Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease affecting 1 in every 3500 boys. The disease is characterized by the absence of the dystrophin protein from the sarcolemma of muscle cells. Muscle cells lacking dystrophin go through cycles of degeneration and regeneration and are considered susceptible to contraction-induced injury 144. Eventually, the satellite cell proliferative capacity is exhausted and the muscle fibers are replaced by connective and adipose tissue that yields a progressive loss of force generating capability. DMD patients typically die by their early 20's, primarily due to respiratory or cardiac failure. The precise role of dystrophin is not presently known. However, its absence suggests that it may play a role in both cellular calcium regulation and oxidative stress 152. Recent studies suggest increased reactive oxygen species (ROS) may precede the initial wave of wasting that marks disease onset 49. Therefore, it is possible oxidative stress may contribute as a pathogenic mechanism of DMD. Strategies to reduce the deleterious effects of oxidative stress could be an effective therapeutic approach. Regular exercise is known to increase antioxidant capacity in humans and mice 146. Green tea extract (GTE) is a powerful antioxidant that is easily supplemented in the diet 83. The purpose of this study was to test the hypotheses that (1) voluntary endurance exercise alone, (2) a diet supplemented with 0.05% (wt/wt) GTE alone, or exercise and GTE combined will blunt the effects of ROS and improve muscle strength and endurance in young mdx mice. Male mdx mice at age 21-days were randomly divided into one of 4 treatment groups: Run Normal diet (RunNorm; n=8), Sedentary Normal diet (SedNorm; n=8), Run GTE diet (RunGTE; n=10), and Sedentary GTE diet (SedGTE; n=8). RunNorm and RunGTE mice were given free access to a running wheel for 3 weeks while SedNorm and SedGTE mice were restricted to normal cage movement. At the end of 3 weeks, mice in each treatment group were sacrificed and assessed for daily and weekly running distances, content of actin and myosin proteins and fiber type distribution (tibialis anterior), contractile/mechanical and fatigue properties (extensor digitorum longus), creatine kinase levels and antioxidant capacity (serum), lipid peroxidation (gastrocnemius), and citrate synthase and beta-hydroxyacyl-CoA dehydrogenase activities (quadriceps and soleus). The key findings of this study were: In normal diet running mice (RunNorm), average daily distance run was increased 300% (from 0.5 to 2.1 km/d, P<0.05) from week 1 to week 3. In GTE diet (RunGTE) compared to RunNorm mice, total distance over the 3 weeks was markedly improved 128% (61.2 vs. 26.8 km, P<0.0001). Running, independent of diet increased EDL muscle tetanic stress (18%), serum antioxidant capacity (22%), citrate synthase activity (35%), and beta-oxidation (37%; all P<0.05). GTE, independent of running decreased lipid peroxidation (gastrocnemius:-64%; heart: -29%) and serum creatine kinase (-36%), and increased citrate synthase activity (59%; all P<0.05). These findings in dystrophic mice suggest that voluntary endurance exercise with or without GTE supplementation blunted the deleterious effects of ROS. If similar positive effects are evident in human DMD patients, then these approaches may be beneficial therapies either alone or in combination. / Master of Science

L’excès de glucocorticoïdes lors d’un stress prolongé perturbe la neurotransmission monoaminergique et mène à des troubles de l’humeur et de la mémoire. La Venlafaxine (Vlx) et l’Agomelatine (Ago) sont utilisés pour traiter ces troubles. L’exercice physique volontaire est aussi bénéfique pour la santé mentale. Nous avons analysé 1. les changements de l’humeur induits par le stress en fonction du temps, 2. l’effet de l’exercice volontaire sur l’axe hypothalamo-pituitaire, 3. l’efficacité de l’Ago, de la Vlx et de l’exercice à prévenir les perturbations liées au stress et 4. la localisation des récepteurs MT1 et MT2 chez des souris rapportrices transgéniques. Nous démontrons que le stress induit des dérèglements physiques, émotionnels et comportementaux chez des rats stressés. Le prétraitement par l’Ago, la Vlx et l’exercice préviennent l’anxiété, la dépression et les déficits de mémoire. La cartographie des récepteurs MT1 et MT2 a identifié des sites d’action potentiels de l’Ago. / In long-term stress exposure, excess glucocorticoids disturb the balance of monoamine neurotransmitters leading to mood disorders and memory impairment. Venlafaxine and Agomelatin are currently used to treat these disorders. Voluntary exercise also has beneficial effects on mental health. In this work, we analyzed 1. the time-dependent changes in stress-induced mood disorders, 2. the modulating effect of voluntary exercise on the hypothalamic pituitary adrenal axis, 3. the effectiveness of Agomelatin, Venlafaxine and exercise to prevent stress-related behaviors and 4. the localization of MT1 and MT2 receptors in transgenic reporter mice. We demonstrate that stress caused physical, emotional and behavioral abnormalities in stressed rats. Pre-treatment with Agomelatin, Venlafaxine and exercise reduced the chronic stress-related behaviors and prevented anxiety, depression and memory deficits. The mapping of MT1 and MT2 receptors identified potential sites of action of Agomelatin.

INTRODUÇÃO: A Peroxisome Proliferator Activated Receptor- - Coactivator 1 ( PGC-1 e ) é proteína responsável pela conexão entre estímulos ambientais e resposta metabólica celular. Sua presença é importante em tecidos adiposo, hepático e muscular esquelético e, em animais, em tecido adiposo marrom. Interage com receptores nucleares modulando a biogênese mitocondrial e mantendo o equilíbrio termo energético celular com o meio ambiente. A redução da expressão de PGC-1 e da oxidação fosforilativa tem sido associada à resistência à insulina em doenças como Diabetes Mellitus tipo 2 e Síndrome Metabólica. OBJETIVOS: Avaliar o efeito do exercício na expressão da PGC-1 em tecidos alvos da insulina, como o fígado e músculos esquléticos soleus ( SOL ) e plantaris ( PLA ) de ratos machos Wistar e correlacioná-lo com a sensibilidade à insulina. METODOLOGIA: Ratos machos Wistar 190±15 g, n = 24, randomizados em 2 grupos: Ex ( exercício físico ) e Sd ( sedentário ) colocados respectivamente, em roda de atividade ou gaiolas comuns durante cinco semanas. Ao final do período, após jejum de quatro horas, foi colhido sangue para dosagens de glicose ( GLI ), insulina ( INS ) e ácidos graxos livres ( AGL ) e, em seguida, foram submetidos ao Teste de Supressão da Glicose e Insulina Endógenas com infusão durante 180 minutos de solução GLI ( 20mg/kg/min ) + INS ( 5 mU/kg/min ); amostras de sangue foram colhidas aos 140, 150, 160, 170 e 180 minutos. Terminado o teste e ainda sob anestesia, foram retirados os tecidos: fígado ( FG ) e músculos esquléticos ( PLA e SOL ), os quais foram imediatamente congelados e mantidos a -70ºC para posteriores análises. A expressão da PGC-1 foi avaliada pelo Western Blot com anticorpo policlonal anti- PGC-1. Análise estatística por teste t Student não-pareado e nível de significância 5%. RESULTADOS: Os dados se referem à média e erro padrão médio dos valores individuais das amostras. A distância percorrida na última semana ( km/dia ) pelo grupo Ex foi eficaz ( 5,61 ± 0,67 ). Não houve diferença no peso ( g ) dos ratos entre os grupos Ex e Sd ( 355,85 ± 9,51 x 375,68 ± 5,30 ) NS. Os valores de GLI jejum ( mg/dl ) foram semelhantes entre os grupos ( 117,6 ± 3,7 x 122,4 ± 2,6 ) NS. Entretanto, INS e AGL foram menores no grupo Ex: INS ( ng/ml ) ( 0,68 ± 0,12 x 1,45 ± 0,14 ) p < 0,001 e AGL ( mEq/L ) ( 1,12 ± 0,11 x 1,60 ± 0,11 ) p < 0,006. Durante o teste de supressão, os valores de GLI e INS na fase de estabilidade foram semelhantes entre grupos ( expressos em área sob a curva ): AUC GLI ( mg/dl/min ) ( 2,77 ± 0,12 x 2,95 ± 0,07 ) NS; AUC INS ( ng/ml/min ) ( 0,81 ± 0,15 x 0,99 ± 0,09 ) NS. A expressão da PGC-1 foi maior no PLA de ratos do grupo Ex e, em FG e SOL foi semelhante entre os grupos. CONCLUSÃO: O exercício físico durante 5 semanas em roda de atividade voluntária, aumentou a sensibilidade à insulina e a oxidação de ácidos graxos livres no jejum. A melhora da sensibilidade à insulina esteve associada à maior expressão da PGC-1 somente em músculo PLA. Estes dados sugerem que o aumento da sensibilidade à insulina no jejum não se relacionou com o aumento da expressão da PGC-1 em outros tecidos alvos da ação insulínica, como FG e SOL, neste modelo de estudo. / INTRODUCTION: The Peroxisome Proliferator-Activated Receptor- - Coactivator 1 ( PGC-1 e ) is a protein responsible for the connection between environmental stimuli and cell metabolic response. Its presence is important in fat tissue, hepatic and skeletal muscle and in animals on brown fat tissue. Interact with nuclear receptors modulating the mitochondrial biogenesis and maintain thermal energy balance with the environment. Diminished of PGC-1 expression and oxidative phophorylation has been associated to insulin resistance in diseases like Type 2 Diabetes and Metabolic Syndrome. OBJECTIVES: To evaluate the effects of exercise on the PGC-1 expression in target tissues of insulin, such as liver and skeletal muscles soleus (SOL) and plantaris (PLA) of male Wistar rats and correlates with insulin sensitivity. METHODOLOGY: Male Wistar rats 190±15g, n = 24, divided randomly into 2 groups: Ex ( physical exercise ) and Sd ( sedentary ), respectively placed in a voluntary running wheel cage or a standard cage for five weeks. At the end of study, after fasting for 4 hours, blood was collected for measurements of glucose ( GLU ), insulin ( INS ) and free fatty acids ( FFA ) then the animals were submitted to Test of Suppression Endogenous Glucose and Insulin, with infusion during 180 minutes of solution GLU ( 20mg/kg/min ) + INS ( 5mU/kg/min ); blood samples was collected at 140, 150, 160, 170 and 180 minutes. Finished the test and still anesthetized, the tissues were removed: liver ( LIV ), skeletal muscle ( SOL and PLA ) that were immediately frozen in liquid nitrogen and stored at -70ºC until analysis. The PGC-1 expression was evaluated by Western Blotting with polyclonal antibody anti-PGC-1. Statistical analysis by unpaired Students t test with significance level 5%. RESULTS: the data refer to the mean and standard error of individual values. The distance covered per day during last week ( km / day ) by Ex group was efficient ( 5,61 ± 0,67 ). There was no difference in weight ( g ) of rats between Ex and Sd groups ( 355,85 ± 9,51 x 375,68 ± 5,30 ) NS. The values of fasting GLU were similar between groups ( mg/dl ) ( 117,6 ± 3,7 x 122,4 ± 2,6 ) NS. However INS and FFA were lower in group Ex: INS ( ng/ml ) ( 0,68 ± 0,12 x 1,45 ± 0,14 ) p < 0,001 and FFA ( mEq/L ) ( 1,12 ± 0,11 x 1,60 ± 0,11 ) p < 0,006. During the suppression test the values of GLU and INS on stability step were similar between groups ( expressed in area under curve ): AUC GlU ( mg/dl/min ) ( 2,77 ± 0,12 x 2,95 ± 0,07 ) NS; AUC INS ( ng/ml/min ) ( 0,81 ± 0,15 x 0,99 ± 0,09 ) NS. The PGC-1 expression was greater in PLA of rats Ex than Sd group, and there was no difference in LIV and SOL between groups. CONCLUSION: The physical exercise during five weeks in voluntary running wheel increased the insulin sensitivity and fasting free fatty acids oxidation. The improvement of insulin sensitivity was associated with higher PGC-1 expression on PLA muscle only. These data suggest that increasing insulin sensibility on fasting is not associated with increasing of the PGC-1 expression in others targets tissues of insulin action, such as LIV and SOL, in this study model.

INTRODUÇÃO: A Peroxisome Proliferator Activated Receptor- - Coactivator 1 ( PGC-1 e ) é proteína responsável pela conexão entre estímulos ambientais e resposta metabólica celular. Sua presença é importante em tecidos adiposo, hepático e muscular esquelético e, em animais, em tecido adiposo marrom. Interage com receptores nucleares modulando a biogênese mitocondrial e mantendo o equilíbrio termo energético celular com o meio ambiente. A redução da expressão de PGC-1 e da oxidação fosforilativa tem sido associada à resistência à insulina em doenças como Diabetes Mellitus tipo 2 e Síndrome Metabólica. OBJETIVOS: Avaliar o efeito do exercício na expressão da PGC-1 em tecidos alvos da insulina, como o fígado e músculos esquléticos soleus ( SOL ) e plantaris ( PLA ) de ratos machos Wistar e correlacioná-lo com a sensibilidade à insulina. METODOLOGIA: Ratos machos Wistar 190±15 g, n = 24, randomizados em 2 grupos: Ex ( exercício físico ) e Sd ( sedentário ) colocados respectivamente, em roda de atividade ou gaiolas comuns durante cinco semanas. Ao final do período, após jejum de quatro horas, foi colhido sangue para dosagens de glicose ( GLI ), insulina ( INS ) e ácidos graxos livres ( AGL ) e, em seguida, foram submetidos ao Teste de Supressão da Glicose e Insulina Endógenas com infusão durante 180 minutos de solução GLI ( 20mg/kg/min ) + INS ( 5 mU/kg/min ); amostras de sangue foram colhidas aos 140, 150, 160, 170 e 180 minutos. Terminado o teste e ainda sob anestesia, foram retirados os tecidos: fígado ( FG ) e músculos esquléticos ( PLA e SOL ), os quais foram imediatamente congelados e mantidos a -70ºC para posteriores análises. A expressão da PGC-1 foi avaliada pelo Western Blot com anticorpo policlonal anti- PGC-1. Análise estatística por teste t Student não-pareado e nível de significância 5%. RESULTADOS: Os dados se referem à média e erro padrão médio dos valores individuais das amostras. A distância percorrida na última semana ( km/dia ) pelo grupo Ex foi eficaz ( 5,61 ± 0,67 ). Não houve diferença no peso ( g ) dos ratos entre os grupos Ex e Sd ( 355,85 ± 9,51 x 375,68 ± 5,30 ) NS. Os valores de GLI jejum ( mg/dl ) foram semelhantes entre os grupos ( 117,6 ± 3,7 x 122,4 ± 2,6 ) NS. Entretanto, INS e AGL foram menores no grupo Ex: INS ( ng/ml ) ( 0,68 ± 0,12 x 1,45 ± 0,14 ) p < 0,001 e AGL ( mEq/L ) ( 1,12 ± 0,11 x 1,60 ± 0,11 ) p < 0,006. Durante o teste de supressão, os valores de GLI e INS na fase de estabilidade foram semelhantes entre grupos ( expressos em área sob a curva ): AUC GLI ( mg/dl/min ) ( 2,77 ± 0,12 x 2,95 ± 0,07 ) NS; AUC INS ( ng/ml/min ) ( 0,81 ± 0,15 x 0,99 ± 0,09 ) NS. A expressão da PGC-1 foi maior no PLA de ratos do grupo Ex e, em FG e SOL foi semelhante entre os grupos. CONCLUSÃO: O exercício físico durante 5 semanas em roda de atividade voluntária, aumentou a sensibilidade à insulina e a oxidação de ácidos graxos livres no jejum. A melhora da sensibilidade à insulina esteve associada à maior expressão da PGC-1 somente em músculo PLA. Estes dados sugerem que o aumento da sensibilidade à insulina no jejum não se relacionou com o aumento da expressão da PGC-1 em outros tecidos alvos da ação insulínica, como FG e SOL, neste modelo de estudo. / INTRODUCTION: The Peroxisome Proliferator-Activated Receptor- - Coactivator 1 ( PGC-1 e ) is a protein responsible for the connection between environmental stimuli and cell metabolic response. Its presence is important in fat tissue, hepatic and skeletal muscle and in animals on brown fat tissue. Interact with nuclear receptors modulating the mitochondrial biogenesis and maintain thermal energy balance with the environment. Diminished of PGC-1 expression and oxidative phophorylation has been associated to insulin resistance in diseases like Type 2 Diabetes and Metabolic Syndrome. OBJECTIVES: To evaluate the effects of exercise on the PGC-1 expression in target tissues of insulin, such as liver and skeletal muscles soleus (SOL) and plantaris (PLA) of male Wistar rats and correlates with insulin sensitivity. METHODOLOGY: Male Wistar rats 190±15g, n = 24, divided randomly into 2 groups: Ex ( physical exercise ) and Sd ( sedentary ), respectively placed in a voluntary running wheel cage or a standard cage for five weeks. At the end of study, after fasting for 4 hours, blood was collected for measurements of glucose ( GLU ), insulin ( INS ) and free fatty acids ( FFA ) then the animals were submitted to Test of Suppression Endogenous Glucose and Insulin, with infusion during 180 minutes of solution GLU ( 20mg/kg/min ) + INS ( 5mU/kg/min ); blood samples was collected at 140, 150, 160, 170 and 180 minutes. Finished the test and still anesthetized, the tissues were removed: liver ( LIV ), skeletal muscle ( SOL and PLA ) that were immediately frozen in liquid nitrogen and stored at -70ºC until analysis. The PGC-1 expression was evaluated by Western Blotting with polyclonal antibody anti-PGC-1. Statistical analysis by unpaired Students t test with significance level 5%. RESULTS: the data refer to the mean and standard error of individual values. The distance covered per day during last week ( km / day ) by Ex group was efficient ( 5,61 ± 0,67 ). There was no difference in weight ( g ) of rats between Ex and Sd groups ( 355,85 ± 9,51 x 375,68 ± 5,30 ) NS. The values of fasting GLU were similar between groups ( mg/dl ) ( 117,6 ± 3,7 x 122,4 ± 2,6 ) NS. However INS and FFA were lower in group Ex: INS ( ng/ml ) ( 0,68 ± 0,12 x 1,45 ± 0,14 ) p < 0,001 and FFA ( mEq/L ) ( 1,12 ± 0,11 x 1,60 ± 0,11 ) p < 0,006. During the suppression test the values of GLU and INS on stability step were similar between groups ( expressed in area under curve ): AUC GlU ( mg/dl/min ) ( 2,77 ± 0,12 x 2,95 ± 0,07 ) NS; AUC INS ( ng/ml/min ) ( 0,81 ± 0,15 x 0,99 ± 0,09 ) NS. The PGC-1 expression was greater in PLA of rats Ex than Sd group, and there was no difference in LIV and SOL between groups. CONCLUSION: The physical exercise during five weeks in voluntary running wheel increased the insulin sensitivity and fasting free fatty acids oxidation. The improvement of insulin sensitivity was associated with higher PGC-1 expression on PLA muscle only. These data suggest that increasing insulin sensibility on fasting is not associated with increasing of the PGC-1 expression in others targets tissues of insulin action, such as LIV and SOL, in this study model.

La drépanocytose est, sous sa forme grave, une pathologie très invalidante pour les personnes qui en sont porteuses. Elle est rythmée par la récurrence de crises vaso-occlusives (CVO) et autres symptomatiques qui en découlent (accident vasculaire cérébral, syndrome thoracique aigu, hypertension pulmonaire, etc.). En participant, chez des sujets sains et pathologiques, à la limitation de certains facteurs impliqués dans les CVO (inflammation, adhésion vasculaire, stress-oxydatif), l'entrainement physique régulier pourrait hypothétiquement être bénéfique au sujets drépanocytaires et participer à améliorer leurs qualités de vie. C'est l'hypothèse qui a été étudiée durant ce travail de thèse chez des sujets porteurs du trait drépanocytaire (PTD) puis sur un modèle de souris transgéniques drépanocytaires, les souris SAD. La première étude a permis de montrer que des sujets PTD entrainés présentaient, au repos et en réponse à un exercice incrémental maximal, des concentrations en molécules solubles d'adhérence vasculaire (sVCAM-1) inférieures à celles des sujets PTD sédentaires. Ces concentrations sont par ailleurs comparables à celles de sujets sains. Ce résultat allant dans le sens d'une limitation de l'activation endothéliale par l'entrainement chez les PTD, des études plus poussées ont été réalisées sur les souris SAD. Deux études de caractérisation ont été nécessaires pour mettre en place notre expérimentation sur les souris SAD. La première a permis de valider un protocole de 8 semaines en roue d'activité physique volontaire sur des souris saines C57Bl/6. La deuxième a permis de caractériser le déclenchement de la CVO chez des souris SAD à l'aide d'un protocole d'hypoxie/réoxygénation (H/R) du point de vue de l'inflammation, l'adhérence vasculaire et du stress-oxydatif. Ces deux études ont alors permis la mise en place du dernier protocole de cette thèse : l'entrainement par roue d'activité physique volontaire des souris SAD. Huit semaines d'activité physique ont induit, chez les souris SAD entrainées, une limitation des dysfonctionnements endothéliaux induit par le stress d'H/R et observé chez les souris SAD sédentaires. Cette thèse tend donc à montrer que l'activité physique régulière pourrait être bénéfique pour les souris SAD en limitant certains facteurs impliqués dans la CVO. D'autres études seront nécessaires sur d'autres modèles murins drépanocytaires plus sévères et dans un plus long terme chez l'Homme pour adapter ces conclusions au sujets drépanocytaires / Sickle cell disease is, in its severe form, a very disabling disease. lt is punctuated by recurrent vaso- occlusive crisis (VOC) which may induce other symptomatics (stroke, acute chest syndrome, pulmonary hypertension, etc ...). By participating, in healthy and pathological subjects, in the limitation of some factors involved in the VOC (inflammation, vascular adhesion, oxidative stress), habitual physical training could hypothetically be beneficial in subjects with sickle cell disease and participate in improving their quality of life. This hypothesis has been investigated during this thesis on sickle cell trait (SCT) carriers and on a transgenic sickle mice model, SAD mice. The first study showed that trained SCT carriers displayed, at rest as in response to an incremental and maximal exercise, lower plasma vascular cell adhesion molecules (sVCAM-1) compared to sedentary SCT carriers. Moreover, these concentrations were similar to that of healthy subject. These results confirm a possible limitation of endothelial activation in SCT carriers. This potential benefic effect has further been investigated in sickle SAD mice. Two characterization studies were necessary to establish our experiments on SAD mice: the first permitted to validate a 8 weeks voluntary wheel running (VWR) protocol on healthy C57Bl/6 mice while the second characterized the VOC induced by a hypoxia/reoxygenation (H/R) stress in SAD mice from the inflammation, vascular adhesion molecule and oxidative stress points of view. Thanks to these studies, a 8 weeks VWR protocol has been performed in SAD mice. Thus, physical activity permitted to reduce in VWR SAD mice the endothelial dysfunction induced by the H/R stress and observed in sedentary SAD mice. This thesis shows that habitual physical activity may be beneficial in sickle SAD mice in limiting some factors involved in VOC. Further studies will be necessary, on more severe sickle mice models and in a longer term in Humans, to adapt these findings to SCD subjects