Le stress oxydatif joue un rôle prépondérant en tant que messager secondaire dans la régulation de nombreux processus cellulaires tels que l’apoptose, la survie et la prolifération et serait impliqué dans l’ensemble des étapes de la carcinogenèse pulmonaire. L’activité physique et la nutrition sont deux facteurs pouvant moduler le stress oxydatif et les mécanismes associés. La bétaïne et la C-Phycocyanine sont deux micronutriments reconnus pour avoir des effets antioxydants, anti-inflammatoires et antiprolifératifs. Récemment notre équipe a montré in vitro qu’un traitement en bétaïne et/ou la C-phycocyanine diminuait la viabilité des cellules A549 (carcinome pulmonaire). L’objectif général de ce travail de thèse était d’évaluer l’effet de facteurs nutritionnels (bétaïne, C-PC ou activité physique) sur la croissance tumorale de cellules A549 implantées chez le rat Nude et de déterminer les mécanismes sous-jacents. Dans un premier temps, nous avons étudié l’effet d’une supplémentation nutritionnelle (bétaïne ou C-phycocyanine) associée ou non à la pratique d’une activité physique volontaire (roue d’activité) sur l’équilibre redox et l’inflammation, chez des rats sains. Nous avons montré que la bétaïne et la C-phycocyanine augmentaient les défenses antioxydantes tandis que l’activité physique volontaire n’avait pas d’effet si elle n’était pas couplée à une supplémentation. Nous avons également mis en évidence que C-phycocyanine inhibait l’augmentation de Cox-2 musculaire induite par l’activité physique. Dans un second temps, nous avons étudié l’effet de la bétaïne et/ou la C-Phycocyanine sur la croissance des cellules A549 implantées chez des rats Nude. Nous avons montré que ces deux micronutriments associés ou non ralentissaient la croissance des tumeurs pulmonaires, au travers de mécanismes communs (activation de NF-kappaB, augmentation de la peroxydation lipidique et de l’expression de cytokine pro-inflammatoire (IL-1-beta, Cox-2 et TNF-alpha) au sein de la tumeur) et de mécanismes propres à chaque micronutriment. La C-phycocyanine a induit une diminution du ratio AKT phosphorylé / AKT total et une augmentation du ratio p38 phosphorylé / p38 total, mécanisme en faveur de l’apoptose et de l’autophagie. La bétaïne associée à la C-phycocyanine a augmenté le ratio caspase-3 / pro-caspase-3. Dans un dernier temps, nous avons évalué l’effet de l’activité physique volontaire sur la croissance tumorale des cellules A549 implantées chez des rats Nude. Nous avons mis en évidence que l’activité physique volontaire ralentissait la croissance des tumeurs pulmonaires induites, sans différence significative avec la bétaïne et/ou la C-phycocyanine. Il apparait que l’augmentation de la peroxydation lipidique, l’activation de la MAPK p38 et de NF-kappaB, et l’inhibition d’AKT, favorisant la mort cellulaire soient impliquées dans cette diminution tumorale. Un régime enrichi en bétaïne et/ou C-phycocyanine ralentit la croissance cellulaire d’adénocarcinome pulmonaire implanté chez le rat, suggérant leur intérêt dans l’action anti-carcinogène pulmonaire. L’activité physique semble jouer sur les mêmes mécanismes. Nos résultats méritent d’être confirmés par des protocoles à plus large échelle et suggèrent de possibles applications chez des patients porteurs de tumeurs pulmonaires. / Oxidative stress seems to play a crucial role as a secondary messenger in the regulation of several cellular processes such as apoptosis, survival and proliferation, and could be involved in all steps of the lung carcinogenesis (i.e. initiation, promotion and progression). Physical activity and nutrition are two factors able to modulate oxidative stress and associated mechanisms. Betaine and C-phycocyanin are two known micronutrients having antioxidant, anti-inflammatory and anti-proliferative effects. Previously, our team showed that betaine and/or C-phycocyanin treatment decreased the viability of A549 cells in vitro (pulmonary adenocarcinoma cell line). The main objective of this work was to evaluate the effect of nutritional factors (betaine, C-phycocyanin) or physical activity on growth of implanted A549 cells in Nude rats and to determine underlying mechanisms.Firstly, we studied the effect of nutritional supplementation (betaine or C-phycocyanin) combined or not with voluntary physical activity (wheel running) on redox balance and inflammation in healthy rats. We showed that betaine and C-phycocyanin increased antioxidant defenses, whereas voluntary physical activity did not have an effect when it was not associated with micronutrient supplementation. We also observed that C-phycocyanin inhibited physical activity-induced muscle Cox-2 activity increase.Secondly, we studied the effect of betaine and/or C-phycocyanin on growth of implanted A549 cells in Nude rats. We showed that these two micronutrients, whether associated or supplied separately, slowed down the lung tumour growth through similar mechanisms (NF-kappaB activation and increase of lipid peroxidation and expression of pro-inflammatory cytokines (IL-1beta, Cox-2 et TNF-alpha) in tumour). Also, some mechanisms were specific for each micronutrient or their combination. C-phycocyanin induced a decrease of phosphorylated AKT / total AKT ratio, and an increase of phosphorylated p38 / total p38 ratio, both mechanisms promoting apoptosis and autophagy. On the other hand, betaine associated with C-phycocyanin increased caspase-3 / pro-caspase-3 ratio.Finally, we studied the effect of voluntary physical activity on growth of implanted A549 cells in Nude rats. We showed that voluntary physical activity slowed down the lung tumour growth, without significant difference if animals were supplied with betaine or/and C-phycocyanin. It seems that the increase of lipid peroxidation, NF-kappaB and p38 activation, and AKT inhibition, all having a role in promotion of a cell death, are responsible for the tumour growth slowdown following the physical activity. In conclusion, diet enriched with betaine or/and C-phycocyanin slows down the growth of pulmonary adenocarcinoma cells implanted in rats, suggesting their interest in anti-cancer activity. Physical activity seems to act on similar mechanisms as these micronutrients. Our results have to be confirmed with further studies, but are already suggesting a potential application in lung cancer patients.

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