ainda, já havia sido descrito que gefirina, parceira funcional de CB, interage com mTOR, uma quinase que desempenha papel fundamental no controle do início da tradução. Contudo, até o momento não havia estudos adicionais investigando o papel de CB neste cenário. Assim sendo, o presente trabalho teve como objetivo investigar o envolvimento da proteína CB no controle do início da síntese proteica mediada pela via de sinalização mTORC1. Foram utilizados dois modelos experimentais: i) um sistema de expressão heteróloga - superexpressão de CB em células HEK293T, e ii) um modelo endógeno de expressão - células neuroprogenitoras derivadas de células-tronco pluripotentes induzidas (iNPCs) provenientes de indivíduos controles e de um paciente com deleção no gene da CB. Por meio de experimentos de coimunoprecipação nós verificamos que CB interage fisicamente com mTOR nos dois modelos experimentais utilizados. Ainda, nossos resultados mostraram que CB parece modular a atividade da via mTORC1, e nas iNPCs derivadas do paciente a ausência de CB leva a um aumento na ativação desta via de sinalização. Em concordância com esses resultados, nós observamos aumento em neo-síntese proteica nas iNPCs provenientes do paciente, o que pode ser um mecanismo patofisiológico contribuindo para as alterações cognitivas e comportamentais observadas no paciente. Embora estudos adicionais sejam necessários para melhor entender os mecanismos moleculares deste controle de início de tradução mediado por CB, nós sugerimos um modelo no qual CB, por interagir fisicamente com mTOR e eIF3, sequestra estas proteínas e impede que mTOR ative seus alvos e desencadeie a formação do complexo de inicio de tradução. Em conclusão, nossos resultados oferecem novas evidências do envolvimento de CB no controle da síntese proteica / Collybistin (CB), a neural specific RhoGEF, plays key roles in inhibitory synapse formation and function that cluster and localize the scaffold protein gephyrin and GABAA receptors to the neural postsynaptic membrane. We have recently reported that CB interacts with a protein complex that controls translation initiation in eukaryotic cells (eIF3 complex), which suggested for the first time that this protein may also act as regulator of protein synthesis in neural cells. Moreover, it has been previously described that gephyrin, the CB functional partner, interacts with mTOR, a kinase that plays a pivotal role in the control of translation initiation. However, until now there were no further studies investigating the role of CB in this scenario. The purpose of this study was to investigate if CB is involved in the control of translational initiation mediated by the mTORC1 signaling pathway. Two experimental models were used: i) a heterologous expression system - overexpression of CB in HEK293T cells, and ii) an endogenous expression model - neural progenitor cells derived from induced pluripotent stem cells (iNPCs) from control individuals and a patient with a deletion of the entire CB gene. We performed coimmunoprecipitation experiments and verified that CB physically interacts with mTOR both in 293T cells and in control iNPCs. In addition, our results show that CB appears to modulate the activity of mTORC1 pathway, and the absence of CB leads to increased mTORC1 signaling activation in patient\' iNPCs. In agreement with these results, we observed increased de novo cap-dependent translation in patient cells, which could be a pathophysiological mechanism contributing to cognitive and behavioral abnormalities observed in the patient. Although further studies are needed to better understand the molecular details of CB-mediated translational control, we suggest a model whereby CB, by physically interacting with mTOR and eIF3, sequesters these proteins, thereby preventing both the ability of mTOR to activate its targets and the formation of the translational initiation complex. In conclusion, our results offer new insights into the role of CB in protein synthesis control