L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles. / The injection of immuno-competent cells into a histo-incompatible host can result in the development of Graft-versus-Host disease (GVHD). GVHD is the most significant barrier to a more widespread use of allogeneic hematopoietic cell transplantation (AHCT), a potent treatment for several diseases. A better understanding of the pathophysiological underpinnings of GVHD would facilitate the design of rational approaches to treat and prevent this complication of AHCT. Gamma-interferon (IFN-γ) and Transforming Growth Factor-beta (TGF-β) are master cytokines of immunity and have a role in the function and homeostasis of transplanted cells. Using a murine model, we show that IFN-γ curtails lympho-hamatopoitic reconstitution in a dose-dependent fashion by increasing apoptosis and by limiting donor cell proliferation. TGF-β is an immunosuppressive cytokine that controls immune cells through multiple signaling pathways. The relative contribution of these pathways in AHCT is unknown. We specifically studied the role of one of these pathways by transplanting SMAD3 deficient cells (SMAD3-KO) in histo-incompatible hosts. SMAD3 is a key mediator of the so-called canonical TGF-β signaling pathway. Although SMAD3-KO donor mice are healthy, the injection of SMAD3-KO cells leads to severe GVHD in the hosts, characterized by intestinal involvement associated with Th1 skewing and massive granulocyte infiltration. These findings hint at a crucial role for SMAD3 in CD4 T-cell and myeloid cell biology. We then focalized on the role of SMAD3 in CD4 T cells knowing that SMAD3 is active in tolerant, resting CD4 T cells. We found that SMAD3 was rapidly inactivated upon T cell activation, suggesting that SMAD3 inactivation was functionally important to break the state of tolerance. Our cDNA microarray experiments show that indeed, SMAD3 regulates the transcript levels of multiple genes known to be involved in T cell tolerance and in biological processes plausibly related to immune tolerance.

La Fibrose Pulmonaire Idiopathique (FPI) est une maladie dévastatrice, d’étiologie inconnue, qui reste pour le moment incurable. Cette maladie est caractérisée par l’accumulation de fibroblastes et de protéines de la matrice extracellulaire dans les espaces aériens distaux aboutissant à une destruction alvéolaire et à une altération des propriétés mécaniques du poumon. La physiopathologie de la FPI est mal connue mais de nombreuses études suggèrent que la réactivation des voies impliquées dans le développement contribue à l’accumulation de la matrice extra-cellulaire et au comportement anormal des cellules épithéliales et des fibroblastes.La voie Hedgehog (HH) joue un rôle crucial dans le développement embryonnaire. Dans le développement pulmonaire fœtal, la voie HH est impliquée dans les interactions épithélium-fibroblaste et contrôle la prolifération et la différenciation du mésenchyme. La voie HH a été impliquée dans la fibrogénèse, notamment dans le foie et le rein.L’objectif de cette thèse a été de caractériser la voie HH dans la fibrose pulmonaire chez l’homme et dans un modèle de fibrose induite par la bléomycine chez la souris.Nous avons démontré que la voie HH est réactivée dans les tissus pulmonaires de patients atteints de FPI et dans le modèle de fibrose pulmonaire chez la souris. Nous avons montré que le TGF-β1 activait la voie HH dans les fibroblastes pulmonaires humains et que l’inhibition pharmacologique de la voie HH au niveau des facteurs GLI inhibait l’effet du TGF-β1 in vitro. Par contre, ces inhibiteurs ne protégent pas les cellules épithéliales alvéolaires de la transition épithélio-mésenchymateuse induite par le TGF-β1. In vivo, chez la souris, nous avons montré que le traitement par des inhibiteurs de Smoothened ne protégeait pas du développement de la fibrose tandis que le GANT61, un inhibiteur de l’interaction des GLI avec l’ADN, inhibait la fibrose.En conclusion, nos résultats démontrent l’implication de la voie HH dans la fibrose pulmonaire et ouvrent des perspectives thérapeutiques nouvelles. / Idiopathic Pulmonary Fibrosis (IPF ) is a devastating disease of unknown etiology, which no efficient treatment. This disease is characterized by the accumulation of fibroblasts and extracellular matrix proteins in the distal airways resulting to the destruction of alveoli and alteration of mechanical properties of the lung. The pathogenesis of IPF is not well known but many studies suggest that reactivation of pathways involved in the development, contributes to the accumulation of extracellular matrix and the abnormal behavior of epithelial cells and fibroblasts.The Hedgehog pathway (HH) plays a crucial role in embryonic development. In the fetal lung development, the HH pathway is involved in the epithelial-fibroblast interactions and controls the proliferation and differentiation of the mesenchyme. The HH pathway has been implicated in the fibrogenesis, particularly in the liver and kidney.The aim of this thesis was to characterize the HH pathway in pulmonary fibrosis in humans and in a model of bleomycin-induced fibrosis in mice.We demonstrated that the HH pathway is reactivated in lung tissue of IPF patients and in the model of pulmonary fibrosis in mice. We have shown that TGF-β1 activated the HH pathway in human lung fibroblasts and that the pharmacological inhibition of the HH pathway at the level of GLI transcription factors, inhibited the effect of TGF-β1 in vitro. By contrast, these inhibitors did not protect alveolar epithelial cells from TGF-β1-induced epithelial-mesenchymal transition. In vivo, we have shown that treatment with Smoothened inhibitors did not protect mice from the development of fibrosis while GANT61, an inhibitor of the GLI interaction with DNA, inhibited fibrosis .In conclusion, our results demonstrate the involvement of the HH pathway in pulmonary fibrosis and open new therapeutic perspectives.

P8 est un gène lié au stress cellulaire qui a été identifié et caractérisé dans notre laboratoire. Il est surexprimé dans diverses pathologies, et plus particulièrement dans l'adénocarcinome pancréatique. Notre étude se focalise sur le rôle de p8 dans la progression tumorale et la formation des métastases du cancer du pancréas. Dans ce travail, nous avons démontré, dans un premier temps, que p8 régule la migration, l'adhésion et l'invasion cellulaire induites par diverses molécules dont le TGF-β1, par le biais de la GTPase CDC42, dont il contrôle l'expression et l'activité. Nous avons prouvé aussi que la présence de p8 est nécessaire pour la mise en place d'une transition épithélio-mésenchymateuse, facilitant ainsi l'action pro-tumorale du TGF-β1. Enfin, une analyse morphologique d'adénocarcinomes pancréatiques humains et murins nous a permis d'identifier la présence de cellules « cannibales », déficientes en p8, capables de phagocyter et ainsi limiter la prolifération d'autres cellules. Nous avons décortiqué ce mécanisme au niveau moléculaire. Son étude nous a permis de conclure, qu'en absence de p8, une nouvelle transition de type épithélio-phagocytaire est instaurée, ayant comme résultat un cannibalisme cellulaire, potentialisé notamment par le TGF-β1, qui agirait dans ce cas comme un agent anti-tumoral. L'avancée de ces résultats donne place à des nouvelles perspectives vis-à-vis de l'importance de p8, d'abord d'un point de vue moléculaire sur les actions pro et anti-tumorales du TGF-β1, ensuite en tant que potentielle cible thérapeutique dans le cancer du pancréas. / P8 is a gene related to cellular stress, identified and characterized in our laboratory, and overexpressed in several diseases, especially in pancreatic cancer (PDAC). Our study focuses on the role of p8 in tumor progression and metastasis formation in PDAC. In this work, we have demonstrated that firstly, p8 regulates pancreatic cancer cell migration, invasion and adhesion, induced by several molecules like TGF-β1, through CDC42, a small GTPase, whose expression and activation is controlled by p8. We also established that p8 is necessary to set up epithelial-to-mesenchymal transition, promoting TGF-β1 pro-tumoral effects. Finally, morphological analysis of human and murine pancreatic cancer, allowed us to identify “cannibal” cells, in which p8 expression was absent, able to phagocytose another cells and in this way limit its proliferation. We dissected the mechanism involved in this process at the molecular level. This study led us to conclude that when p8 is absent, a new epithelial-to-phagocytic transition takes place, resulting in cell cannibalism, maximized by TGF-β1 action that will play an anti-tumoral role. These results underscore, on one hand, the crucial role of p8, at the molecular level, over the pro and anti-tumoral effects of TGF-β1 and on the other hand its potential role in pancreatic cancer therapy.

A cirurgia bariátrica confere proteção cardiometabólica à indivíduos obesos, contribuindo para uma redução do risco de mortalidade. No entanto, a extensão do benefício metabólico pode estar sujeita a mudanças no estilo de vida do paciente após a intervenção cirúrgica. Embora o exercício físico pareça melhorar os efeitos da cirurgia na sensibilidade à insulina, o mecanismo de ação subjacente permanece em grande parte sem explicação. Especula-se que mudanças potenciais na matriz extracelular do músculo esquelético (ECM) poderiam estar associadas à melhora da sensibilidade à insulina induzida pelo exercício físico em pacientes pós-bariátricos. Além disso, não se sabe se os benefícios da cirurgia bariátrica sobre a função endotelial, importante marcador precoce de aterosclerose, são sustentáveis sem alterações no estilo de vida, como a inclusão de exercícios físicos. Dessa forma, foram objetivos do presente estudo, investigar os efeitos do exercício físico sobre a sinalização intracelular envolvida na sensibilidade à insulina e remodelamento da matriz extracelular do músculo esquelético (Estudo 1) e sobre a função endotelial da artéria braquial de pacientes submetidos à cirurgia bariátrica (Estudo 2). Sessenta e duas mulheres foram randomizados após a cirurgia bariátrica para um programa de exercícios físicos de 6 meses ou tratamento padrão. No início do estudo, 3 e 9 meses após a cirurgia, a sensibilidade à insulina foi avaliada pelo teste oral de tolerância à glicose (TOTG), análise da função endotelial e amostras de músculo esquelético foram obtidas a partir do vasto lateral. As amostras de músculo esquelético foram submetidas a análises abrangentes, incluindo expressão de genes e proteínas, fenótipo do músculo esquelético, transcriptoma e identificação de novas vias de sinalização celular. O treinamento físico após a cirurgia bariátrica melhorou a sensibilidade à insulina no músculo esquelético. Esta resposta foi mediada por alterações moleculares e fenotípicas na ECM. A cirurgia bariátrica per se foi incapaz de solucionar completamente a resistência à insulina e a expansão da ECM no músculo esquelético. Candidatos relevantes modulados pelo exercício emergiram como alvos terapêuticos para o tratamento da resistência à insulina do músculo esquelético, nomeadamente a via TGF \'beta\' 1 SMAD 2/3 e seu antagonista folistatina. Em resumo, empregamos uma abordagem \"top-down approach\" para fornecer evidências de que a ECM do músculo esquelético desempenha um papel fundamental nos efeitos sobrepostos da cirurgia bariátrica e do exercício físico sobre a sensibilidade à insulina em mulheres obesas. Além disso, este estudo demonstrou que o treinamento físico evitou a reversão da melhora da função endotelial por meio da melhora do padrão de fluxo sanguíneo e redução de marcadores inflamatórios. Em conclusão, ao revelar um novo mecanismo pelo qual o exercício pode contrabalançar a resistência à insulina em pacientes pós-bariátricos (isto é, atenuar a espessura da ECM) e preservar a função endotelial, este estudo endossa que o exercício físico deve ser adotado como relevante medida terapêutica a fim de garantir os melhores resultados cardiometabólicos em pacientes submetidos à cirurgia bariátrica / Bariatric surgery provides cardiometabolic protection to obese individuals, contributing to a reduction in mortality risk. However, the extent of metabolic benefit may be subject to changes in the patient\'s lifestyle after surgical intervention. Although exercise seems to improve the effects of surgery on insulin sensitivity, the underlying mechanism of action remains largely unexplained. It is speculated that potential changes in the skeletal muscle extracellular matrix (ECM) could be associated with improved insulin sensitivity induced by physical exercise in post-bariatric patients. In addition, it is not known whether the benefits of bariatric surgery on endothelial function, an important marker of early atherosclerosis, are sustainable without changes in lifestyle, such as the inclusion of physical exercise. Thus, the aims of the present study were to investigate the effects of exercise on intracellular signaling involved in insulin sensitivity and skeletal muscle ECM remodeling (Study 1) and the effects of exercise on the brachial artery vasodilator response of patients undergoing bariatric surgery (Study 2). Sixty-two women were randomized after bariatric surgery to a 6-month exercise program or standard of treatment. At the beginning of the study, 3 and 9 months after surgery, insulin sensitivity was assessed by the oral glucose tolerance test (OGTT), endothelial function analysis and skeletal muscle samples were obtained from the vastus lateralis. Skeletal muscle samples were subjected to comprehensive analysis, including gene and protein expression, skeletal muscle phenotype, transcriptome and identification of new cell signaling pathways. Exercise training after bariatric surgery improved insulin sensitivity in skeletal muscle. This response was mediated by molecular and phenotypic changes in ECM. Bariatric surgery per se was unable to completely resolve insulin resistance and skeletal muscle ECM expansion. Relevant exercise-modulated candidates emerged as therapeutic targets for the treatment of skeletal muscle insulin resistance, namely the TGFβ1/SMAD 2/3 pathway and its follistatin antagonist. In summary, we employed a \"top-down approach\" to provide evidence that skeletal muscle ECM plays a key role in the overlapping effects of bariatric surgery and exercise on insulin sensitivity in obese women. In addition, this study demonstrated that physical training avoided reversal of endothelial function improvement by improving blood flow pattern and reducing inflammatory markers. In conclusion, in revealing a new mechanism by which exercise can counterbalance insulin resistance in post-bariatric patients (i.e., attenuate ECM thickness) and preserve endothelial function, this study endorses that exercise should be adopted as a relevant therapeutic measure in order to guarantee the best cardiometabolic results in patients undergoing bariatric surgery.

Rôle de la NADPH oxydase NOX4 dans la régulation de l'expression du symporteur sodium/iode (NIS) dans le cas du cancer papillaire de la thyroïde (PTC). L’activation autocrine de la voie TGF-β induite par BRAFV600E régule négativement l’expression du symporteur sodium/iode (NIS) via une production de ROS dépendante de la NOX4 dans le cancer papillaire de la thyroïde. Résumé : Le cancer papillaire de la thyroïde (PTC) est la pathologie thyroïdienne la plus répandue. Les mutations ponctuelles de BRAF sont retrouvées dans 40 à 60 % des cas de PTC. La transversion BRAFT1799A est la mutation de BRAF la plus fréquente. Les tumeurs porteuses de la mutation BRAFV600E sont souvent associées avec une diminution significative de l’expression du transporteur sodium/iode (NIS). Les résultats cliniques sur les patients atteints d’un cancer de la thyroïde porteur de la mutation BRAFV600E ont montré que l’inhibition de la voie MAPK ne permet pas de rétablir de manière assez importante l’expression du NIS induite par BRAFV600E. L’expression de BRAFV600E induit la sécrétion de TGF-β fonctionnel, qui inhibe l’expression des protéines thyroïdiennes impliquées dans le métabolisme de l’iode, et particulièrement le NIS. La NOX4 est surexprimée dans un nombre croissant de tumeurs, et particulièrement dans les cas de PTC. Dans le cas du cancer du sein, les mécanismes critiques pour le développement du cancer impliquent la régulation par le TGF-β de la NOX4 au niveau transcriptionnel via le facteur de transcription Smad3. Ces données nous mènent à considérer la NOX4 comme un candidat sérieux pour le rôle de système générateur de ROS contrôlé par la boucle autocrine TGF-β induite par BRAFV600E. Dans cette étude, nous avons tout d’abord observé une corrélation entre la présence de l’oncogène BRAFV600E, la surexpression de la NOX4 et l’inhibition de l’expression du NIS dans les cancers papillaires de la thyroïde. Puis, en utilisant la lignée BCPAP comme modèle in vitro de PTC, nous avons démontré BRAFV600E contrôle l’expression de la NOX4 et de la p22phox par l’intermédiaire de la signalisation TGF-β/Smads. La boucle TGF-β induite par BRAFV600E induit l’expression de la NOX4 et de la p22phox au niveau transcriptionnel via phosphorylated SMAD3. L’expression constitutive de la NOX4 et de la p22phox, qui forment ensemble un complexe NADPH oxydase fonctionnel, contribue au stress oxydatif observé dans les cellules BCPAP. Le traitement des cellules BCPAP par des scavengers de ROS comme le N-acetyl cysteine (NAC) et le Tiron permettent d’augmenter l’expression du NIS au niveau transcriptionnel et de rétablir l’expression d’une protéine fonctionnelle permettant la captation d’iode, ce qui indique que les ROS sont impliqués dans l’inhibition de l’expression du NIS. L’inhibition spécifique de la NOX4 par siRNA permet de réinduire l’expression de l’ARN messager et de la protéine NIS. Ces résultats montrent pour la première fois que les ROS produits par la NOX4 jouent un rôle critique dans l’inhibition de l’expression du NIS induite par BRAFV600E via la signalisation TGF-β/SMAD3. / BRAFV600E induced-TGF-β secretion down-regulates sodium iodide symporter (NIS) expression via NOX4-dependent ROS generation in papillary thyroid carcinoma. Abstract : Papillary thyroid cancer (PTC) is the most common thyroid pathology and BRAF point mutations account for 40-60% of tumors. BRAFT1799A is the most frequent BRAF mutation and BRAFV600E positive tumors are often associated with a significant loss of sodium/iodide symporter (NIS) expression. Clinical results on patients harboring thyroid cancer with BRAF mutation have recently shown that MAPK pathway inhibition does not fully reverts the BRAF-induced NIS repression. BRAFV600E expression induces secretion of functional TGF-β which is a repressor of thyroid specific genes such as NIS. Importantly, NOX4 has been shown to be prominently expressed in an increasing number of tumors, in particular in PTCs. In breast cancer cells, a critical mechanism for cancer development involves the transcriptional regulation of NOX4 by TGF-β. This result prompted us to test NOX4 as a ROS-producing candidate induced by BRAF-induced TGF-β. In this report, we first show in PTCs a correlation between BRAFV600E status, NOX4 overexpression, and low NIS expression level. Then, using BCPAP cells as an in vitro PTC model, we demonstrate that BRAFV600E controls NOX4 and p22phox expression via TGF-β signalling. The TGF-β autocrine loop activated by BRAFV600E induces NOX4 and p22phox expression at the transcriptional level via phosphorylated SMAD3. Both constitutively expressed proteins form a functional NADPH oxidase which produces high intracellular ROS levels. ROS scavengers increase the NIS expression at both mRNA and protein levels, and rescue a functional NIS, indicating that ROS are involved in the repression of NIS. Knocking down NOX4 with specific siRNAs reinduces NIS expression at both mRNA and protein levels. Altogether, these results show for the first time that NOX4-dependent ROS generation has a critical role in BRAF-induced NIS repression via the TGF-β/SMAD3 oncogenic signalling.

A nefropatia diabética (ND) é uma das complicações microvasculares do diabetes e consiste no dano ao parênquima renal por consequência de uma série de fatores hemodinâmicos e moleculares. A ocorrência de ND e de outras complicações mesmo em indivíduos sob adequado controle glicêmico tem sido associada a um fenômeno conhecido como memória metabólica. Neste trabalho foram investigadas vias bioquímicas e moleculares persistentemente alteradas no rim de animais diabéticos tratados após um período inicial de hiperglicemia, com o propósito de entender os mecanismos envolvidos na memória metabólica. Para tanto, ratos com diabetes induzida por estreptozotocina foram mantidos hiperglicêmicos durante 4 semanas (período curto) ou 12 semanas (período longo) e posteriormente tratados com insulina isoladamente ou combinada com metformina (100mg/kg/dia) durante as 4 (período curto) ou 12 (período longo) semanas seguintes. Todos os animais tratados tiveram os seus níveis glicêmicos e função renal normalizados. Os tratamentos também foram capazes de normalizar os níveis elevados de malonaldeído no rim, bem como a excreção aumentada dos adutos de DNA 8-oxo-2\'-desoxiguanosina (8-oxodG) e N2-carboxietil-2\'- desoxiguanosina (CEdG) na urina observados nos animais diabéticos. Níveis aumentados de 8-oxodG foram detectados em DNA mitocondrial (mtDNA), mas não em DNA nuclear, de animais diabéticos apenas no período curto de estudo e também foram normalizados após o controle glicêmico. Nós identificamos uma via gradualmente alterada durante o curso do diabetes que permanece persistentemente alterada após o controle glicêmico tardio. Essa via compreende um declínio precoce do clearance de ácido úrico e expressão da pAMPK, seguida pelo acúmulo de fumarato, expressão aumentada de TGF-β, expressão reduzida de PGC-1α e redução da metilação e hidroximetilação do mtDNA. A redução persistente do clearance de ácido úrico em animais diabéticos tratados pode sustentar as alterações bioquímicas renais prolongadas observadas após o controle glicêmico, e essa regulação é provavelmente mediada pela redução sustentada da expressão de pAMPK e pela indução de inflamação. Este trabalho propõe a primeira consideração do possível papel da hiperuricemia e das alterações bioquímicas subjacentes como parte da memória metabólica na nefropatia diabética. / Diabetic nephropathy is one of the diabetes microvascular complications, and it consists on the damage to the renal parenchyma due to several hemodynamic and molecular factors. The occurrence of diabetic nephropathy and other complications even in those individuals under tight glycemic control has been associated to a phenomenon known as metabolic memory. Here we investigated biochemical and molecular pathways persistently altered in the kidney of diabetic animals treated after a previous period of hyperglycemia, aiming to understand underlying mechanisms in metabolic memory. Streptozotocin-induced diabetic rats were maintained hyperglycemic during 4 (short period) or 12 weeks (long period), and then they were treated with insulin alone or combined with metformin (100 mg/kg/day) for the following 4 or 12 weeks, respectively. All the treated animals had them glycemic levels and renal function normalized. The treatments were also able to control enhanced kidney malondialdehyde levels, as well as the increased urine excretion of the DNA adducts 8-oxo-2\'- deoxyguanosine (8-oxodG) and N2-carboxyethyl-2\'-deoxyguanosine seen in diabetic animals. Increased levels of 8-oxodG were detected in mitochondrial DNA, but not in nuclear DNA of diabetic animals in the short period, and were also recovered after glycemic control. We have identified a kidney pathway that is gradually altered during the course of diabetes and remains persistently changed after late glycemic control. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-β expression, reduced PGC-1α expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This work proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy.

Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural. / Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

Genes homeobox, vitais para muitos aspectos relacionados com crescimento e diferenciação celular, têm sido descritos desregulados em alguns cânceres. Seu papel na carcinogênese, principalmente de carcinomas epidermóides de boca, permanence pouco claro e pobremente caracterizado. Desse modo, esse estudo objetivou avaliar, em cultura de células, o perfil de expressão de seis genes homeobox (ASH2L, HOXA7, HHEX, PKNOX1, PITX1, TGIF) selecionados dentre aqueles previamente identificados no Projeto Genoma Câncer de Cabeça e Pescoço (2001) sob estímulo de EGF e TGF-beta1. Para tal, linhagens celulares de carcinoma epidermóide de cabeça e pescoço primário (HN6) e metastático (HN31) e uma linhagem não-tumoral (HaCat) foram cultivadas sob condições-padrão. Após a confecção dos cDNAs de cada linhagem, por meio de RT-PCR, os transcritos foram amplificados e quantificados pela técnica de PCR em tempo real. Os dados foram normalizados com o gene HPRT e a quantificação relativa foi realizada seguindo o método do delta Ct. De acordo com os resultados foi possível verificar que o EGF produziu uma modulação variável da expressão dos genes avaliados em todas as linhagens celulares, enquanto que, em geral, o TGF-beta1 foi capaz de aumentar significantemente (ANOVA, p<0,05) a expressão dos transcritos de 5 genes homeobox (HOXA7, HHEX, PKNOX1, PITX1, TGIF). Particularmente transcritos dos genes PITX1 e TGIF foram signicantemente mais expressos nas linhagens tumorais (HN6 e HN31) frente à linhagem não-tumoral quando tratados com TGF-beta1. Desse modo, sugere-se que os genes homeobox estudados desempenhem diferentes funções na carcinoma epidermóide de boca, e que, especialmente PITX1 e TGIF atuem como oncogenes inibindo a resposta anti-proliferativa dependente de TGF-beta e levando a progressão tumoral. / Homeobox genes, vital to many aspects related with cellular growth and differentiation, had been described as deregulated in some cancers. Their role in carcinogenesis, mainly oral squamous cell carcinomas, remains unclear and poorly characterized. Thus, this study had the purpose to evaluate, in cell cultures, the expression profile of six homeobox genes (ASH2L, HOXA7, HHEX, PKNOX1, PITX1, TGIF) selected among genes previously identified in the Head and Neck Cancer Genoma Project (2001), under stimulation with EGF and TGF-beta1. Oral squamous cell carcinoma cell lines from primary tumour (HN6) and from methastasis (HN31), and a non-tumoral cell line (HaCat) were cultured under standard procedures. CDNAs were obtained by RT-PCR and the transcripts were amplified and quantified by real-time PCR. Data were normalized by HPRT gene and the relative quantification was made by the delta Ct method. According to the results, it was possible to observe that EGF produced a variable modulation of the analyzed genes, in all cell lines. Generally, TGF-beta1 was able to significantly increase (ANOVA, p<0,05) the expression of the transcripts of 5 homeobox genes (HOXA7, HHEX, PKNOX1, PITX1, TGIF). Transcripts of PITX1 and TGIF genes were particularly more expressed in the tumoral cell lines (HN6 e HN31), when compared to the non-tumoral cell line, when treated with TGF-beta1. It is suggested that the studied homeobox genes play different roles in oral squamous cell carcinoma and that, especially the PITX1 and TGIF act as oncogenes, inhibitting the TGF-dependent anti-proliferative response, leading to tumour progression.

Arkadia participa do processo de amplificação da sinalização de TGF-b mediada por Smads, via degradação do I-Smad. O objetivo desse estudo foi caracterizar e investigar a influência de Arkadia em linhagens celulares de cânceres de tiróide. A expressão gênica de Arkadia em linhagens celulares de carcinomas papílifero (NPA), folicular (WRO) e anaplásico (ARO), foi avaliada por PCR quantitativo. Em ARO, que apresenta a maior expressão de Arkadia, foram identificados subclones (ARO_1 e ARO_2) com expressão diferencial de Arkadia, ARO_2>ARO_1. A expressão gênica de SMAD2, 3, 4, 7 e de genes do ciclo celular modulados por TGF-b, foi maior em ARO_2. Os subclones respondem ao tratamento com peptídeo de TGF-b1 e activina A. O crescimento in vivo (xenotransplante) mostra que ARO_2 desenvolve um tumor de menor volume. Recentemente a origem de ARO foi questionada e comprovamos sua origem por análises de expressão gênica e morfologias. Desta maneira, observamos que a expressão diferencial de Arkadia indica que ela está envolvida na modulação inibitória da via de TGF-b. / Arkadia is involved in the process of amplification of the TGF-b signaling mediated by Smads, by degradation of I-Smad. The aim of this study was to characterize and investigate the influence of Arkadia in thyroid cancers cell lines. Arkadia gene expression in the papillary (NPA), follicular (WRO) and anaplastic carcinoma cell lines (ARO) was evaluated by quantitative PCR. In ARO, which presents the highest Arkadia expression, we identified subclones (ARO_1 and ARO_2) with differential Arkadia expression ARO_2> ARO_1. The expression of SMAD2, 3, 4, 7 and the cell cycle genes modulated by TGF-b, was also higher in ARO_2. However both the subclones responded to treatment with peptide of TGF-b1 and activin A. The in vivo growth (evaluated by xenotransplant), showed that ARO_2 developed tumors of lower volume. Recently the ARO origin was questioned and we proved its origin by gene expression and morphological analysis. This way, the differential Arkadia expression indicates that it is involved in modulation of the inhibitory TGF-b pathway.

Les cellules souches neurales quiescentes (CSN) sont le réservoir de la neurogenèse adulte, permettant de produire des nouveaux neurones tout au long de la vie. Cependant, la neurogenèse décroit au cours du vieillissement, provoquant des déclins cognitifs incurables. Afin de mieux comprendre les mécanismes qui contrôlent la prolifération des CSN, nous avons mis en place une méthode de tri par cytométrie en flux qui permet pour la première fois d’isoler les CSN quiescentes et leurs cellules filles dans la ZSV adulte murine. Cette technique nous a permis de prouver que le blocage de la voie GABAAR in vivo provoque l’entrée en cycle des CSN quiescentes. Ainsi, les signaux GABA produits par les neuroblastes dans la ZSV permettent de maintenir les CSN dans leur état de quiescence. Au cours du vieillissement, nous montrons que la production progressive de TGFβ1 par les cellules endothéliales de la niche allonge la phase G1 des CSN activées, diminuant sensiblement la production de nouveaux neurones, sans toutefois diminuer le stock de CSN. Nous mettons ainsi en évidence deux voies majeures contrôlant la prolifération des CSN in vivo, la voie du GABAAR et la voie TGF-β/Smad-3. En vue d’une application thérapeutique, nous prouvons que leur blocage pharmacologique permet de stimuler efficacement la neurogenèse in vivo. / Quiescent neural stem cells (NSCs) are considered the reservoir for adult neurogenesis, generating new neurons throughout life. However, neurogenesis decreases during aging, causing a progressive decline that is currently untreatable. To study the regulatory mechanisms of NSCs proliferation, we set up a new technique allowing the isolation of quiescent NSCs and their progeny. We show that GABAAR directly regulates NSCs quiescence in vivo as the depletion of GABA-producing neuroblasts or GABAAR pathway pharmacological blockade provoked NSCs cell cycle entry in the SVZ. During aging, the stock of NSCs is not perturbed, but we show that an over-production of TGFβ1 by brain endothelial cells directly lengthens activated NSCs G1 phase, strongly decreasing the production of new neurons. These findings highlight GABAAR and TGF-β/Smad-3 as two major pathways controlling NSCs proliferation. In line with a future therapeutic application, we also prove that their blocking stimulates endogenous neurogenesis in vivo.