O infarto do miocárdio é atualmente considerado a etiologia que mais contribui para o aparecimento de insuficiência cardíaca (IC) em humanos. Em detrimento a hiperativação de fatores neuro-humorais, a progressão da IC é caracterizada por uma série de anormalidades celulares associadas à disfunção ventricular. Dentre estas anormalidades, alterações na função e dinâmica mitocondrial merecem destaque, uma vez que a homeostase da organela é essencial para a viabilidade celular e o bom funcionamento da bomba cardíaca. No presente estudo, caracterizamos em modelo animal de disfunção cardíaca associada ao infarto do miocárdio: a) fenótipo cardíaco; b) função mitocondrial; c) equilíbrio redox; e d) dinâmica mitocondrial. Nossos resultados nos permitem afirmar que doze semanas após a cirurgia de infarto do miocárdio, os animais desenvolveram importantes alterações fenotípicas como aumento da massa cardíaca, dilatação ventricular, hipertrofia do cardiomiócito e maior deposição de tecido fibroso cardíaco, que contribuíram para o estabelecimento da disfunção ventricular. Além disso, foi possível confirmar a instalação do quadro de disfunção mitocondrial cardíaca, representada pela redução na capacidade respiratória e perda da homeostase redox. Por fim, encontramos um aumento no número de mitocôndrias cardíacas com menor diâmetro, alterações que vieram acompanhadas de uma menor atividade das enzimas relacionadas à fusão mitocondrial. Uma vez caracterizada a função e a dinâmica mitocondrial na disfunção cardíaca, avaliamos o efeito do treinamento físico aeróbico (TF) nessas variáveis. O TF, atualmente utilizado como um adjuvante no tratamento das doenças cardiovasculares, foi eficaz em promover o remodelamento cardíaco reverso e melhorar a função cardíaca nos animais infartados. Além disso, melhorou a capacidade respiratória e reduziu o estresse oxidativo, restaurando a função mitocondrial. Aliado a esses achados, o TF normalizou a atividade das enzimas relacionadas à dinâmica mitocondrial, fato associado à normalização do número e tamanho da organela. Esses resultados demonstram que a disfunção cardíaca induzida por infarto do miocárdio está associada à um quadro de mitocondriopatia em ratos, com alterações tanto na função quanto estrutura mitocondrial, e que o TF desencadeia efeitos benéficos na manutenção da integridade/função mitocondrial e melhora da função contrátil cardíaca / Myocardial infarction is considered the etiology that most contributes to the onset of heart failure in humans. Among the ventricular dysfunction-associated cellular abnormalities, changes in mitochondrial function and dynamics are critical, since the organelle homeostasis is crucial in maintaining the metabolic, electrical and mechanical properties of the heart. In the present study, we characterized in cardiac dysfunction- induced myocardial infarction in rats: a) cardiac phenotype; b) mitochondrial metabolism; c) redox balance, and d) mitochondrial dynamics. Our results show that twelve weeks after myocardial surgery, the animals developed pathological cardiac remodeling-associated ventricular dysfunction. Furthermore, we observed a reduced mitochondrial respiratory capacity and loss of redox homeostasis. Finally, we found a lower activity of enzymes related to mitochondrial fusion, these changes were accompanied by an increase in the number of small mitochondria. Once characterized mitochondrial function and dynamics, we evaluated the effect of exercise training in these variables in rats with cardiac dysfunction. The exercise training, currently established as an important non-pharmacological treatment for cardiovascular diseases, reversed the pathological cardiac remodeling and minimized the ventricular dysfunction in infarcted animals. Furthermore, exercise training restored the mitochondrial function by increasing respiratory capacity and reducing oxidative stress. Finally, exercise training restored the activity of mitochondrial dynamics-related enzymes and morphology. Taken together, our findings uncover the potential benefits of exercise training in reversing the cardiac mitochondriopathy observed in failing hearts, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy

Le processus conduisant à la prolifération et différenciation des cellules souches hématopoïétiques (CSH) en cellules de toutes les lignées sanguines s’appelle l’hématopoïèse. Bien que l'engagement des CSH soit régi par les cytokines, les facteurs de transcription, les modificateurs épigénétiques et la niche des CSH, notre groupe a constaté que leur engagement vers la lignée érythroïde dépendait aussi du métabolisme de la glutamine. La glutaminolyse contribue à la biosynthèse des nucléotides de novo ainsi qu’à la production de l'alpha-kétoglutarate (αKG), intermédiaire métabolique du cycle TCA (Oburoglu et al. 2014). Il est cependant important de noter que la différenciation érythroïde est un processus unique, où chaque cellule fille est structurellement et fonctionnellement différente de sa cellule mère. Chaque division définit un stade de différenciation précis avec un dernier cycle de division produisant un réticulocyte énucléé. Ainsi, nous avons émis l'hypothèse que les réseaux métaboliques mobilisés dans les progéniteurs érythroïdes changent en fonction du stade de différenciation et que ces réseaux régulent la transition des progéniteurs d'un stade à l'autre.Au cours de ma thèse, j’ai caractérisé les états métaboliques associés aux différents stades de différenciation des progéniteurs érythroïdes. Nous avons ainsi montré qu'aux stades précoces de différenciation érythroïde, avant la différenciation terminale, les progéniteurs hématopoïétiques présentent une activité métabolique accrue avec un niveau de phosphorylation oxydative (OXPHOS) plus élevé. Ces données sont en corrélation avec l'augmentation de la génération de l’αKG à ces stades de différenciation. De plus, nous avons constaté une augmentation de l’OXPHOS de ces progéniteurs en présence d’αKG exogène. Cependant, la différenciation terminale des précurseurs érythroïdes, caractérisée par la perte de la masse mitochondriale et de leur potentiel membranaire, est associée à une diminution du niveau d'OXPHOS. Ainsi, l'administration exogène d’αKG, a fortement atténué la différenciation érythroïde terminale et l'énucléation, sans affecter la différenciation des pro-érythroblastes. Inversement, un antagoniste de l’αKG (diméthyloxalylglycine, DMOG) n'a pas altéré la différenciation terminale ou l'énucléation, malgré l'abrogation de l'OXPHOS dans les érythroblastes.Ces données suggèrent que la production d’αKG et sa contribution à l’OXPHOS perturbent l'énucléation des globules rouges. C'est pourquoi, dans le but de réduire les niveaux intracellulaires d’αKG, nous avons inhibé l’expression de l'isocitrate déshydrogénase I (IDH1), enzyme cytosolique catalysant la conversion de l'isocitrate en αKG. Cependant, comme IDH1 peut catalyser les réactions dans les deux sens, la diminution de son expression pourrait également augmenter les niveaux d’αKG. En effet, nous avons constaté que le knockdown d'IDH1 entraînait une forte atténuation de la différenciation terminale et de l'énucléation des précurseurs érythroïdes. Cet effet est probablement dû à un déséquilibre de la disponibilité des substrats ; ainsi l’administration ectopique de l’αKG ainsi que du citrate renforce l’altération de la différenciation terminale des précurseurs érythroïdes IDH1-/- ainsi que leur énucléation. Cette étude identifie donc un rôle crucial pour le métabolite αKG dans la régulation de la fonction mitochondriale et de l’OXPHOS, processus qui sont une condition sine qua non pour la différenciation des précurseurs érythroïdes au stade proérythroblaste. Nous montrons en outre que la suppression d’OXPHOS et la catalyse d’intermédiaires du TCA, substrats d’IDH1, sont requis pour les phases terminales de la différenciation érythroïde et l'énucléation.En conclusion, les résultats obtenus au cours de ma thèse mettent en évidence la nature dynamique des réseaux métaboliques qui régulent la progression des précurseurs érythroïdes tout au long des différents stades de la différenciation érythroïde. / Hematopoiesis is the process whereby hematopoietic stem cells (HSCs) proliferate and differentiate to all blood cell lineages. While HSC commitment is known to be regulated by cytokines, transcription factors, epigenetic modifiers and the HSC niche, our group found that specification of HSCs to the red cell lineage is dependent on glutamine metabolism. Glutaminolysis contributes to de novo nucleotide biosynthesis and to the generation of the alpha-ketoglutarate (αKG) TCA cycle metabolite (Oburoglu et al. 2014). Importantly though, erythroid differentiation is a unique process as each daughter cell is structurally and functionally different from its parent cell. Each division defines a stage of differentiation with the final division cycle resulting in the production of an enucleated reticulocyte which further matures to a biconcave erythrocyte. Thus, we hypothesized that progenitor metabolic networks change as a function of the erythroid differentiation stage and moreover, that they regulate the transition of progenitors from one stage of differentiation to the next.During my PhD, I assessed the metabolic alterations that occur as a function of the erythroid differentiation stage. We showed that at early stages of human red cell development, prior to terminal differentiation, hematopoietic progenitors exhibited an increased metabolic activity with a significantly higher level of oxidative phosphorylation (OXPHOS). This correlated with the increased generation of αKG and indeed, we found that ectopic αKG directly augmented OXPHOS in these progenitors. However, the terminal differentiation of erythroid precursors, characterized by the loss of mitochondrial mass and membrane potential, was associated with a decreased level of OXPHOS. Notably, ectopic αKG, which did not alter pro-erythroblast erythroid differentiation, severely attenuated terminal differentiation and enucleation. Conversely, an αKG antagonist (dimethyloxalyl glycine, DMOG) did not negatively impact on terminal differentiation or enucleation despite abrogating OXPHOS in erythroblasts.These data suggested that the production of αKG and its subsequent contribution to oxidative phosphorylation perturb red cell enucleation. We therefore downregulated isocitrate dehydrogenase I (IDH1), the cytosolic enzyme that catalyzes the conversion of isocitrate to αKG, by an shRNA approach in an attempt to decrease αKG levels. However, because IDH1 can catalyze both the forward and reverse reactions, its downregulation could also increase αKG levels. Indeed, we found that IDH1 knockdown resulted in a severe attenuation of terminal erythroid differentiation and enucleation. This effect was likely due to an imbalance in substrate availability––both ectopic αKG as well as citrate further decreased polychromatic to orthochromatic erythroblast differentiation and the subsequent enucleation of IDH1-knockdown erythroid precursors. Thus, the present study identifies a crucial role for the αKG metabolite in regulating mitochondrial function and oxidative phosphorylation, processes that are a sine qua non for erythroid precursors at the pro-erythroblast stage. We further show that terminal erythroid differentiation and enucleation requires OXPHOS suppression and the IDH1-mediated enzymatic catalysis of its TCA substrates.To conclude, the results generated during my PhD highlight the dynamic nature of the metabolic networks that regulate the progression of erythroid precursors through the distinct stages of erythroid differentiation.

Orientadora: Marion Burkhardt de Koivisto / Banca: Gisele Zoccal Mingoti / Banca: Sony Dimas Bicudo / Resumo: O objetivo deste trabalho foi avaliar os efeitos da gota citoplasmática proximal (GCP) no sêmen de bovinos quanto à integridade do DNA, das membranas citoplasmática, acrossomal, no potencial mitocondrial e verificar a taxa de produção de embriões in vitro. Três amostras descongeladas de cinco (Controle: espermiograma normal), e oito touros Bos indicus (Gota: GCP ≥15%) foram avaliadas. Foram realizados os seguintes testes: motilidade e vigor pós-descongelação, concentração, morfologia espermática, teste de termo-resistência lento (TTL), integridade da membrana acrossomal, plasmática e potencial mitocondrial utilizando sondas fluorescentes (PI, FITC-PSA e JC-1) e integridade da cromatina pelo método de coloração com laranja de acridina. Dois touros com índices elevados de GCP e três animais controle foram selecionados para fertilização in vitro (FIV). As análises estatísticas foram efetuadas empregando-se o programa Statistical Analysis System. O nível de significância foi de 5%. Os resultados obtidos demonstraram que altos índices de GCP não afetaram a motilidade e o vigor, antes e após o TTL, assim como não interferiram na porcentagem de acrossomas intactos. Os resultados destas avaliações mostraram que a alta incidência de GCP afetou a integridade da membrana acrossomal e plasmática bem como a presença de potencial mitocondrial. No entanto, a alta incidência de GCP não promoveu aumento na porcentagem de injúrias à cromatina após descongelação, mas os resultados sugerem que podem ser mais sensíveis à desnaturação quando incubados por três horas. No experimento II, os índices de produção de embriões in vitro podem ter sido afetados pela interação da alteração morfológica e o efeito individual do touro. / Abstract: The objective of this study was to evaluate the effects of the proximal cytoplasmic droplets (PCD) in bovine semen, on the integrity of DNA, cytoplasmic membrane, acrossome, mitochondrial function and the rate of in vitro embryo production. Three batches of five (control group G1: normal sperm parameters) and eight Bos indicus bulls (G2: PCD ≥15%) were analysed. The following tests were carried out: post thaw motility and, vigor, concentration, sperm morphology, slow thermo-resistance (TRT), membrane integrity, acrossome status, mitochondrial function through fluorescent probes (FITC-PSA, PI and JC-1) and integrity of chromatin was accessed by acridine orange stain. Two bulls with high rates of PCD and three animals (control group) were selected for in vitro fertilization (IVF). Statistical analyses were performed using the Statistical Analysis System. The significance level was 5%. The results showed that high rates of PCD did not affect motility and vigor, before and after the TRT, and did not affect the percentage of intact acrossome. The results showed that the high incidence of PCD affected membrane integrity, acrossome status and mitochondrial function when compared to the G1 group due. However, the high incidence of PCD did not affect the percentage of chromatin injury after thawing, but results suggest that spermatozoa may be more susceptible to damage when incubated for three hours. In experiment II the embryo production rate may have been affected by the interaction of the morphology traits and the bull effect. / Mestre

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A insuficiência cardíaca (IC) é a evolução final das várias formas de doenças cardiovascular, sendo resultado de modificações estruturais, metabólicas e de contratilidade miocárdica. A fim de compreender o papel na dinâmica do metabolismo cardíaco no estado basal e na sobrecarga de pressão, utilizamos os modelos de cre-lox com deleção específica no coração para substrato do receptor de insulina (IRS) e co-ativador do PPAR (PGC-1β) e analisamos a estrutura cardíaca (histologia e estereologia), função cardíaca (ecocardiograma e técnica de Working heart), o metabolismo (isolamento de cardiomiócito e captação de glicose), ação hormonal (Western Blotting), expressão gênica (PCR-RT) de enzimas do metabolismo (lipídico, glicídico, da cadeia respiratória fatores transcricionais e hipertróficos) e a função mitocondrial. Verificamos, nos CIRS12KO, disfunção cardíaca grave, disfunção mitocondrial e prejuízo na expressão gênica das enzimas do metabolismo energético. Nos PGC-1βKO observamos disfunção mitocondrial e alteração de expressão gênica das enzimas do metabolismo energético quando submetidos à sobrecarga de pressão. Através do estudo do metabolismo cardíaco e da expressão gênica nestes diferentes modelos conseguimos explorar as vias metabólicas que levam a hipertrofia compensada à IC. Sugerimos que o mecanismo responsável pela descompensação seja a disfunção mitocondrial em conseqüência à alteração da expressão gênica. E que IRS e o PGC-1β são fatores chaves da dinâmica cardíaca, e que são indispensáveis para a estrutura e funcionamento cardíaco. Além de representar alvo promissor para limitar a transição da hipertrofia cardíaca compensada a insuficiência cardíaca. / Heart failure (HF) is the end stage of different types of cardiovascular diseases and it is characterized by changes in the metabolic and myocardial contractility. We use the models cre-lox with specific knockout for insulin receptor substrate (IRS) and co-activator of PPAR (PGC-1b) (basal and pressure overload). The objective was understood the role in the dynamics of cardiac metabolism. We analyzed cardiac structure (histology and stereology), cardiac function (echocardiography and the working heart technique), metabolism (glucose uptake), hormonal action (Western Blotting), gene expression (RT-PCR) from enzyme metabolism (lipid, carbohydrates, respiratory chain, transcriptional and hypertrophic factors) and mitochondrial function. We found in CIRS12KO, severe cardiac dysfunction, mitochondrial dysfunction and reduction of gene expression. And in the PGC-1bKO when subjected to pressure overload, the progression to heart failure, with mitochondrial dysfunction, and alteration of gene expression from enzyme metabolism. The data show that changes on cardiac metabolism and gene expression in both models explain the metabolic pathways that lead to compensated hypertrophy to HF. We suggest that the mitochondrial dysfunction and the gene expression was possible mechanisms for HF. We conclude that IRS and PGC-1b are key factors of cardiac dynamics, which are essential to the structure and heart function. IRS and PGC-1b represent a promising target for limiting the transition from compensated cardiac hypertrophy to heart failure.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A insuficiência cardíaca (IC) é a evolução final das várias formas de doenças cardiovascular, sendo resultado de modificações estruturais, metabólicas e de contratilidade miocárdica. A fim de compreender o papel na dinâmica do metabolismo cardíaco no estado basal e na sobrecarga de pressão, utilizamos os modelos de cre-lox com deleção específica no coração para substrato do receptor de insulina (IRS) e co-ativador do PPAR (PGC-1β) e analisamos a estrutura cardíaca (histologia e estereologia), função cardíaca (ecocardiograma e técnica de Working heart), o metabolismo (isolamento de cardiomiócito e captação de glicose), ação hormonal (Western Blotting), expressão gênica (PCR-RT) de enzimas do metabolismo (lipídico, glicídico, da cadeia respiratória fatores transcricionais e hipertróficos) e a função mitocondrial. Verificamos, nos CIRS12KO, disfunção cardíaca grave, disfunção mitocondrial e prejuízo na expressão gênica das enzimas do metabolismo energético. Nos PGC-1βKO observamos disfunção mitocondrial e alteração de expressão gênica das enzimas do metabolismo energético quando submetidos à sobrecarga de pressão. Através do estudo do metabolismo cardíaco e da expressão gênica nestes diferentes modelos conseguimos explorar as vias metabólicas que levam a hipertrofia compensada à IC. Sugerimos que o mecanismo responsável pela descompensação seja a disfunção mitocondrial em conseqüência à alteração da expressão gênica. E que IRS e o PGC-1β são fatores chaves da dinâmica cardíaca, e que são indispensáveis para a estrutura e funcionamento cardíaco. Além de representar alvo promissor para limitar a transição da hipertrofia cardíaca compensada a insuficiência cardíaca. / Heart failure (HF) is the end stage of different types of cardiovascular diseases and it is characterized by changes in the metabolic and myocardial contractility. We use the models cre-lox with specific knockout for insulin receptor substrate (IRS) and co-activator of PPAR (PGC-1b) (basal and pressure overload). The objective was understood the role in the dynamics of cardiac metabolism. We analyzed cardiac structure (histology and stereology), cardiac function (echocardiography and the working heart technique), metabolism (glucose uptake), hormonal action (Western Blotting), gene expression (RT-PCR) from enzyme metabolism (lipid, carbohydrates, respiratory chain, transcriptional and hypertrophic factors) and mitochondrial function. We found in CIRS12KO, severe cardiac dysfunction, mitochondrial dysfunction and reduction of gene expression. And in the PGC-1bKO when subjected to pressure overload, the progression to heart failure, with mitochondrial dysfunction, and alteration of gene expression from enzyme metabolism. The data show that changes on cardiac metabolism and gene expression in both models explain the metabolic pathways that lead to compensated hypertrophy to HF. We suggest that the mitochondrial dysfunction and the gene expression was possible mechanisms for HF. We conclude that IRS and PGC-1b are key factors of cardiac dynamics, which are essential to the structure and heart function. IRS and PGC-1b represent a promising target for limiting the transition from compensated cardiac hypertrophy to heart failure.

L’huile de palme est l’huile végétale la plus consommée au monde. Du fait de sa teneur élevée en acides gras saturés (AGS), notamment en acide palmitique, cette huile est considérée par certains auteurs comme potentiellement nocive pour la santé. Cette étude avait pour objectif de comparer les effets de l’huile de palme (rouge ou oléine), à l’huile d’olive (réputée bonne pour la santé) et aux saindoux (riche en AGS), sur la santé. Pour réaliser cette étude, 40 rats mâles Wistar ont été répartis en 5 groupes de 8 rats chacun : 1 groupe contrôle et 4 groupes nourris par des régimes obésogènes contenant respectivement de l’huile de palme rouge, de l’oléine de palme, de l’huile d’olive ou du saindoux. Après 12 semaines de régime, les rats ont été sacrifiés et les tissus prélevés. Les examens réalisés sur les tissus ont montré que l’huile de palme (rouge ou oléine) induit un statut antioxydant et un profil lipidique superposables à ceux de l’huile d’olive. Tous les régimes obésogènes ont favorisé la prise de poids, l’altération de la fonction mitochondriale et la perturbation du métabolisme glucidique par l’induction d’une insulino-résistance. Il ressort de cette étude que l’huile d’olive est plus délétère pour le foie que l’huile de palme (rouge ou oléine) et le saindoux. Hormis l’huile de palme rouge, l’oléine de palme, l’huile d’olive et le saindoux influencent négativement les tissus adipeux. Les études menées sur l’aorte ont montré que les effets vasculaires de l’huile de palme sont moins délétères pour l’aorte que le saindoux et l’huile d’olive.Les résultats de cette étude indiquent que globalement, l’huile de palme (rouge ou oléine) n’a pas d’effets délétères supérieurs à ceux de l’huile d’olive concernant les organes qui ont été étudiés / Palm oil is the most consumed vegetable oil in the world. Because of its high content of saturated fatty acids (SFA), particularly palmitic acid, this oil is considered by some authors as potentially harmful to health. The aim of this study was to compare the effects of palm oil (red or olein), olive oil (considered good for health) and lard (rich in SFA), on health. To do this, 40 male Wistar rats were divided into 5 groups of 8 rats each: 1 control group et 4 groups fed by high fat diet (HFD) containing respectively red palm oil, palm olein, olive oil or lard. After 12 weeks of diet, the rats were sacrificed and the tissues removed. Tissue tests have shown that palm oil (red or olein) induces an antioxidant status and a lipid profile superimposed on those of olive oil. All HFD contributed to weight gain, impaired mitochondrial function, and disturbance of carbohydrate metabolism by the induction of insulin resistance. The study shows that olive oil is more deleterious to the liver than palm oil (red or olein) and lard. Apart from red palm oil, palm olein, olive oil and lard negatively influence adipose tissue. Studies on the aorta have shown that the vascular effects of palm oil are less deleterious to the aorta than lard and olive oil.Overall, the results of this study show that harmfull effects of palm oil (red or olein) were not worse than that of olive oil on organ that were analyzed

O infarto do miocárdio é atualmente considerado a etiologia que mais contribui para o aparecimento de insuficiência cardíaca (IC) em humanos. Em detrimento a hiperativação de fatores neuro-humorais, a progressão da IC é caracterizada por uma série de anormalidades celulares associadas à disfunção ventricular. Dentre estas anormalidades, alterações na função e dinâmica mitocondrial merecem destaque, uma vez que a homeostase da organela é essencial para a viabilidade celular e o bom funcionamento da bomba cardíaca. No presente estudo, caracterizamos em modelo animal de disfunção cardíaca associada ao infarto do miocárdio: a) fenótipo cardíaco; b) função mitocondrial; c) equilíbrio redox; e d) dinâmica mitocondrial. Nossos resultados nos permitem afirmar que doze semanas após a cirurgia de infarto do miocárdio, os animais desenvolveram importantes alterações fenotípicas como aumento da massa cardíaca, dilatação ventricular, hipertrofia do cardiomiócito e maior deposição de tecido fibroso cardíaco, que contribuíram para o estabelecimento da disfunção ventricular. Além disso, foi possível confirmar a instalação do quadro de disfunção mitocondrial cardíaca, representada pela redução na capacidade respiratória e perda da homeostase redox. Por fim, encontramos um aumento no número de mitocôndrias cardíacas com menor diâmetro, alterações que vieram acompanhadas de uma menor atividade das enzimas relacionadas à fusão mitocondrial. Uma vez caracterizada a função e a dinâmica mitocondrial na disfunção cardíaca, avaliamos o efeito do treinamento físico aeróbico (TF) nessas variáveis. O TF, atualmente utilizado como um adjuvante no tratamento das doenças cardiovasculares, foi eficaz em promover o remodelamento cardíaco reverso e melhorar a função cardíaca nos animais infartados. Além disso, melhorou a capacidade respiratória e reduziu o estresse oxidativo, restaurando a função mitocondrial. Aliado a esses achados, o TF normalizou a atividade das enzimas relacionadas à dinâmica mitocondrial, fato associado à normalização do número e tamanho da organela. Esses resultados demonstram que a disfunção cardíaca induzida por infarto do miocárdio está associada à um quadro de mitocondriopatia em ratos, com alterações tanto na função quanto estrutura mitocondrial, e que o TF desencadeia efeitos benéficos na manutenção da integridade/função mitocondrial e melhora da função contrátil cardíaca / Myocardial infarction is considered the etiology that most contributes to the onset of heart failure in humans. Among the ventricular dysfunction-associated cellular abnormalities, changes in mitochondrial function and dynamics are critical, since the organelle homeostasis is crucial in maintaining the metabolic, electrical and mechanical properties of the heart. In the present study, we characterized in cardiac dysfunction- induced myocardial infarction in rats: a) cardiac phenotype; b) mitochondrial metabolism; c) redox balance, and d) mitochondrial dynamics. Our results show that twelve weeks after myocardial surgery, the animals developed pathological cardiac remodeling-associated ventricular dysfunction. Furthermore, we observed a reduced mitochondrial respiratory capacity and loss of redox homeostasis. Finally, we found a lower activity of enzymes related to mitochondrial fusion, these changes were accompanied by an increase in the number of small mitochondria. Once characterized mitochondrial function and dynamics, we evaluated the effect of exercise training in these variables in rats with cardiac dysfunction. The exercise training, currently established as an important non-pharmacological treatment for cardiovascular diseases, reversed the pathological cardiac remodeling and minimized the ventricular dysfunction in infarcted animals. Furthermore, exercise training restored the mitochondrial function by increasing respiratory capacity and reducing oxidative stress. Finally, exercise training restored the activity of mitochondrial dynamics-related enzymes and morphology. Taken together, our findings uncover the potential benefits of exercise training in reversing the cardiac mitochondriopathy observed in failing hearts, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy

Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia associated with amyloid-beta peptide deposition and loss of mitochondrial function and regulation. Currently, there is no cure for AD, thus, there is a need to continuously develop therapeutic strategies that could address the complex multifactorial causes of AD development. Due to this necessity, this study has investigated the role of vitamin B2 as a disease modifying drug for AD by employingamyloid-beta and mitochondrial based AD therapeutic strategies. Using a transgenic C. elegans AD worm model expressing amyloid-beta (Aβ1-42) in muscle cells at temperature upshift to 25°C, we screened for protective effect of dose-dependent concentrations of active forms of vitamin B2, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), against amyloid-beta mediated paralysis. Protective concentrations were then assayed for improvement of mitochondrial metabolic functions by performing ATP, oxygen consumption and reactive oxygen species (ROS) production assays. Consequently, we investigated for drug protective mechanisms of FMN and FAD using RNAi genetic screening technique. FMN and FAD significantly delayed amyloid-beta mediated paralysis and improved mitochondrial metabolic functions at final concentrations of 0.74mM and 0.74µM respectively. More so, both compounds induced activation of stress response FOXO transcription factor, daf-16. Specifically, FMN treatment induced mitochondrial unfolded protein response (UPRmt) pathway through ubiquitin-like protein (ubl-5) activation as well as other stress response pathway signature such as Activating Transcription Factor Associated with Stress (atfs-1). This study will be useful in understanding the importance of micronutrients such as vitamin B2 in normal cellular function as related to neurodegenerativediseases and aging. Therefore, vitamin B2 supplementation could be an important source of Alzheimer’s disease therapeutic strategy.

<p>This thesis examined the biochemical role of skeletal muscle mitochondria and metabolic consequences of mitochondrial adaptations to exercise in individuals with poor glycemic control. Mitochondrial dysfunction and/or ectopic lipid accumulation has been implicated in the pathogenesis of metabolic-related diseases such as obesity and type 2 diabetes (T2D). However, whether mitochondrial dysfunction is the cause of insulin resistance and T2D or is a consequence of this disorder remains controversial. Alternatively, pro-inflammatory stress signals initiated through altered secretion of adipocytokines and oxidative stress may be a unifying mechanism underlying insulin resistance and T2D. Furthermore, the impact of exercise on muscle adaptation in insulin-resistant states is not well defined. At rest and prior to exercise training, no evidence of mitochondrial dysfunction or disproportionate intramyocellular lipid (IMCL) accretion was detected in obese, insulin-resistant skeletal muscle biopsy samples <em>vs.</em> healthy, lean age-, and fitness-matched men. In response to exercise training (12 weeks, consisting of 32 sessions of 30-60 min @ 50-70% maximal oxygen uptake [VO₂peak]), there was an increase in mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial content, and IMCL deposition with sub-cellular specificity. Exercise training also reduced both skeletal muscle and systemic oxidative damage, already elevated in the obese. The improved adipocytokine profile associated with obesity after training also coincided with improvements in glycemic regulation. Patients with genetic mitochondrial mutations, resulting in skeletal muscle mitochondrial dysfunction have an increase prevalence of dysglycemia/T2D. However, when evaluated against age- and activity-matched normoglycemic myopathy controls, no differences in mitochondrial electron transport chain protein subunits, mitochondrial or IMCL density, or level of whole-body insulin resistance was detected. In fact, dysglycemic mitochondrial myopathy patients demonstrated <em>higher </em>skeletal muscle OXPHOS capacity and Akt activation, a key step in insulin-stimulated glucose transport activity as compared with normoglycemic mitochondrial myopathy patients. Interestingly, a significant impairment in β-cell function (defective insulin secretion), in the dysglycemic patients was observed coincident with elevated glucose levels during the oral glucose tolerance test (OGTT). These findings indicate that insulin resistance does not cause skeletal muscle mitochondrial dysfunction/IMCL accumulation or <em>vice versa</em> and provides evidence against a direct link between mitochondrial dysfunction and the development of insulin resistance/T2D. Perhaps, oxidative stress/inflammation and pancreatic β-cell erosion mediate the observed obesity-induced insulin resistance and mitochondrial myopathy-associated T2D, respectively? Twelve weeks of moderate endurance exercise is an effective strategy to improve mitochondrial capacity, oxidative damage, inflammation, and glycemic regulation in insulin-resistant, obese individuals, but an improvement in muscle insulin sensitivity did not appear to be required.</p> / Doctor of Philosophy (Medical Science)

A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.