The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt-deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt iv activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V-ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer.

O overreaching não funcional (NFOR) induzido por consecutivas sessões de treinamentos intensos intercaladas por períodos insuficientes de recuperação, está associado com inflamação e consequente prejuízo da via de sinalização insulínica em músculos esqueléticos de camundongos. Sabe-se que o miocárdio também é capaz de produzir tais proteínas inflamatórias associadas ao comprometimento da via hormonal e que alterações na atividade do receptor insulínico cardíaco levam à forçadas modificações na utilização dos substratos energéticos com prejuízos na mecanoenergética cardíaca predispondo o miocárdio à diversas injúrias. No entanto os efeitos do NFOR nas vias inflamatórias e insulínica cardíaca ainda não foram investigados. Assim, o presente estudo tem como objetivo avaliar os efeitos do NFOR no conteúdo de glicogênio cardíaco e ativação de proteínas relacionadas às vias insulínica e inflamatória. Os animais foram divididos em 6 grupos: Naive, Controle, Treinado, e os grupos submetidos ao protocolo de overtraining em declive (OTR/down), aclive (OTR/up) e sem inclinação (OTR). As especificidades das contrações musculares induziram diferentes adaptações cardíacas. Os grupos OTR e OTR/up não apresentaram sinais de inflamação além de superexpressarem a via insulínica, por outro lado, o grupo OTR/down apresentou inflamação cardíaca de baixo grau, contudo, sem queda no conteúdo de pIR. Todos os protocolos de overtraining induziram elevação no conteúdo de glicogênio cardíaco acompanhado de expressiva queda da pAMPK. Os resultados do presente trabalho nos trazem, portanto, a hipótese de que o tecido cardíaco apresente uma maior resistência à inflamação viabilizando dessa forma a melhora da resposta insulínica e acúmulo do glicogênio cardíaco a fim de fornecer a energia necessária ao extenuante exercício físico evitando a lipotoxicidade cardíaca. Por outro lado, a queda da AMPK consequente do excessivo acúmulo de glicogênio cardíaco pode predispor o miocárdio à diversas injúrias, sendo necessários mais estudos na área. / Non-functional overreaching (NFOR) induced by consecutive intense training sessions interspersed by insufficient periods of recovery is associated with inflammation and a consequent impairment of the insulin signaling pathway in skeletal muscle of mice. It is known that the myocardium is also capable of producing such inflammatory proteins associated with the impairment of the hormonal pathway and that changes in cardiac insulin receptor activity lead to forced modifications in the use of energetic substrates with losses in cardiac mecanoenergética predisposing the myocardium to various injuries. However, the effects of NFOR on inflammatory and cardiac insulin pathways have not been investigated yet. Thus, the present study aims to evaluate the effects of NFOR on cardiac glycogen content and activation of proteins related to insulin and inflammatory pathways. The animals were divided into 6 groups: Naïve, Control, Trained, and the groups submitted to the overtraining protocol in decline (OTR/down), uphill (OTR /up) and without inclination (OTR). The specificities of muscle contractions induced different cardiac adaptations. OTR and OTR/up groups showed no signs of inflammation and an over expressive of the insulin pathway; on the other hand, the OTR/down group presented low-grade cardiac inflammation, however, without any decrease in the pIR content. All overtraining protocols induced elevation in cardiac glycogen content accompanied by significant drop in pAMPK. The results of the present work hypothesize that the cardiac tissue presents a greater resistance to inflammation, thus enabling the improvement of the insulin response and the accumulation of cardiac glycogen in order to provide the necessary energy to the strenuous physical exercise avoiding cardiac lipotoxicity. On the other hand, the decrease in AMPK due to the excessive accumulation of cardiac glycogen may predispose the myocardium to several injuries, and further studies in the area are required.

O overreaching não funcional (NFOR) induzido por consecutivas sessões de treinamentos intensos intercaladas por períodos insuficientes de recuperação, está associado com inflamação e consequente prejuízo da via de sinalização insulínica em músculos esqueléticos de camundongos. Sabe-se que o miocárdio também é capaz de produzir tais proteínas inflamatórias associadas ao comprometimento da via hormonal e que alterações na atividade do receptor insulínico cardíaco levam à forçadas modificações na utilização dos substratos energéticos com prejuízos na mecanoenergética cardíaca predispondo o miocárdio à diversas injúrias. No entanto os efeitos do NFOR nas vias inflamatórias e insulínica cardíaca ainda não foram investigados. Assim, o presente estudo tem como objetivo avaliar os efeitos do NFOR no conteúdo de glicogênio cardíaco e ativação de proteínas relacionadas às vias insulínica e inflamatória. Os animais foram divididos em 6 grupos: Naive, Controle, Treinado, e os grupos submetidos ao protocolo de overtraining em declive (OTR/down), aclive (OTR/up) e sem inclinação (OTR). As especificidades das contrações musculares induziram diferentes adaptações cardíacas. Os grupos OTR e OTR/up não apresentaram sinais de inflamação além de superexpressarem a via insulínica, por outro lado, o grupo OTR/down apresentou inflamação cardíaca de baixo grau, contudo, sem queda no conteúdo de pIR. Todos os protocolos de overtraining induziram elevação no conteúdo de glicogênio cardíaco acompanhado de expressiva queda da pAMPK. Os resultados do presente trabalho nos trazem, portanto, a hipótese de que o tecido cardíaco apresente uma maior resistência à inflamação viabilizando dessa forma a melhora da resposta insulínica e acúmulo do glicogênio cardíaco a fim de fornecer a energia necessária ao extenuante exercício físico evitando a lipotoxicidade cardíaca. Por outro lado, a queda da AMPK consequente do excessivo acúmulo de glicogênio cardíaco pode predispor o miocárdio à diversas injúrias, sendo necessários mais estudos na área. / Non-functional overreaching (NFOR) induced by consecutive intense training sessions interspersed by insufficient periods of recovery is associated with inflammation and a consequent impairment of the insulin signaling pathway in skeletal muscle of mice. It is known that the myocardium is also capable of producing such inflammatory proteins associated with the impairment of the hormonal pathway and that changes in cardiac insulin receptor activity lead to forced modifications in the use of energetic substrates with losses in cardiac mecanoenergética predisposing the myocardium to various injuries. However, the effects of NFOR on inflammatory and cardiac insulin pathways have not been investigated yet. Thus, the present study aims to evaluate the effects of NFOR on cardiac glycogen content and activation of proteins related to insulin and inflammatory pathways. The animals were divided into 6 groups: Naïve, Control, Trained, and the groups submitted to the overtraining protocol in decline (OTR/down), uphill (OTR /up) and without inclination (OTR). The specificities of muscle contractions induced different cardiac adaptations. OTR and OTR/up groups showed no signs of inflammation and an over expressive of the insulin pathway; on the other hand, the OTR/down group presented low-grade cardiac inflammation, however, without any decrease in the pIR content. All overtraining protocols induced elevation in cardiac glycogen content accompanied by significant drop in pAMPK. The results of the present work hypothesize that the cardiac tissue presents a greater resistance to inflammation, thus enabling the improvement of the insulin response and the accumulation of cardiac glycogen in order to provide the necessary energy to the strenuous physical exercise avoiding cardiac lipotoxicity. On the other hand, the decrease in AMPK due to the excessive accumulation of cardiac glycogen may predispose the myocardium to several injuries, and further studies in the area are required.

<p>RanBPM is a conserved putative scaffold protein of unknown function. Loss-of-function in <em>RanBPM</em> leads to pleiotropic phenotypes such as reduced locomotion, decreased size and larval lethality in the <em>Drosophila melanogaster</em>.</p> <p><em>dRanBPM</em> mutants have decreased branching and boutons at the neuromuscular junction, which may contribute to their locomotory defect. To investigate if dRanBPM is involved in controlling synaptic architecture at the neuromuscular junction, levels of two cytoskeletal proteins, Futsch and profilin, were assessed in <em>dRanBPM</em> mutants.</p> <p>Due to time constraints, immunoblots for Futsch were not fully optimized for protein measurement. Immunoblots for profilin, on the other hand, were successfully carried out. However, results from the reproduction of a blot demonstrating the negative regulation of <em>Drosophila</em> FMRP on profilin did not agree with that of the literature. In addition, results from an epistatic experiment demonstrated that profilin levels were not affected in FMRP deficient flies when compared to those with additional decrease in dRanBPM function.</p> <p>Targeted expression of <em>dRanBPM</em> to neurosecretory cells is able to rescue size and lethality of <em>dRanBPM</em> mutants, suggesting a common pathway through which both phenotypes operate is disrupted in these mutants. Activation of the insulin signaling pathway was indeed found to be downregulated in <em>dRanBPM</em> mutants. A longevity assay was alternatively carried out to demonstrate decreased insulin pathway activation in <em>dRanBPM</em> mutants. Unfortunately, due to inappropriate controls used for this experiment, no conclusive points can be made. Together, these findings contribute to the knowledge that RanBPM plays and to designing future experiments to test for RanBPM function.</p> / Master of Science (MSc)

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-04-27T15:00:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-05-16T19:24:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) / Made available in DSpace on 2017-05-16T19:24:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) mariana_aguiar_matos.pdf: 2901378 bytes, checksum: 40dbd704043d49a1eee587bb086c4eb4 (MD5) Previous issue date: 2016 / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O excesso de gordura corporal caracter?stico da obesidade est? relacionado a diversas altera??es metab?licas, que incluem a resist?ncia ? insulina. Dentre as medidas n?o farmacol?gicas empregadas para a melhora da sensibilidade ? insulina est? o treinamento f?sico aer?bio, como o treinamento intervalado de alta intensidade (HIIT, do ingl?s high intensity interval training). Sendo assim, esse estudo avaliou os efeitos do HIIT em componentes bioqu?micos, celulares e moleculares relacionados ? resist?ncia ? insulina em obesos. Indiv?duos obesos sens?veis (n=9) e resistentes ? insulina (n=8) foram submetidos a 8 semanas de HIIT, em cicloerg?metro, realizado 3 vezes por semana, com intensidade e volume progressivos (8 a 12 est?mulos; 80 a 110% da pot?ncia m?xima). Amostras de sangue venoso e do m?sculo vasto lateral foram obtidas antes e ap?s o programa de HIIT. Ap?s o programa de treinamento houve aumento da sensibilidade ? insulina nos obesos resistentes ? insulina, mas n?o houve redu??o da massa de gordura. A concentra??o de citocinas no soro, o estresse oxidativo sist?mico e frequ?ncia das c?lulas imunes n?o foram modificadas ap?s o treinamento. No m?sculo esquel?tico, o HIIT promoveu aumento da fosforila??o do substrato do receptor de insulina (IRS) (Tyr612), da Akt (Ser473) e da prote?na quinase dependente de c?lcio/calmodulina (CAMKII) (Thr286), e aumento do conte?do da ?-hidroxiacil-CoA desidrogenase (?-HAD) e citocromo C oxidase (COX-IV). Houve ainda, redu??o da fosforila??o da quinase regulada por sinal extracelular (ERK1/2) nos obesos resistentes ? insulina. Conclu?mos que 8 semanas de HIIT promoveram melhora da sensibilidade ? insulina, modificou componentes da via de sinaliza??o da insulina e do metabolismo oxidativo no m?sculo esquel?tico. Essas altera??es ocorreram independentes de mudan?as na gordura corporal total e de par?metros inflamat?rios sist?micos. / Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / Obesity is characterized by excess of body fat, and its development can lead to a variety of metabolic disorders, including insulin resistance. Exercise is recognized as a non-pharmacological approach to increasing skeletal muscle insulin sensitivity, although the mechanisms are not elucidated. Additionally, the understanding of high intensity interval training (HIIT, high intensity interval training) treat insulin resistance is less understood. Therefore, this study evaluated the effects of HIIT on biochemical, molecular, and cellular markers related to insulin resistance in sedentary obese individuals. Sensitive (n=9) and insulin resistant (n=8) obese individuals (body mass index ? 30 kg/m-2) were engaged in 8 weeks of HIIT using a cycle ergometer. The HIIT was performed 3 times a week, and its intensity and volume progressively increased throughout the training period (from 8 to 12 stimuli; from 80 to 110% of the maximum power). Venous blood and the vastus lateralis muscle samples were obtained before and after the HIIT. HIIT enhanced insulin sensitivity in insulin-resistant obese individuals without changing body fat mass. Cytokine concentration in serum, blood oxidative stress, and frequency of some immune cells were not altered by HIIT. In skeletal muscle, HIIT increased the phosphorylation of insulin receptor substrate (IRS) (Tyr612), Akt (Ser473), and protein kinase dependent calcium/calmodulin (CaMKII) (Thr286). HIIT also increased the expression of ?-hydroxyacyl-CoA dehydrogenase (?-HAD) and cytochrome C oxidase (COX-IV). A reduction of the kinase phosphorylation of extracellular signal-regulated (ERK1/2) was only seen in obese insulin resistant individuals. The results show that 8 weeks of HIIT enhanced insulin sensitivity, modified components of the insulin-signaling pathway, and improved skeletal muscle oxidative metabolism. These changes were independent of alterations in body fat and inflammatory parameters.

Le réticulum endoplasmique et la mitochondrie sont deux organites majeurs impliqués dans la régulation du métabolisme glucido-lipidique. Ces structures interagissent au niveau de points de contact étroits appelés Mitochondria-Associated Endoplasmique Reticulum Membranes (MAMs). Les MAMs sont une zone de communication et d’échanges, de lipides et de calcium entre autre, indispensables à l’activité des deux organites et au maintien de l’homéostasie cellulaire. Des connexions physiques sont assurées par l’interaction de protéines complémentaires, comme le canal anionique voltage-dépendant (VDAC)-1, la protéine chaperonne (Grp)-75 et le récepteur de l'inositol 1,4,5-triphosphate (IP3R)-1, constituant un complexe impliqué dans le transfert de calcium. D’autres acteurs comme les mitofusines 1 et 2 (MFN1/2) assurent également un rapprochement entre les deux organites et semblent jouer un rôle dans les échanges des lipides. Récemment, les MAMs sont apparues comme un nouveau carrefour de la signalisation de l’insuline dans le foie. Le monoxyde d’azote (NO) participe également au contrôle de la réponse à l’insuline hépatique et a une action spécifique sur la mitochondrie. Mes travaux de thèse ont montré que le NO à des concentrations physiologiques module les interactions entre le RE et la mitochondrie dans le foie et que son action sur les MAMs implique la voie de signalisation sGC/cGMP/PKG. J’ai également démontré que la modulation des MAMs par le NO semble jouer un rôle clé dans la régulation de la voie de signalisation à l’insuline (projet1). Par ailleurs, j’ai exploré l’importance des MAMs dans la régulation du métabolisme lipidique. Pour cela, j’ai modulé l’expression protéique de Grp75 et Mfn2 sur un modèle d’hépatocarcinome humain (Huh7). Mes résultats ont montré qu’une surexpression des deux protéines améliore les MAMs et la β-oxydation mitochondriale mais conduit à une accumulation intracellulaire de lipides. Ceci serait dû à un défaut de sécrétion des lipides dans les lipoprotéines VLDL et pourrait impliquer l’apparition d’un stress mitochondrial et une altération des échanges de phospholipides entre les deux organites (projet 2). Par conséquence mon travail confirme le rôle physiologique des MAMs et éclaire les mécanismes d’actions de cette plateforme cellulaire dans la régulation du métabolisme glucido-lipidique hépatique. A plus long terme ces connaissances participeront peut-être à l’identification de potentielles cibles thérapeutiques afin de prévenir la stéatose et la résistance à l’insuline hépatiques et leurs complications / The endoplasmic reticulum and mitochondria are two major organelles involved in the regulation of glucose and lipid metabolism. These structures interact at close contact points called Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs). MAMs constitute an area of communication and exchange, of lipids and calcium among others, essential for the activity of both organelles and the maintenance of cellular homeostasis. Physical connections are ensured by the interaction of complementary proteins, such as the voltage-dependent anionic channel (VDAC)-1, the chaperone protein (Grp)-75 and the inositol 1,4,5-triphosphate receptor (IP3R)-1, constituting a complex involved in calcium transfer. Other actors such as mitofusins 1 and 2 (MFN1/2) also connect the two organelles and are involved in lipid exchanges. Recently, MAMs have emerged as a new carrefour for insulin signaling in the liver. Nitric oxide (NO) also helps control the response to hepatic insulin and has a specific action on mitochondria. My thesis work showed that NO at physiological concentrations modulates the interactions between RE and mitochondria in the liver and that its action on MAMs involves the sGC/cGMP/PKG signalling pathway. I also demonstrated that NO modulation of MAMs plays a key role in regulating the insulin signaling pathway (project 1). In addition, I explored the importance of MAMs in the regulation of lipid metabolism. For that purpose, protein expression of Grp75 and Mfn2 was modulated in a human hepatocarcinoma model (Huh7). Results showed that overexpression of both proteins improves MAMs and mitochondrial β-oxidation but leads to intracellular lipid accumulation. This could be due to a defect in lipid secretion in VLDL lipoproteins and could imply the appearance of mitochondrial stress and an alteration of phospholipid exchanges between the two organelles (project 2). Consequently, my work confirms the physiological role of MAMs and sheds light on the mechanisms of action of this cellular platform in the regulation of glucose and lipid metabolism in the liver. In the longer term, this knowledge may contribute to the identification of potential therapeutic targets to prevent steatosis and hepatic insulin resistance and their complications