Reproduction de : Thèse de doctorat : Immunologie : Lille 2 : 2008. / Résumé en français et en anglais. Titre provenant de l'écran-titre. Bibliogr. f. 60-79.

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La perte de l’expression de la sous-unité β4 des canaux sodiques dépendants du voltage NaV dans les tumeurs mammaires est associée à un grade cancéreux élevé et au développement des métastases. L’extinction de son expression dans les cellules MDA-MB-231 augmente de plus de deux fois leur invasivité. Au cours de cette thèse, nous avons montré que la sous-expression de β4 favorise la transition mésenchymato-amoeboïde et augmente l’invasion cancéreuse indépendante de NaV. Cette transition se caractérise par l’acquisition d’une morphologie plus arrondie, par la présence de blebs à la surface cellulaire et par une augmentation de l’activité RhoA-GTPase. Cette transition est inhibée par la surexpression du domaine intracellulaire C-terminal de la sousunité β4. L’expression de β4 peut être augmentée par un apport en acide docosahexaènoïque (22:6n-3), qui augmente l’activité du promoteur de son gène SCN4B. Le DHA augmente également l’expression de β4 en modulant l’expression des récepteurs nucléaires PPAR, sensibles aux lipides. / The loss of voltage gated sodium channel NaVβ4 subunit expression in breast cancer biopsies is associated with high grade tumors and metastatic development. The inhibition of β4 expression in MDA-MB-231 breast cancer cells enhanced their invasiveness by two fold. During this thesis, we have shown that β4 underexpression promotes mesenchymal-amoeboid transition and increases NaV-independent invasion. This transition is characterized by rounded morphology, the presence of blebs at the cell surface and an increased RhoAGTPase activity. This transition is inhibited by β4 C-terminal intracellular domain overexpression. Expression of β4 can be enhanced by a DHA supplementation that increases the encoding SCN4B promoter activity. DHA also increases β4 expression through the modulation of PPARs lipid-sensitive nuclear receptors expression.

Les récepteurs nucléaires PPAR sont régulés par les acides gras polyinsaturés n-3 (AGPI n-3), dont l’acide docosahexaènoïque (DHA, 22:6n-3) qui inhibe la croissance des lignées de cellules cancéreuses mammaires. Des siRNA et des ligands pharmacologiques des PPAR (α, β et γ) ont permis de montrer que PPARβ régule cet effet inhibiteur du DHA. Dans un modèle murin de tumeur mammaire, la supplémentation en AGPI n-3 diminue l’expression de PPARβ et les tumeurs qui régressent le plus, sous chimiothérapie, sont celles qui expriment le moins PPARβ. L’invasivité des cellules cancéreuses mammaires et l’activité du canal sodique NaV1.5, qui promeut l’invasivité, sont inhibées par le DHA. Des antagonistes de PPARβ, comme l’inhibition de son expression, diminuent l’invasivité, le courant NaV1.5 et l’activité de son partenaire, l’échangeur Na+/H+ de type 1 (NHE-1). Les activités NaV1.5 et NHE-1 sont aussi diminuées dans les cellules qui n'expriment plus PPARβ. Cette étude montrant un lien mécanistique entre DHA, PPARβ, NaV1.5 et NHE-1, indique que l’inhibition de PPARβ par des AGPI n-3 à longue chaîne pourrait compléter les traitements actuels du cancer du sein. / Peroxisome proliferator-activated receptors (PPAR) are nuclear receptors for n-3 polyunsaturated fatty acids (n-3 PUFA), such as docosahexaenoic acid (DHA, 22:6n-3) which inhibits breast cancer cell growth. Using siRNA and pharmacological ligands of PPAR (α, β or γ) in breast cancer cell lines, we showed that PPARβ regulated DHA-induced inhibition of cell growth. In a rat mammary tumor model supplemented with n-3 PUFA, PPARβ expression diminished and tumors that regressed most, under docetaxel treatment, displayed the lowest PPARβ expression. Breast cancer cells invasiveness and activity of NaV1.5 voltage-gated sodium channel, known to promote invasiveness, were both inhibited by DHA. Antagonizing PPARβ, or inhibiting its expression, reduced invasiveness and NaV1.5 current as well as its accompanying Na+/H+ exchanger type 1 (NHE-1) activity. NaV1.5 and NHE-1 activities were also reduced in cells knocked-down for PPARβ. This study shows a mechanistic link between DHA, PPARβ, NaV1.5 current and NHE-1 pro-invasive activities, advocating for the inhibition of PPARβ with long chain n-3 PUFA to complement current breast cancer therapies.

Acredita-se que a diminuição progressiva da massa de células observada durante a evolução do diabetes mellitus tipo 2 (DM 2) ocorra por apoptose deste tipo celular. As tiazolidinedionas (TZDs), uma classe de medicamentos utilizada no tratamento do DM 2, atuam como ligantes dos receptores ativados por proliferadores de peroxissomos (PPAR) e e promovem diminuição da resistência periférica à insulina. Embora existam estudos controversos, tem se especulado que as TZDs possam exercer efeitos diretos sobre as células pancreáticas, prevenindo a perda por apoptose e melhorando a sua viabilidade. O objetivo deste estudo foi avaliar os efeitos diretos da Pioglitazona (PIO) na concentração de 10 M sobre a viabilidade funcional e o índice de apoptose de ilhotas pancreáticas isoladas de ratos Wistar expostas a concentrações fisiológica (5,6 mM) e suprafisiológica (23 mM) de glicose durante 24, 48 e 72 horas. A viabilidade funcional foi avaliada pela análise da secreção de insulina estimulada por glicose e do conteúdo total de insulina nas ilhotas. O índice de apoptose foi avaliado pela medida da fragmentação do DNA, da expressão do RNAm dos genes Bcl2 (anti-apoptótico) e Bax (pró-apoptótico) e da atividade proteolítica da caspase-3 em ilhotas tratadas e não tratadas com a PIO. Em 5,6 mM de glicose, não se observou efeito significativo sobre a secreção de insulina, mas a avaliação do conteúdo total de insulina evidenciou uma diminuição transitória nas ilhotas tratadas com PIO por 24 horas, seguida por um aumento no conteúdo de insulina quando as ilhotas foram cultivadas por 48 e 72 horas em presença da droga. Em relação à avaliação da apoptose, observou-se uma diminuição na expressão do RNAm do gene Bax nas ilhotas tratadas com PIO por 24 horas, entretanto, após 48 e 72 horas, houve um aumento da expressão do RNAm deste gene nas ilhotas tratadas com a droga. Não foram observadas diferenças estatisticamente significativas na expressão do RNAm do gene Bcl2 em nenhum dos tempos estudados e a avaliação da apoptose determinada pela medida da fragmentação do DNA somente demonstrou uma diminuição do índice de apoptose após 48 horas de tratamento com a PIO. Em 23 mM de glicose, a PIO promoveu um aumento transitório na secreção de insulina estimulada por glicose e no conteúdo total de insulina (após 48 horas), no entanto, após 72 horas, observou-se diminuição significativa no conteúdo total de insulina. Em relação à apoptose, o tratamento com PIO determinou um aumento do índice de apoptose medido pela fragmentação do DNA e da atividade proteolítica da caspase-3 após 48 e 72 horas e uma diminuição da expressão do RNAm do gene Bcl2 nos tempos 24 e 48 horas. Os resultados do presente estudo sugerem que os efeitos diretos da PIO sobre as ilhotas pancreáticas murídeas em cultura variam de acordo com a concentração de glicose a qual as ilhotas estão expostas: em concentração fisiológica de glicose, a PIO parece exercer efeitos diretos benéficos, enquanto em concentração suprafisiológica de glicose, ela exerce efeitos diretos deletérios sobre a viabilidade funcional e o índice de apoptose de ilhotas pancreáticas murídeas em cultura. / The progressive decrease in -cell mass observed during the evolution of type 2 diabetes (T2DM) is believed to occur due to cell apoptosis. Thiazolidinediones (TZDs), a class of agents used for the treatment T2DM, act as ligands of the peroxisome proliferator-activated receptor (PPAR) and and decrease peripheral insulin resistance. Although still controversial, some studies have shown a direct effect of TZDs on pancreatic -cell, preventing cell loss due to apoptosis and improving their viability. The objective of this study was to evaluate the direct effects of 10 M Pioglitazone (PIO) on functional viability and apoptosis rate of islets isolated from Wistar rats exposed to physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations during 24, 48 and 72 hours. The functional viability was evaluated by the analysis of insulin secretion after glucose challenge and of islet total insulin content. Apoptosis rate was evaluated by measurement of DNA fragmentation, of Bcl2 (antiapoptotic) and Bax (proapoptotic) mRNA expression and of proteolytic activity of caspase-3 in pancreatic islets treated or not with PIO. At 5.6 mM glucose concentration, no significant effects in insulin secretion were observed, while a transitory decrease (after 24 hours) followed by an increase in total insulin content was observed in islets treated with PIO for 48 and 72 hours. Regarding apoptosis, a lower expression of Bax mRNA was detected in islets treated with PIO for 24 hours, followed, however, by an increase in the expression of this gene after 48 and 72 hours of drug exposition. PIO treatment did not promote significant changes in Bcl2 mRNA expression, while decreased the apoptosis rate measured by DNA fragmentation only after 48 hours of exposition. At 23 mM glucose concentration, PIO treatment elicited a transitory increase in insulin secretion after glucose challenge and in islet total insulin content after 48 hours followed by a decrease in the islet total insulin content after 72 hours. Concerning apoptosis, PIO treatment determined an increase in the apoptose rate measured by DNA fragmentation and by proteolytic activity of caspase-3 after 48 and 72 hours and a decrease in Bcl2 mRNA expression after 24 and 48 hours. These findings suggest that the direct effects of PIO on pancreatic islets depend on glucose concentration to which they are exposed: while under physiological glucose concentration the direct effects seem to be beneficial, under supraphysiological glucose concentration, PIO exerts direct deleterious effects on the functional viability and on the apoptosis rate of murine pancreatic islets.

Orientador: Hernandes Faustino de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T08:33:52Z (GMT). No. of bitstreams: 1 Escobar_EsdrasLauniOliveira_M.pdf: 992060 bytes, checksum: 84890f8de9a24a3d1c8b06e7da089112 (MD5) Previous issue date: 2007 / Resumo: A próstata é uma glândula acessória do trato reprodutor masculino. Muitos são os fatores envolvidos no carcinogênese prostática. Os nutricionais são usualmente associados com a etiopatogenia do câncer de próstata, sendo que os ácidos graxos estão entre os mais importantes. O objetivo deste trabalho foi avaliar o efeito dos ácidos graxos presentes na dieta sobre o crescimento da próstata ventral de ratos, sobre a organização do tecido e sobre a expressão do receptor de andrógeno (AR) e do receptor dos ativadores da proliferação dos peroxissomos ? (PPAR?). Ratos Wistar machos foram utilizados, divididos em 5 grupos que receberam dietas isocalóricas por 10 semanas após o desmame. O grupo I foi tratado com dieta cuja porção lipídica continha óleo de soja. O grupo II recebeu óleo de soja e óleo de linhaça (1:1). O grupo III foi alimentado com óleo de linhaça. O grupo IV teve na dieta óleo de soja e banha de porco (1:1). O grupo V foi tratado com banha de porco. Ao fim do tratamento, as próstatas foram removidas e processadas para imunohistoquímica ou congeladas para análises bioquímicas. Os níveis circulantes de testosterona e do estradiol foram medidos. Os ensaios demonstraram que a banha de porco e o óleo de linhaça causaram efeitos opostos em relação ao crescimento prostático. Enquanto o primeiro promoveu um aumento no peso prostático, o outro causou uma significativa diminuição no órgão. A análise histológica revelou nítida hiperplasia epitelial, que foi confirmada pela análise estereológica, em resposta ao tratamento com dieta contendo apenas banha de porco como componente lipídico. Esta estrutura epitelial está associada a um aumento na taxa de proliferação epitelial, como demonstrado pela quantificação de células em proliferação, com o uso do antígeno Ki67. Experimentos de imunohistoquímica e Western blotting demonstraram um aumento na expressão de AR e PPAR? nos grupos que receberam banha de porco na dieta, enquanto o óleo de linhaça (contido) promoveu uma diminuição nos níveis de ambas as proteínas nucleares. Estes resultados sugerem que o crescimento prostático é influenciado por ácidos graxos presentes na dieta, e que esta influência pode ocorrer através de um aumento da expressão de AR e de PPAR?. Estes dados sugerem que PPAR? pode representar o elo de ligação entre a dieta e a predisposição ao câncer do próstata, sendo que a expressão e a função do AR podem ser alvos de regulação pelo PPAR?. Como os níveis circulantes de testosterona mostraram-se alterados é também possível que as alterações prostáticas sejam secundárias aos efeitos sistêmicos da dieta / Abstract: The prostate is an accessory gland of the male reproductive tract. Many factors are involved in prostate carcinogenesis. Nutritional factors are usually associated with the etiopathogenesis of the prostate cancer and fatty acids are among the most important ones. The objective of this work was to evaluate the effect of dietary fatty acid on the rat ventral prostate growth, tissue organization, and expression of androgen receptor (AR) and peroxisome proliferation-activator receptor ? (PPAR?). Male Wistar rats were used and divided in 5 groups that received isocaloric diets for 10 weeks after weaning. Group I was treated with soybean oil. Group II received soybean oil and linseed oil (1:1). Group III was fed on linseed oil. Group IV had soybean oil and rendered pork fat (1:1). Group V was treated with rendered pork fat. At the end of treatment, the prostates were removed and processed for immunohistochemistry or frozen for biochemical analyses. Circulating levels of testosterone and estradiol were measured. Experiments demonstrated that lard and linseed oil caused opposite effects on prostatic growth. While the former promoted an increase in prostatic weight, the latter resulted in a significantly lighter organ. Epithelial hyperplasia was observed by histological inspection and confirmed by stereology as the major tissue response to the rendered pork fat diet. This aspect was associated with higher proliferative rates as demonstrated by counting proliferating cell expressing the KI-67 antigen. Immunohistochemistry and Western blotting demonstrated increased expression of AR and PPAR? in groups receiving lard as the source of fatty acids, while linseed oil promotes a decrease on the protein levels of both nuclear receptors. These results suggest that prostate growth is influenced by dietary fatty acids and that this influence might occur via enhanced expression of PPAR? and AR. One suggests that PPAR? might represent the link between diet and predisposition to prostate cancer and that AR expression and function could be targeted by PPAR? activation. Since the levels of circulating testosterone were altered it is also possible that prostatic changes are secondary to systemic effects of the diet / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Sandra Martha Gomes Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:13:51Z (GMT). No. of bitstreams: 1 Gomes_EmersonRodrigoMachi_M.pdf: 4424058 bytes, checksum: 4547741a163b53b4836c32f103096cd3 (MD5) Previous issue date: 2013 / Resumo: Células tumorais apresentam uma autonomia metabólica aumentada em comparação a células não-transformadas, incorporando nutrientes e metabolizando-os através de vias que suportam o seu crescimento e proliferação. O foco deste trabalho foi a enzima glutaminase, a qual processa glutamina em glutamato para posterior produção de alfa-cetoglutarato pela enzima glutamato desidrogenase, reabastecendo o ciclo do TCA e suportando seu funcionamento e geração de metabólitos essenciais para a síntese de macromoléculas. O gene GLS1 codifica para as isoformas glutaminase kidney-type (KGA) e glutaminase C (GAC). Estas proteínas apresentam outros domínios além do catalítico, e, no caso da KGA, repetições do tipo ankirin, sabidamente envolvidas em contatos proteínas-proteínas. Os objetivos deste projeto foram de encontrar parceiros de interação para a glutaminase kidney-type (KGA) e avaliar o impacto desta interação para o metabolismo tumoral. Um candidato inicialmente avaliado, a Aldolase A, não foi confirmado como parceiro de interação. Outro candidato, a BNIP-H, apesar de ter sido mostrado interagir com a KGA em células nervosas, não mostrou indícios de interação com a KGA em linhagem de células de câncer de mama. Por fim, estudos de duplo-híbrido em levedura revelaram o receptor nuclear PPAR? (Peroxisome proliferator-activated receptor gamma) como forte candidato a parceiro de interação. Realizou-se um mapeamento dos domínios responsáveis pela interação entre estas duas proteínas, também por duplo híbrido, tendo sido identificado o domínio LBD da proteína PPAR? como envolvido na interação. Mesmo estudos realizados com fragmento da KGA, apesar de incompletos, mostraram que a interação não ocorre pelo domínio carboxi-terminal da enzima. Ensaios de anisotropia de fluorescência com as proteínas KGA e PPAR? purificadas indicaram que a interação é favorecida pela presença do produto da reação glutaminolítica, glutamato, e apresenta um Kd de 4,6 ± 0,5 ?M. Microscopia confocal de imunofluorescência mostrou que ambas as proteínas se co-localizam no citoplasma, mas não no núcleo. Mais se verificou que em células HEK 293T a presença de KGA diminui a capacidade de transativação de PPAR? induzida pelo ativador roziglitazona, enquanto que celular PC3 com superexpressão de KGA apresentam níveis diminuídos de expressão da proteína ACADL, alvo do PPAR?. Da mesma maneira, ensaios in vitro de atividade da KGA mostraram que a presença de PPAR? inibe a atividade da glutaminase. Nossos resultados mostram a interação in vitro entre as proteínas KGA-PPAR? e a potencial influência funcional que uma proteína exerce sobre a outra. Dado a participação de ambas as proteínas no processo tumoral, especula-se que esta interação possa ter impacto no desenvolvimento do câncer / Abstract: Tumor cells have an increased metabolic autonomy compared to non-transformed cells, metabolizing nutrients and incorporating them through pathways that support cell growth and proliferation. The focus of this study was the glutaminase enzyme, which processes glutamine to glutamate for subsequent production of alpha-ketoglutarate, by the glutamate dehydrogenase enzyme, replenishing TCA cycle and bearing its function and the generation of metabolites essential for the synthesis of macromolecules. The gene GLS1 codes for the isoforms kidney-type glutaminase (KGA) and glutaminase C (GAC). These proteins exhibit other domains besides the catalytic, and in the case of KGA, ankirin repeats, known to be involved in protein-protein contacts. The goal of this project was to investigate potential interacting partners of KGA and contextualize the interaction within the metabolic demands of tumor cells. A candidate initially evaluated, the Aldolase A, was not confirmed as a partner of interaction. Another candidate, the BNIP-H, despite having been shown to interact with the KGA in nervous cells, showed no evidence of interaction with KGA in one tested breast cancer cell lines. Finally, yeast two-hybrid studies revealed the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR?) as a strong interaction partner candidate. We mapped the domains responsible for the interaction between these two proteins, also by two-hybrid and identified the LBD domain of PPAR? as involved in the interaction. The same studies with KGA fragments, although incomplete, showed that the interaction did not involve the carboxy-terminal domain of the enzyme. KGA and PPAR? proteins were expressed in E. coli, purified and their interaction was analyzed by pull-down, fluorescence anisotropy, electrophoresis under native conditions, gel filtration chromatography and crosslinking. The assays indicated that the interaction is favored by the presence of the reaction product glutamate and has a Kd of 4,6 ± 0,5 ?M and disfavored by phosphate. Immunogold labeling followed by transmission electron microscopy of SKBR3 cells revealed a curious nuclear staining pattern probably heterochromatic of KGA. Immunofluorescence confocal microscopy showed that both proteins co-localize in the cytoplasm but not in the nucleus. Moreover, it was found that in HEK 293T cells, the presence of KGA decreases PPAR? ability of inducing transactivation of a reporter gene, while PC3 cell overexpressing KGA have low levels of protein expression ACADL target this receptor. Likewise, activity in vitro assays in the presence of KGA showed that PPAR? receptor inhibits the glutaminase activity. Our results demonstrate the in vitro interaction between proteins KGA-PPAR? and the potential functional influence that these proteins exerts on each other. Given the involvement of both proteins in the tumor growth, it is speculated that this interaction may have impact on the development of cancer / Mestrado / Clinica Medica / Mestre em Ciências

Orientador: Munir Salomão Skaf / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T04:47:12Z (GMT). No. of bitstreams: 1 Mottin_Melina_M.pdf: 3350054 bytes, checksum: 28a03021273db312bbcba2e77fe7b2eb (MD5) Previous issue date: 2012 / Resumo: O Receptor Ativador da Proliferação de Peroxissomos g (PPARg) e membro de uma família de receptores nucleares cuja atividade é regulada por ligantes. O PPARg atua no metabolismo de lipídios e promove a sensibilização sistêmica à insulina, sendo, portanto, um alvo em potencial para o desenvolvimento de fármacos antidiabéticos. Dentre os ligantes desse receptor, o mais conhecido é a rosiglitazona (RSG), um agonista total que tem a utilização limitada por seus efeitos adversos: toxicidade cardiovascular, ganho de peso e retenção hídrica. A utilização de agonistas parciais é uma alternativa promissora para a redução desses efeitos, já que, apesar de apresentarem menor efeito farmacológico, conseguem desacoplar a sensibilização à insulina do acúmulo de triglicerídeos. Neste trabalho, realizamos simulações de dinâmica molecular do PPARg ligado a um agonista parcial recentemente descoberto, o GQ16. O complexo PPARg-RSG também foi simulado e utilizado como comparativo, para estudar os modos de ligação dos ligantes e suas influências sobre a dinâmica do PPARg. O mecanismo clássico de ativação do receptor é através da estabilização de uma de suas hélices (H12). Analisando-se a estabilidade da H12 durante as simulações observamos que esta permaneceu mais estável em presença da RSG em relação ao GQ16. As simulações revelaram que enquanto a RSG interage diretamente com um resíduo da H12, o GQ16 interage através de uma molécula de água, estabilizando mais fracamente a H12. Essa diferença de comportamento entre os ligantes pode estar por trás da menor ativação promovida pelo GQ16 em relação a RSG, o que está de acordo com os estudos funcionais que mostraram que o GQ16 atua como agonista parcial. Estudos recentes mostram que características estruturais do receptor são importantes na ativação ligante-específica. Um desses fatores auxiliares da ativação do PPARg está relacionado a fosforilação de uma serina (S245) do receptor, mediada por uma proteína quinase, a Cdk5. As simulações revelaram que há uma maior estabilização do loop que contém a S245 em presença do GQ16. Além disso, vimos que o resíduo K244, vizinho ao alvo da fosforilação pela Cdk5, varre um espaço conformacional menor em relação ao complexo PPARg-RSG. Esses resultados sugerem que o GQ16 possibilitaria um bloqueio mais efetivo da fosforilação, ao estabilizar esse loop como um todo, incluindo o resíduo K244 e deixando a S245 menos suscetível a acção da Cdk5 / Abstract: The Peroxisome Proliferator-Activated Receptors gamma (PPARg) is a member of a family of nuclear receptors whose activity is regulated by ligands. The PPARg acts on lipid metabolism and promotes systemic insulin sensitization, therefore being a potential target for the development of antidiabetics agents. Among the ligands of this receptor, the most popular is rosiglitazone (RSG), a full agonist which has restricted use by its side effects: cardiovascular toxicity, weight gain and water retention. The use of partial agonists is a promising alternative to reduce these effects because they can uncouple insulin sensitization from the triglyceride accumulation. In this work, we performed molecular dynamics simulation to investigate the dynamics of PPARg in presence of a partial agonist recently discovered, GQ16 and rosiglitazone (RSG). The classic mechanism of receptor activation is through the stabilization of one of its helixes (H12). Analyzing the stability of H12 during the simulations we found that it remained more stable in the presence of RSG in relation to GQ16. The simulations revealed that while RSG interacts directly with a residue of H12, GQ16 interacts through a water molecule, thus destabilizing somewhat the productive conformation of H12. This difference in behavior promoted by the ligands may underlie the lower GQ16-induced activation in relation to the RSG, which is consistent with the functional studies that showed that GQ16 acts as partial agonist. Recent studies show that structural characteristics are important in receptor ligand-specific activation. One of these auxiliary factors of the activation of PPARg is related to the phosphorylation of a serine (S245) receptor mediated by a protein kinase, Cdk5. The simulations revealed that there is a greater stabilization of the loop containing the S245 in the presence of GQ16. In addition, we found that the residue K244, neighboring the target of phosphorylation by Cdk5, sweeps a narrower conformational space in relation to complex PPARg-RSG. These results suggest that GQ16 enable a more effective blocking of the phosphorylation by stabilizing this loop as a whole, including the residue K244, letting S245 less susceptible to the action of Cdk5 / Mestrado / Físico-Química / Mestre em Química

L‟athérosclérose est un processus inflammatoire chronique à l‟origine des accidents cardiovasculaires quiconstitue l‟une des premières causes de mortalité en France. L‟inflammation est le facteur essentiel dansl‟initiation, la progression et l'instabilité des lésions athéromateuses à l‟origine des accidents aigus. Lesdonnées récentes suggèrent que l‟activation des récepteurs nucléaires PPAR (Peroxysome-ProliferatorActivated Receptor) par des ligands pharmacologiques prévient le développement et la progression del‟athérosclérose et diminue de manière importante la mortalité cardiovasculaire. À côté de ces traitementspharmacologiques, l‟exercice physique prévient aussi la mortalité cardiovasculaire de manière significative.L‟objectif de notre premier travail a été d‟explorer les effets de l‟exercice physique de natation, sur le développementdes lésions athéromateuses d‟une part et d‟autre part, sur l‟expression des récepteurs nucléairesPPAR. Nos résultats montrent que l’exercice physique de natation diminue la progression del’athérosclérose et stimule l’expression des PPAR-γ vasculaires. De manière intéressante, lorsque lePPAR-γ est inhibé avec l'antagoniste BADGE, les effets antiathérogènes de l’exercice physique sontabolis.L‟hypertension est à l‟origine des complications graves telles que la rupture de plaque d‟athérosclérose.L‟objectif de notre deuxième travail a été d‟explorer l‟implication des PPAR dans la progression et la stabilitédes lésions athéromateuses chez des souris ApoE-/- hypercholestérolemiques et hypertendues (2K1C),soumises à des exercices physiques (volontaire ou imposé) ou traités avec le telmisartan, un antihypertenseur.Nos résultats montrent que l’exercice physique possède différents mécanismes protecteurs. Demanière similaire, l‟exercice physique favorise la stabilité de lésions athéromateuses de manière comparableau traitement pharmacologique. De plus, nos résultats montrent que les souris traitées avec l‟exerciceimposé ou le telmisartan présentent un mécanisme comparable qui permet de réduire significativementl‟expression des cytokines pro-inflammatoire et d‟activer les PPAR-γ vasculaires. L‟exercice volontairefavorise l‟expression des marqueurs des macrophages alternatifs Mβ et des cytokines anti-inflammatoires(CD 206, IL-1Ra). L‟exercice volontaire diminue significativement l‟extension des lésions athéromateuses demanière comparable au telmisartan. Ces résultats montrent que l’exercice physique volontaire etl’exercice physique imposé ont deux mécanismes d’actions distincts. De plus, la surexpression des M2en réponse à l‟exercice volontaire modifie la balance inflammatoire en faveur des Mβ. Ce renversement dela balance au profit des macrophages alternatifs M2 est significativement corrélé à la diminution dela progression des lésions athéromateuses.Les exercices imposé et volontaire possèdent des mécanismes d‟action distincts. L‟exercice soumis diminuel‟expression des cytokines pro-inflammatoires tandis que l‟exercice volontaire augmente l‟expression descytokines anti-inflammatoires et favorise un phénotype anti-inflammatoire des macrophages M2 quis‟accompagne d‟une réduction des lésions athéromateuses / Atherosclerosis is a complex inflammatory process, leading cause of morbidity and mortality in France.Inflammation is essential in initiation, progression and atherosclerosis plaque destabilization leading to acutecardiovascular events. Recent studies suggest that pharmacological PPAR activation prevents ATH developpementand progression and decreased cardiovascular mortality. Compared to pharmacological treatment,physical exercise also significantly prevents cardiovascular mortality.The aim of the first study was to investigate the influence of physical exercise on ATS development andPPAR expression in arterial wall. Our results had shown that physical exercise decrease ATH progressionand increase PPAR-γ expression in arterial wall. Interestingly, PPAR-γ inhibition with BADGE, a PPAR-γantagonist abolishes these antiatherogenic effects. Hypertension increase ATH complication such as plaque rupture. The aim of the second study were to investigatePPAR-γ implication in progression and stabilization of ATH lesions in hypercholesterolemic and hypertensiveApoE-/- mice (2K1C) submitted to different exercises (voluntary wheel running and submitted treadmillrunning) or treated with telmisartan an anti-hypertensive drug. Our results shown that, physical exerciseprevents ATS cardiovascular events by several mechanisms. Similarly to telmisartan, physical exercisesstabilize ATH lesion. Moreover results shown that, submitted exercise and telmisartan have an comparablemechanism. In fact, they significantly decrease pro-inflammatory cytokines expression and in the same timeactivated PPAR-γ expression in arterial wall. Contrary to submitted exercise, voluntary exercises increasesexpression of anti-inflammatory cytokines IL-1ra and increase M2 marker CD206. These results suggestthat voluntary and submitted exercise have two different mechanism of action. Moreover, M2 surexpressionin response to voluntary exercise shift the inflammatory balance in favor to M2. Further, this change of balancein favor to M2, is significantly correlated to decrease of ATH progression. Voluntary exercises significantlydecreases ATH progression in the same levels like telmisartan treatment.Voluntary and submitted exercise has two different mechanisms, submitted exercise decrease proinflammatory

Au cours des dix dernières années, il a été observé une augmentation des maladies métaboliques (obésité, diabète de type 2…) avec des conséquences dramatiques en santé humaine. Un intérêt scientifique a émergé pour mieux comprendre comment les bactéries lactiques (BLs) régulent la l’équilibre énergétique de leur hôte. PPAR- (peroxisome proliferator activated receptor ), un récepteur nucléaire, et FIAF (fasting – induced adipose factor) une adipokine apparaissent comme deux régulateurs centraux dans l’homéostasie énergétique. Dans cette étude, nous avons examiné les mécanismes de régulation de Fiaf par les BLs. Nous avons identifié une souche L.rhamnosus CNCMI–4317 induisant l’expression de Fiaf dans la cellule épithéliale intestinale. Nous avons déterminé que cet effet était probablement du à une protéine de surface agissant de façon indépendante de PPAR-. Nous avons confirmé cet effet dans un model in vivo. De plus, nous avons réalisé une transcription du génome complet des cellules HT-29 en contact avec la bactérie confirmant une régulation de Fiaf et suggérant une régulation supplémentaire dans le métabolisme des lipides.Nous avons caractérisé un model HT-29 PPAR- rapporteur à la luciférase. Nous avons appliqué une approche de métagénomique fonctionnelle développée au sein de l’équipe pour cribler des banques génomiques de bactéries lactiques (BLs). Nous n’avons pas réussi à identifier des clones d’intérêt parmi les banques testées. Nous avons également développé une méthode de criblage des BLs sur le modèle HT-29 PPAR-mais après avoir caractériser l'effet bactérien, nous n’avons pas pu confirmer celui-ci par une approche classique de RT-qPCR. Nous avons donc émis l’hypothèse que l’effet observé était un effet direct sur le signal luciférase.Ce travail contribue à une meilleure compréhension des mécanismes de régulation du métabolisme de l’hôte par les bactéries. / Over the last decades, an increase of metabolic diseases (obesity, type-2 diabetes…) has been observed with dramatic consequences on human health. Scientific interest has extended for a better understanding of lactic acid bacteria (LAB) regulation of host energy balance. PPAR- (peroxisome proliferator activated receptor ), a nuclear receptor, and FIAF (fasting – induced adipose factor), a secreted adipokine, appear as two major regulators of energy homeostasis.In this study we examined the mechanisms of Fiaf regulation by LAB. We identified a lactobacillus rhamnosus (L.rhamnosus) CNCMI-4317 strain up-regulating Fiaf expression in intestinal epithelial cells (HT-29). We determined that the effect was probably due to a surface exposed protein acting in a PPAR- independent manner. We confirmed this regulation in an in vivo model. Furthermore, we performed a whole genome transcription (of HT-29 in contact with bacteria) confirming the Fiaf regulation and suggesting an additional lipids metabolism regulation. We characterized a HT-29 PPAR- luciferase reporter model. We applied functional metagenomic approach developed inside the team to screen bacteria genomic libraries. We failed to identify clones of interest among tested libraries. We also performed a screening of LABs on PPAR- luciferase reporter model but after characterization of bacterial effect we failed to confirm it using another approach based on RT-qPCR and speculated that it was a direct effect on luciferase activity. This work contributed to a better knowledge of host metabolism regulation by bacteria.