Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2012-2013. / Le récepteur activé par les proliférateurs de peroxysomes de type y (PPARy) est un puissant régulateur des gènes impliqués dans le métabolisme du tissu adipeux et l'adipogenèse. Son activation pharmacologique, par des ligands de type thiazolidinedione (TZD), diminue la résistance à l'insuline et est utilisée en clinique dans le traitement du diabète de Type 2. Les agonistes de PPARy exercent leurs effets bénéfiques en favorisant la séquestration des lipides et leur entreposage dans les tissus adipeux périphériques métaboliquement plus sûrs, en stimulant la production adipocytaire d'adipokines telles l'adiponectine et en réduisant l'infiltration de macrophages dans les réserves de graisses. En détournant les lipides des organes tels que le muscle squelettique et le foie, les TZD préviennent la lipotoxicité. Bien que l'élucidation des mécanismes par lesquels PPARy contrôle l'entreposage des lipides dans le tissu adipeux progresse, les connaissances en ce domaine demeurent imcomplètes. Les travaux décrits dans cette thèse montrent que les agonistes de PPARy favorisent l'entreposage des graisses en périphérie en modulant de façon dépôt-spécifique et coordonnée la lipase lipoprotéique et les protéines impliquées dans sa maturation, les transporteurs d'acides gras et les enzymes lipogènes. Les résultats présentés dans cette thèse mettent également en évidence le rôle de la cible de la rapamycine chez les mammifères (mTOR, mammalian target of rapamycin) dans le contrôle de la lipogenèse exercé par PPARy in vivo. Lorsque cette protéine au coeur d'un réseau de signalisation contrôlant le métabolisme cellulaire est inactivée, l'activité transcriptionnelle de PPARy est significativement réduite et les TZD perdent leur capacité à stimuler l'entreposage des lipides circulants. Finalement, les études décrites dans cet ouvrage montrent que l'activation de PPARy peut à son tour influencer le sentier métabolique de mTOR en stimulant le catabolisme des acides aminés à chaîne ramifiée, l'un de ses principaux activateurs, dans les tissus adipeux blanc et brun. L'étude des interactions complexes entre ces deux systèmes a le potentiel de contribuer au développement de nouvelles avenues thérapeutiques pour prévenir et guérir l'obésité et le diabète.

Doctor en Farmacología / Diversos estudios clínicos han revelado que las tiazolidinedionas, fármacos para el tratamiento de la diabetes de tipo 2 y resistencia a insulina, podrían reducir la morbimortalidad cardiovascular. Su mecanismo de acción es a través de la activación de los Receptores Activados por Proliferadores Peroxisomales (PPARs), los cuales son factores transcripcionales activados por ligandos. En el sistema cardiovascular, los PPARs se expresan de forma variable y juegan un importante papel en la regulación del metabolismo energético y en la respuesta inflamatoria. Durante diversos estados patológicos como por ejemplo en el infarto al miocardio, el tratamiento con tiazolidinedionas ha mostrado efectos cardioprotectores ya que reducen la hipertrofia y el área infartada y atenúan la respuesta inflamatoria cardiaca. Estos antecedentes sugieren un importante papel de PPARγ durante el remodelado cardiaco, proceso fisiopatológico que consiste en un cambio estructural y funcional del tejido, caracterizado por fibrosis, hipertrofia y pérdida progresiva de los cardiomiocitos. Se ha sugerido que la apoptosis es el principal mecanismo de muerte celular en el corazón pero últimamente se ha avanzado en los estudios de la participación de la autofagia o “muerte programada de tipo II”. Sin embargo, la autofagia se describió inicialmente como un proceso fisiológico clave para la sobrevida celular durante la privación de aminoácidos, diferenciación celular y desarrollo. Consiste en un proceso dinámico y programado que procede con el secuestro de proteínas citoplasmáticas y organelos enteros dentro de vacuolas de doble membrana, que posteriormente se fusionan con los lisosomas formando los autolisosomas. Todos estos elementos capturados en las vacuolas son degradados por proteasas lisosomales y removidos de la célula por exocitosis. Evidencias recientes han mostrado que los agonistas de PPARγ podrían inducir la autofagia en algunas líneas celulares. Sin embargo, aún no queda claro si la autofagia es realmente un proceso de muerte o un mecanismo de sobrevida celular. Dado que prácticamente se desconoce si la activación de PPARγ regula la autofagia cardiaca, en esta tesis se postuló como hipótesis que “El agonista farmacológico de PPARγ rosiglitazona induce la autofagia del cardiomiocito, protegiéndolo de la muerte”. Los objetivos específicos propuestos fueron: • Estudiar in vitro el efecto de agonistas farmacológicos de PPARα y/o PPARγ en la viabilidad del cardiomiocito de rata. • Determinar si rosiglitazona induce autofagia en el cardiomiocito y si ésta se relaciona con sobrevida celular. • Investigar si la estimulación con rosiglitazona afecta la viabilidad de cardiomiocitos expuestos a estrés nutricional, estrés hiperosmótico o a isquemia/reperfusión simulada. El modelo experimental utilizado fue cultivo primario de cardiomiocitos de ratas neonatas tratados con rosigllitazona en un rango creciente de concentraciones y de tiempo. La autofagia se evaluó mediante procesamiento de la proteína LC3 endógena, cambio en la distribución y degradación de la proteína GFP-LC3 en cardiomiocitos transducidos con el adenovirus GFP-LC3. Los resultados mostraron que PPARγ está presente en cardiomiocitos de ratas y que es transcripcionalmente activo, lo cual se demostró mediante un plasmidio reportero que contiene el elemento de respuesta para este factor transcripcional. Además, rosiglitazona estimuló temprana y progresivamente la autofagia en los cultivos primarios de cardiomiocitos, determinada por el procesamiento de la proteína endógena LC3-I, efecto similar al observado en repuesta al tratamiento con rapamicina. Rosiglitazona también incrementó la distribución punteada de LC3-GFP, sin embargo no disminuyó la fluorescencia de la proteína LC3-GFP en los cardiomiocitos transducidos con el adenovirus LC3-GFP. Por otra parte, rosiglitazona no modificó de forma significativa los niveles intracelulares de ATP y ni afectó la viabilidad basal del cardiomiocito. El tratamiento con gemfibrozilo, tampoco modificó su viabilidad. Para determinar si la inducción de autofagia tiene un efecto en la viabilidad del cardiomiocito, los cultivos celulares se expusieron a estrés mecánico por hiperosmolaridad y se midió la viabilidad. El estrés hiperosmótico indujo de manera rápida y potente la muerte de las células cardiacas. Sin embargo, rosiglitazona y gemfibrozilo no previnieron este efecto. La muerte de las células cardiacas inducida por el estrés hiperosmótico es mediante apoptosis, lo que se demostró la evaluación por citometría de flujo de la subpoblación G1 en células permeabilizadas y tratadas con yoduro de propidio y determinación de potencial mitocondrial. Rosiglitazona y gemfibrozilo no previnieron la apoptosis del cardiomiocito inducida por estrés hiperosmótico. Rosiglitazona tampoco bloqueó la muerte celular inducida por isquemia y reperfusión simulada. Finalmente, los resultados obtenidos con el desarrollo de esta tesis permiten concluir que rosiglitazona induce la autofagia del cardiomiocito pero que ésta es insuficiente para modificar la viabilidad celular / Clinical studies showed that thiazolidinediones, drugs used for type 2 diabetes and insulin resistance treatment, can reduce cardiovascular morbid and mortality. These compounds are highly specific ligands of peroxisome proliferator-activator receptor gamma (PPARγ), a nuclear hormone receptor superfamily member. PPARs are variably expressed in the cardiovascular system and play an important role in both energetic metabolism regulation and inflammation response. In myocardial infarct, treatment with thiazolidinediones has cardioprotective effects reducing cardiac hypertrophy, infarcted area and inflammatory response. These data suggest an important role of PPARγ during cardiac remodeling. Remodeling is a physiopathological alteration in heart structure and function characterized by cardiomyocytes fibrosis, hypertrophy and death. Apoptosis has been described as the main cardiac cell death mechanism. However, recent studies have also described the participation of autophagy, also known as type II programmed cell death. Autophagy was first described as an adaptative physiological process during amino acids starvation. It has also been described its participation in cellular differentiation and development. Autophagy consists in the sequestration of cytoplasm portions and organelles within double membrane vesicles, named autophagosomes. These vesicles were subsequently fused with lysosomes forming the autofagosomes. All elements captured in these vesicles are degraded by lysosomal proteases and removed by exocytosis. Recent evidence has shown that PPARγ agonists could induce autophagy in some cells lines. However, is not clear whether autophagy is a mechanism for cell survival or death. Based on these antecedents we postulated the following hypothesis: “The pharmacological PPARγ agonist, rosiglitazone, induces cardiomyocyte autophagy protecting them from cell death”. The specific aims were: • To study in vitro the effects of PPARα and PPARγ pharmacological agonists on neonatal rat cardiomyocytes. • To determine whether rosiglitazone induces autophagy in cardiomyocyte and whether this process is related with cell viability. • To investigate if the stimulation with rosiglitazone affects cardiomyocyte viability when exposed to nutritional stress, hyperosmotic stress and simulated ischemia/reperfusion. The experimental models were primary cultures of neonatal rat cardiomyocytes treated with rosiglitazone at different concentrations and times. Autophagy was evaluated by endogenous LC3-I processing, and by change in adenoviral expressing GFP-LC3 distribution and degradation. Results showed that PPARγ is expressed and is transcriptionally active in neonatal rat cardiomyocytes as determined by western blot and activity of PPAR reporter plasmid. Furthermore, rosiglitazone stimulated early and progressively cardiac autophagy as determined by endogenous LC3-I processing. This effect was similar to that induced by rapamycin. Rosiglitazone also increased the GFP-LC3 punctuated pattern, but without decreasing GFP-LC3 fluorescence. On the other hand, rosiglitazone neither affects ATP levels nor viability of cardiomyocytes. Gemfibrozil treatment, also did not affect cardiomyocyte viability. To determine whether autophagy affects cardiomyocyte viability, cultured cells were exposed to hyperosmotic stress in the presence or absence of rosiglitazone or gemfobrozil, and viability was measured. Hyperosmotic stress induced a rapid decrease in cardiomyocyte viability. Cardiomyocyte death was also achieved by simulated ischemia/reperfusiom. Neither rosiglitazone nor gemfibrozil prevented cardiomyocyte death induced by both procedures. Hyperosmotic stress-induced cell death was characterized as apoptosis, as determined by mitochondrial potential decay and DNA fragmentation visualized by sub G1 population in propidium iodide-treated cells followed flow cytometry. Both rosiglitazone and gemfibrozil did not prevent the hyperosmotic stress-induced apoptosis. Finally, these results allow us to conclude that rosiglitazone induces cardiomyocyte autophagy but this process does not affect cardiomyocyte viability

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2016-2017. / L’obésité et ses complications telles le diabète et la stéatose hépatique non alcoolique sont des enjeux de santé qui prennent de plus en plus d’ampleur partout sur la planète et la compréhension approfondie des mécanismes physiopathologiques impliqués est essentielle pour mieux contrer ces maladies. La protéine peroxisome proliferator activated receptor gamma (PPARγ) est reconnue pour ses propriétés antiinflammatoires, insulino-sensibilisantes et pro-adipogéniques. Des résultats antérieurs ont démontré qu’en l’absence de la protéine tyrosine phosphatase Src homology region 2 domain-containing phosphatase- 1 (Shp1), l’activité de PPARγ est augmentée. L’activation de PPARγ par ses agonistes, les thiazolidinediones (TZD), est favorable au contrôle du diabète, mais entraîne certains effets indésirables. Nos recherches ont porté sur l’investigation d’une nouvelle voie de régulation de PPARγ. Nous avons montré que Shp1 et PPARγ interagissent et que PPARγ est phosphorylé sur ses résidus tyrosine. Les résultats suggèrent que la déphosphorylation de PPARγ par Shp1 diminue son activité. Des analyses de modélisation moléculaire suggèrent que cette interaction entre Shp1 et PPARγ dépend de la présence de phosphorylation sur un acide aminé particulier, la tyrosine 355. Ce même résidu est aussi important dans la liaison avec la rosiglitazone, médicament de la classe des TZD. Des expériences de mutagénèse ont montré que l’absence de phosphorylation sur la tyrosine 355 diminue grandement l’activité de la protéine et, qu’à l’inverse, la présence de phosphorylation tend à augmenter son activité. Bien que plusieurs modifications post-traductionnelles aient été décrites dans la littérature, la phosphorylation sur tyrosine de PPARγ demeure très peu étudiée. Nos résultats suggèrent une nouvelle voie de régulation de PPARγ qui pourrait mener à l’élaboration de nouveaux ligands qui exploitent ce mécanisme afin de favoriser les propriétés bénéfiques de ce facteur transcriptionnel pour mieux traiter le diabète de type 2 et diminuer les risques d’effets secondaires. / Obesity and its complications such as type 2 diabetes and non-alcoholic fatty liver disease are becoming worldwide health concerns and more insights into the underlying physiopathological mechanisms are necessary to improve the treatment of these conditions. Peroxisome proliferator activated receptor gamma (PPARγ) protein is well known for its anti-inflammatory, insulin sensitizing and pro-adipogenic roles. Previous results showed that in absence of the protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (Shp1), PPARγ activity is increased. PPARγ activation by thiazolidinediones (TZD) is used in the control of diabetes but is also linked to unwanted side effects. We investigated a new mechanism of regulation of PPARγ activity. We show that Shp1 and PPARγ interact and that PPARγ is tyrosine phosphorylated. Our results suggest that Shp1-mediated dephosphorylation of PPARγ reduces its activity. Molecular modeling analyses further suggest that the interaction between Shp1 and PPARγ depends upon the phosphorylation of one specific residue, tyrosine 355. This residue is also important for the binding with rosiglitazone, a member of the TZD drug class. Mutagenesis experiments showed that the absence of phosphorylation on tyrosine residue 355 decreases PPARγ activity, while its phosphorylation tends to increase it. Despite the fact that many post-translational modifications have been reported in the literature, tyrosine phosphorylation of PPARγ remains mostly unexplored. These results suggest a new PPARγ regulating mechanism that could be exploited to elaborate new PPARγ ligands to improve the treatment of type 2 diabetes and to limit side effects.

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.

Many studies have shown that peroxisome-proliferator-activated receptor [gamma] (PPAR[gamma]) plays an important role in adipose tissue formation by activating genes implicated in adipogenesis. PPAR[gamma] heterodimerizes with retinoid X receptor [alpha] (RXR[alpha]), in the presence of ligand, on PPAR response elements (PPREs) in the promoter of target genes involved in adipocyte differentiation. General receptor for phosphoinositides 1 (GRP1) is a corepressor of thyroid hormone receptors (TRs), a nuclear receptor like PPAR[gamma]. GRP1 decreases TRs' transcriptional activity by lowering dimerisation on DNA. Since PPARs and TRs have important structural similarities and that GRP1 interacts with PPARs in vitro, we hypothesized that GRP1 could be a coregulator of PPARs and, thus be implicated in adipogenesis. To better understand GRP1's effect on PPAR[gamma]2, transcriptional activity assays have been done and show that increasing concentrations of GRP1 decrease the transcriptional activity of PPAR[gamma]2. We also studied GRP1 expression by Western blots of total protein extracts from 3T3-L1 cells at different times during differentiation: GRP1 is present in 3T3-L1 preadipocytes and its expression decreases during adipogenesis. According to those results, GRP1 may be a PPAR[gamma] corepressor. After those observations, GRP1 effects on adipogenesis were studied by modulating its expression with lentiviral particles. Interestingly, GRP1 knock-down before inducing 3T3-L1 differentiation, almost abrogates adipogenesis and adipocytes markers, PPAR[gamma] and aP2, while its overexpression increases lipid storage without affecting PPAR[gamma] expression. On the opposite, GRP1 modulation after differentiation induction shows that expression knock-down slightly promotes adipogenesis by increasing PPAR[gamma], aP2 and lipid accumulation and that overexpression weakly decreases lipid storage. Our results suggest that GRP1 implication during adipogenesis occurs at two distinct and precise moments. It seems to be a key factor in the early stages of adipocyte differentiation and to be implicated as a PPAR[gamma] transcriptional activity modulator as a corepressor. Future experiments will help detail modulation of protein expression and underlying mechanisms to better understand the role of GRP1 in adipogenesis and, eventually, comorbidities linked to obesity like cardiovascular diseases and type 2 diabetes mellitus.

Les thiazolidinediones (TZDs) sont des médicaments antidiabétiques (agonistes des récepteurs nucléaires de type PPAR[gamma]) utilisés au cours des dix dernières années pour le traitement du diabète de type II. Malheureusement leur utilisation peut provoquer, chez certains patients, une rétention accrue de fluides et une formation d?oedèmes rénaux. Des études récentes suggèrent l'implication d'un canal sodique épithélial (ENaC), exprimé au niveau du tubule collecteur rénal, dans ces effets secondaires. En effet, la stimulation des PPAR[gamma] par les TZDs activent les canaux sodiques épithéliaux probablement via l'expression et l'activation de SGK1 (Serum and Glucocorticoid-regulated Kinase 1). Sachant que les transports des ions sodiques et potassiques sont étroitement liés au niveau rénal, notre objectif est de déterminer si les TZDs seraient impliqués dans la régulation des canaux potassiques (ROMK1). Nous montrons qu'en traitant les ovocytes de xénopes exprimant ROMK et PPAR[gamma], avec un TZDs comme la rosiglitazone (RGZ), le courant potassique mesuré par voltage-clamp (TEVC) est augmenté de deux fois. Cette augmentation est bloquée par l'utilisation d'un antagoniste de PPAR[gamma], le GW9662. Nous démontrons aussi l'implication de SGK1 dans la régulation de l'activité des canaux ROMK1 d'une part en mutant son site de phosphorylation sur ROMK1 (sérine 44) et d'autre part en utilisant son inhibiteur, GSK. Finalement les expériences d'immunofluorescences ont montré un recrutement de ROMK1 à la membrane des ovocytes traités à la RGZ. L'ensemble des données présentées dans ce travail suggère que la RGZ augmente le courant potassique en augmentant l'expression de SGK1.

A proteína Anexina A1 (ANXA1), sintetizada e liberada por fagócitos pela ação de glicocorticóides, é uma proteína anti-inflamatória, pois inibe o influxo de neutrófilos para o foco da inflamação, e induz os mecanismos de eferocitose em neutrófilos e macrófagos. Nosso grupo mostrou que a ANXA1 regula a expressão do receptor ativado por proliferadores de peroxissomos (PPAR) em macrófagos. Em continuidade, o presente trabalho investigou o mecanismo da ANXA1 sobre a expressão de PPARγ em macrófagos, e se este controle ocorre em demais leucócitos e tecido adiposo. Para tanto, macrófagos, neutrófilos peritoneais, linfócitos do baço, tecido adiposo epididimal foram obtidos de camundongos machos Balb/c selvagens (wild type, WT) ou geneticamente deficientes para ANXA1 (ANXA1-/-). Os resultados obtidos mostraram que a ANXA1 controla a expressão proteica e gênica de PPARγ em macrófagos, já que os níveis proteico (Western Blot, WB) e de RNAm (Real-time PCR) para PPARγ constitutivo, bem como induzidos pelos tratamentos in vitro com bezafibrato ou pioglitazona estavam reduzidos em macrófagos de animais ANXA1-/- em comparação com os níveis de macrófagos de animais WT, e o efeito parece ser dependente de CREB (WB), já que os níveis constitutivos deste fator de transcrição estavam maiores em macrófagos de animais ANXA1-/-. O tratamento in vitro com cicloheximida (CHX), um inibidor da síntese proteica, reduziu a expressão de PPARγ estimulada por bezafibrato ou LYSO-7 em macrófagos de animais WT, reforçando o papel da ANXA1 na expressão gênica de PPARγ. O FPR2 parece não estar envolvido no efeito, uma vez que o pré-tratamento de macrófagos com o antagonista de FPR2 (WRW4) não modificou a expressão de PPARγ em macrófagos de animais WT. O efeito modulador da ANXA1 ocorre em neutrófilos, mas não em tecido adiposo e linfócitos de animais ANXA1-/-. Ademais, a deficiência de ANXA1 não alterou a apoptose espontânea de neutrófilos. Em conjunto, os resultados obtidos mostram uma possível via adicional da ANXA1 sobre a resolução da inflamação, controlando a expressão de PPARγ em fagócitos. / Annexin A1 (ANXA1), is a protein synthetized and released by phagocytes due to the action of glucocorticoids, and an anti-inflammatory protein that inhibits neutrophil influx to site of inflammation and induces the mechanisms of efferocytosis in neutrophils and macrophages. Our group has already demonstrated that ANXA1 regulates the expression of peroxisome proliferator-activated receptor (PPAR) in macrophages. The present work aimed to investigate the role of ANXA1-dependent mechanisms on the expression of PPARγ in macrophages, and if said role also extends to other leukocytes and adipose tissue. For such, macrophages, peritoneal neutrophils, spleen lymphocytes, epididymal adipose tissue were obtained from male Balb/c wild type mice or from mice lacking ANXA1 genetically (ANXA-/-). Obtained results have demonstrated that ANXA1 regulates both proteic and genic expression of PPARγ in macrophages, as protein (Western Blotting, WB) and mRNA (Real-Time PCR) levels of constitutive PPARγ were reduced in macrophages from ANXA1-/- mice in comparison with the observed levels of macrophages from WT mice; the same is true for increased protein and mRNA levels as induced by in vitro treatments with bezafibrate or pioglitazone. This effect appears to be CREB-dependent (WB), as the constitutive levels of this transcription factor were found to be increased in macrophages from ANXA1-/- mice. In vitro treatment with cycloheximide (CHX), an inhibitor of proteic synthesis, reduced the bezafibrate or LYSO-7 (PPAR pan agonist, 10 µM / 2h) induced expression of PPARγ in WT mice, which further suggests a role for ANXA1 in PPARγ genic expression. FPR2 does not seem to be involved with these effects of ANXA1, as pre-treatment of macrophages from WT mice with an FPR2-antagonist (WRW4) did not alter expression of PPARγ. The modulating effect of ANXA1 can be verified in neutrophils of ANXA-/- mice, but not in adipocytes and lymphocytes from the same animals. Moreover, deficiency of ANXA1 did not affect spontaneous apoptosis of neutrophils. Altogether, the obtained results show the existence of a probable additional pathway with which ANXA1 promotes inflammation resolution, also controlling the expression of PPARγ in phagocytes.

A proteína Anexina A1 (ANXA1), sintetizada e liberada por fagócitos pela ação de glicocorticóides, é uma proteína anti-inflamatória, pois inibe o influxo de neutrófilos para o foco da inflamação, e induz os mecanismos de eferocitose em neutrófilos e macrófagos. Nosso grupo mostrou que a ANXA1 regula a expressão do receptor ativado por proliferadores de peroxissomos (PPAR) em macrófagos. Em continuidade, o presente trabalho investigou o mecanismo da ANXA1 sobre a expressão de PPARγ em macrófagos, e se este controle ocorre em demais leucócitos e tecido adiposo. Para tanto, macrófagos, neutrófilos peritoneais, linfócitos do baço, tecido adiposo epididimal foram obtidos de camundongos machos Balb/c selvagens (wild type, WT) ou geneticamente deficientes para ANXA1 (ANXA1-/-). Os resultados obtidos mostraram que a ANXA1 controla a expressão proteica e gênica de PPARγ em macrófagos, já que os níveis proteico (Western Blot, WB) e de RNAm (Real-time PCR) para PPARγ constitutivo, bem como induzidos pelos tratamentos in vitro com bezafibrato ou pioglitazona estavam reduzidos em macrófagos de animais ANXA1-/- em comparação com os níveis de macrófagos de animais WT, e o efeito parece ser dependente de CREB (WB), já que os níveis constitutivos deste fator de transcrição estavam maiores em macrófagos de animais ANXA1-/-. O tratamento in vitro com cicloheximida (CHX), um inibidor da síntese proteica, reduziu a expressão de PPARγ estimulada por bezafibrato ou LYSO-7 em macrófagos de animais WT, reforçando o papel da ANXA1 na expressão gênica de PPARγ. O FPR2 parece não estar envolvido no efeito, uma vez que o pré-tratamento de macrófagos com o antagonista de FPR2 (WRW4) não modificou a expressão de PPARγ em macrófagos de animais WT. O efeito modulador da ANXA1 ocorre em neutrófilos, mas não em tecido adiposo e linfócitos de animais ANXA1-/-. Ademais, a deficiência de ANXA1 não alterou a apoptose espontânea de neutrófilos. Em conjunto, os resultados obtidos mostram uma possível via adicional da ANXA1 sobre a resolução da inflamação, controlando a expressão de PPARγ em fagócitos. / Annexin A1 (ANXA1), is a protein synthetized and released by phagocytes due to the action of glucocorticoids, and an anti-inflammatory protein that inhibits neutrophil influx to site of inflammation and induces the mechanisms of efferocytosis in neutrophils and macrophages. Our group has already demonstrated that ANXA1 regulates the expression of peroxisome proliferator-activated receptor (PPAR) in macrophages. The present work aimed to investigate the role of ANXA1-dependent mechanisms on the expression of PPARγ in macrophages, and if said role also extends to other leukocytes and adipose tissue. For such, macrophages, peritoneal neutrophils, spleen lymphocytes, epididymal adipose tissue were obtained from male Balb/c wild type mice or from mice lacking ANXA1 genetically (ANXA-/-). Obtained results have demonstrated that ANXA1 regulates both proteic and genic expression of PPARγ in macrophages, as protein (Western Blotting, WB) and mRNA (Real-Time PCR) levels of constitutive PPARγ were reduced in macrophages from ANXA1-/- mice in comparison with the observed levels of macrophages from WT mice; the same is true for increased protein and mRNA levels as induced by in vitro treatments with bezafibrate or pioglitazone. This effect appears to be CREB-dependent (WB), as the constitutive levels of this transcription factor were found to be increased in macrophages from ANXA1-/- mice. In vitro treatment with cycloheximide (CHX), an inhibitor of proteic synthesis, reduced the bezafibrate or LYSO-7 (PPAR pan agonist, 10 µM / 2h) induced expression of PPARγ in WT mice, which further suggests a role for ANXA1 in PPARγ genic expression. FPR2 does not seem to be involved with these effects of ANXA1, as pre-treatment of macrophages from WT mice with an FPR2-antagonist (WRW4) did not alter expression of PPARγ. The modulating effect of ANXA1 can be verified in neutrophils of ANXA-/- mice, but not in adipocytes and lymphocytes from the same animals. Moreover, deficiency of ANXA1 did not affect spontaneous apoptosis of neutrophils. Altogether, the obtained results show the existence of a probable additional pathway with which ANXA1 promotes inflammation resolution, also controlling the expression of PPARγ in phagocytes.

Ho Wing Man. / On t.p. "gamma" appears as the Greek letter. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 95-106). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENTS --- p.V / ABBREVIATIONS --- p.VI / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.X / CONTENTS --- p.XII / Chapter CHAPTER ONÉؤ --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.1.1 --- Thyroid cancer --- p.2 / Chapter 1.1.2 --- Apoptosis and thyroid cancer --- p.4 / Chapter 1.2 --- Estrogen receptors and apoptosis --- p.5 / Chapter 1.2.1 --- Estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) --- p.5 / Chapter 1.2.2 --- Differential roles of estrogen receptor-α(ERα) and estrogen receptor-β (ERβ) in apoptosis --- p.6 / Chapter 1.2.3 --- Bcl-2 family --- p.8 / Chapter 1.3 --- Peroxisome proliferator-activated receptor-γ (PPARγ) --- p.9 / Chapter 1.3.1 --- Molecular aspects of PPAR --- p.9 / Chapter 1.3.2 --- PPAR/RXR complex --- p.13 / Chapter 1.3.3 --- PPARγ ligands --- p.16 / Chapter 1.3.4 --- PPARγ and apoptosis in thyroid cancer --- p.19 / Chapter 1.3.5 --- PPARγ ligands-mediated apoptosis pathway --- p.21 / Chapter 1.4 --- Previous results from our laboratory --- p.25 / Chapter 1.5 --- Summary of previous studies --- p.27 / Chapter 1.6 --- Perspectives --- p.28 / Chapter 1.7 --- Objectives of this project --- p.29 / Chapter CHAPTER TWÓؤ --- GENERAL MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Cell lines --- p.31 / Chapter 2.1.2 --- Plasmid vectors used in this study --- p.31 / Chapter 2.1.3 --- Antibodies --- p.32 / Chapter 2.1.4 --- Culture media and transfection reagents --- p.32 / Chapter 2.1.5 --- Materials for protein manipulation --- p.33 / Chapter 2.1.6 --- Drugs for treatment --- p.34 / Chapter 2.1.7 --- Kits --- p.35 / Chapter 2.1.8 --- Instruments --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Cell culture --- p.36 / Chapter 2.2.2 --- Cell viability analysis --- p.36 / Chapter 2.2.3 --- Preparation of protein extract --- p.37 / Chapter 2.2.4 --- Determination of the concentration of target protein --- p.37 / Chapter 2.2.5 --- Gel electrophoresis and protein transfer --- p.38 / Chapter 2.2.6 --- Immunoblotting --- p.39 / Chapter 2.2.7 --- Apoptosis detected by Cell Death ELISAplus --- p.41 / Chapter 2.2.8 --- PPARγ-ligand Enzyme Immunoassay --- p.45 / Chapter 2.2.8.1 --- 15d-PGJ3 Enzyme Immunoassay --- p.45 / Chapter 2.2.8.2 --- 15(S)-HETE Enzyme Immunoassay --- p.46 / Chapter 2.2.8.3 --- 13(S)-HODE Enzyme Immunoassay --- p.46 / Chapter 2.2.9 --- Transient tranfection and luciferase activity assay --- p.47 / Chapter 2.2.10 --- Statistical Analysis --- p.52 / Chapter CHAPTER THREÉؤ --- ESTROGEN RECEPTORa (ERa) AND ESTROGEN RECEPTORP(ERP) MEDIATE THE PROLIFERATION AND APOPTOSIS OF HUMAN THYROID PAPILLARY CARCINOMA CELLS --- p.53 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Cell culture and treatment --- p.56 / Chapter 3.2.2 --- Western Blot --- p.56 / Chapter 3.2.3 --- Cell proliferation determined by MTT assay --- p.57 / Chapter 3.2.4 --- Apoptosis detected by Cell Death ELISAplus assay --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- "The expression of ERα, ERβ and PPARγ in NPA, FRO, ARO and WRO thyroid cancer cell lines" --- p.59 / Chapter 3.2.2 --- Effects of PPT and DPN on cell viability --- p.61 / Chapter 3.3.3 --- Apoptotic cells quantification by Cell Death ELISAplus assay --- p.64 / Chapter 3.4 --- Discussion --- p.67 / Chapter CHAPTER FOUŔؤ --- THE RELATIONSHIP BETWEEN PPARγ AND ESTROGEN RECEPTOR AND THE REGULATION OF THE APOPTOSIS IN THYROID CANCER CELL LINES --- p.70 / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Material and Methods --- p.74 / Chapter 4.2.1 --- Transient transfection --- p.74 / Chapter 4.2.2 --- Luciferase assay --- p.74 / Chapter 4.2.3 --- 15d-PGJ2 ELISA assay --- p.75 / Chapter 4.2.4 --- 15S-HETE ELISA assay --- p.76 / Chapter 4.2.5 --- 13S-HODE ELISA assay --- p.77 / Chapter 4.3 --- Results --- p.78 / Chapter 4.3.1 --- "PPT, ERα-agonist, increased thyroid cancer cell proliferation and caused the decrease the level of PPARγ ligands" --- p.78 / Chapter 4.3.2 --- "DPN, ERβ-agonist, inhibited thyroid cancer cell proliferation, induced apoptosis and caused the increase the level of PPARγ ligands" --- p.83 / Chapter 4.3.3 --- PPT did not alter the transcriptional activity of PPARγ --- p.88 / Chapter 4.4 --- Discussion --- p.90 / Chapter CHAPTER FIVÉؤ --- CONCLUSIONS AND FUTURE PROSPECT --- p.92 / Chapter 5.1 --- Summary of results --- p.93 / Chapter 5.2 --- Conclusion --- p.94 / Chapter 5.3 --- Future prospects --- p.94 / REFERENCE LIST --- p.95

Microarrays have been widely used to study differential gene expression at the genomic level. They can also provide genome-wide co-expression information. Robust approaches are needed for integration and validation of independently-collected datasets which may contribute to a common hypothesis. Previously, attempts at meta-analysis have contributed to solutions to this problem. As an alternative, for microarray data from multiple highly similar biological experimental designs, a more direct combined approach is possible. In this thesis, a novel approach is described for microarray combined analysis, including gene-level unification into a virtual platform followed by normalization and a method for ranking candidate genes based on co-expression information - called Coex-Rank. We applied this approach to our Sppar (a PPARγ mutant) dataset, which illustrated an improvement in statistical power and a complementary advantage of the Coex-Rank method from a biological perspective. We also performed analysis to other PPARγ-related microarray datasets. From the perspective of gene sets, we observed that up-regulated genes from mice treated with the PPARγ ligand rosiglitazone were significantly down-regulated in mice with a global knock-in dominant-negative mutation of PPARγ. Integrated with publicly available PPRE (PPAR Response Element) datasets, we found that the genes which were most up-regulated by rosiglitazone treatment and which were also down-regulated by the global knock-in mutation of PPARγ were robustly enriched in PPREs near transcription start sites. In addition, we identified several potential PPARγ targets in the aorta and mesenteric artery for further experimental validation, such as Rhobtb1 and Rgs5.