Les papillomavirus humains (HPV) sont des petits virus non enveloppés à ADN double brin, qui infectent les épithéliums cutanés et muqueux. Très largement répandus dans la population humaine, les infections sont la plupart du temps asymptomatiques, tandis qu'une minorité provoquent des verrues cutanées, des condylomes acuminés et des carcinomes. Les cancers induits par HPV représentent 640 000 cas en 2012 dans le monde et sont localisés au niveau anogénital et au niveau des voies aérodigestives supérieures.La transformation des cellules est induite en particulier par la surexpression de deux oncoprotéines virales : E6 et E7, qui sont nécessaires à l'initiation, la promotion et la progression du phénotype cancéreux. Leur extinction provoque d'ailleurs la mort des cellules cancéreuses. E6 et E7 favorisent par exemple la dégradation de p53 et pRb, induisant ainsi une prolifération non contrôlée et accrue des cellules infectées. Mais les deux oncoprotéines virales interagissent aussi avec plus de 150 partenaires protéiques et dérégulent l'homéostasie cellulaire. Fait intéressant, E6 et E7 modulent l'expression de nombreuses protéines impliquées dans l'établissement des marques épigénétiques, impliquées dans la régulation de l'expression des gènes. D'ailleurs, la surexpression des oncoprotéines virales peut être liée à la méthylation de l'ADN d'une partie du génome viral.Puisque E6 et E7 détournent la machinerie épigénétique de la cellule, l'objectif du travail de thèse a été d'étudier les mécanismes moléculaires régulant l'expression des oncoprotéines lors du traitement des cellules de cancers du col par un agent déméthylant de l'ADN, le 5-aza-2'-déoxycytidine (5azadC).Dans des cellules issues de cancers du col de l'utérus, nous avons observé qu'un traitement déméthylant induisait une répression de la protéine E6 et une déstabilisation de ses transcrits, suggérant l'implication d'un miARN. Parmi les miARN ciblant les transcrits viraux, le miR-375 était le plus susceptible d'être impliqué dans la déstabilisation des transcrits car (i) la méthylation de son promoteur augmente avec la gravité des lésions du col de l'utérus, (ii) il cible les transcrits viraux au niveau de cinq régions différentes, et (iii) son expression est augmentée lors du traitement de cellules dérivées du cancer du col de l'utérus avec le 5azadC. Il s'est avéré que le miR-375 était effectivement impliqué dans la répression partielle des transcrits viraux lors du traitement. Les résultats de cette étude suggèrent par ailleurs qu'un autre mécanisme moléculaire pourrait être impliqué dans cette répression. C'est pourquoi, dans un deuxième temps, nous avons étudié l'implication possible d'un facteur de transcription, TBX2, dans la répression des transcrits viraux. Bien que le taux transcriptionnel de TBX2 soit augmenté lors du traitement des cellules avec des fortes concentrations de 5azadC, nos résultats suggèrent que la protéine n'est pas exprimée dans les cellules, même après traitement, et que TBX2 ne régule pas l'activité du promoteur viral intégré au génome cellulaire.Le traitement des cellules par l'agent déméthylant entraîne aussi une perte de viabilité en particulier des cellules Ca Ski. En effet, le 5azadC induit un blocage du cycle en G2/M dans ces cellules, une augmentation du pourcentage de cellules en Sub-G1 et un clivage de la PARP. Si le 5azadC est déjà utilisé en clinique pour le traitement d'autres cancers, nos données apportent un éclairage nouveau sur les mécanismes moléculaires du 5azadC dans les cancers induits par HPV, qui pourraient ainsi être traités par cette molécule. / Human papillomaviruses (HPV) are small, non-enveloped double-stranded DNA viruses and infect cutaneous and mucosal epithelia. Highly widespread in the human population, infections are mostly asymptomatic, while a minority causes cutaneous and genital warts, and carcinomas. HPV-induced cancers represent 640 000 cases in 2012 worldwide and are localized at the anogenital and head and neck sites.Cell transformation is induced by the overexpression of two viral oncoproteins E6 and E7, which are necessary for the initiation, promotion and progression of the cancerous phenotype. Indeed, their extinction causes cancer cell death. E6 and E7, for example, promote the degradation of p53 and pRb, thus inducing uncontrolled and increased proliferation of infected cells. But the two viral oncoproteins also interact with more than 150 protein partners and deregulate cell homeostasis. Interestingly, E6 and E7 modulate the expression of many proteins involved in the establishment of epigenetic marks, regulating gene expression. Furthermore, the overexpression of viral oncoproteins may be related to viral genome DNA methylation.Since E6 and E7 hijack epigenetic mechanisms, the aim of the thesis was to study the molecular mechanisms regulating the oncoprotein expression during the treatment of cervical cancer cells by a DNA demethylating agent, the 5-aza-2'-deoxycytidine (5azadC).In cervical cancer cells, we observed that a demethylating treatment induced E6 protein repression and destabilization of its transcripts, suggesting the involvement of a miRNA. Among miRNAs targeting viral transcripts, miR-375 was most likely to be involved this destabilization because (i) its promoter methylation increases with the severity of cervical lesions, (ii) it targets viral transcripts at five different regions, and (iii) its expression is increased in cervical cancer cells treated with 5azadC. In our experiences, miR-375 was indeed involved in the partial repression of viral transcripts during treatment. The results of this study further suggest that another molecular mechanism might be implicated in this repression. This is why, in a second step, we studied the possible involvement of a transcription factor, TBX2, in the viral transcript repression. Although the transcriptional rate of TBX2 is increased in cells treated with high concentrations of 5azadC, our results suggest that the protein is not expressed in cells, even after treatment, and that TBX2 does not regulate the viral promoter activity integrated into the cellular genome.Cell treatment with demethylating agent also leads to loss of viability, in particular in Ca Ski cells. Indeed, 5azadC induces a G2/M cycle arrest in these cells, an increase of Sub-G1 cells percentage and a PARP cleavage. While 5azadC is already used in clinical practice for the treatment of other cancers, our data shed new light on the molecular mechanisms of 5azadC in cancers induced by HPV, which could be treated by this molecule or analogues.

Les mutations germinales activatrices de la voie RAS sont responsables de maladies rares regroupées sous le nom de RASopathies : le Syndrome de Noonan, le Syndrome de Noonan avec de Multiples Lentigines, la Neurofibromatose de type 1, le Syndrome de Malformations Capillaires et Malformations Artério-Veinseuses, le Syndrome Cardio-Facio-Cutané, le Syndrome de Legius et le Syndrome de Costello. Cette thèse s’intéresse au syndrome de Costello causé par une mutation hétérozygote de novo du gène HRAS. Ce syndrome est révélé dans les premiers mois de la vie et se caractérise par un retard de croissance postnatal, des traits du visage épais, un déficit intellectuel, des anomalies cutanées, ainsi qu’une prédisposition à développer des tumeurs. De plus, les patients atteints du syndrome de Costello développent une cardiomyopathie hypertrophique, de l’hypertension, une hypotonie et une myopathie d'origine moléculaire inconnue. En lien avec une association de malade et le service de génétique du CHU de Bordeaux, nous avons mené une exploration des anomalies protéomiques dans les tissus d’une souris modèle du syndrome de Costello ainsi que dans des fibroblastes de patients et des cellules modèles exprimant les formes mutées de HRASG12S et HRASG12A. Cette analyse globale et sans a priori a révélé des altérations au niveau du métabolisme énergétique et plus particulièrement de la composition des mitochondries. Le déficit fonctionnel des mitochondries, centrale énergétique du corps humain, a été caractérisé par des approches de biochimie, de bioénergétique et de biologie cellulaire. De plus, l’analyse des données ‘omiques’ a permis de suggérer une nouvelle hypothèse dans la physiopathologie du syndrome de Costello. Cette hypothèse considère l’implication d’un micro-ARN, le miR-221* dans l’inhibition du métabolisme oxydatif. Les analyses génétiques réalisées sur les cellules de patients et les cellules modèles ont démontré l’inhibition de l’expression de la protéine AMPK, un régulateur majeur du métabolisme mitochondrial, par le miR-221* sous le contrôle de HRASG12S et HRASG12A. Ces découvertes ont permis d’élaborer une stratégie thérapeutique visant à réduire la cardiomyopathie dans le syndrome de Costello. Les analyses précliniques effectuées sur les modèles cellulaires et le modèle murin ont permis d’évaluer l’efficacité d’une stimulation pharmacologique du métabolisme mitochondrial. Cette thèse révèle donc l’implication des mitochondries dans le syndrome de Costello et l’analyse moléculaire réalisée propose une série de données ‘Omiques’ qui permettront de progresser dans la compréhension de cette maladie rare. / Germline activating mutations of the RAS pathway are responsible for rare diseases grouped under the name of RASopathies: Noonan Syndrome, Noonan Syndrome with multiple Lentigines, Type 1-neurofibromatosis, Capillaries malformations and arteriovenous malformations syndrome, Cardio-Facio-Cutaneous Syndrome, Legius Syndrome and Costello Syndrome. This Ph.D thesis focuses on Costello syndrome that is caused by a heterozygous de novo mutation of the HRAS gene. This syndrome is revealed in the first months of life and is characterized by postnatal growth retardation, thick facial features, intellectual deficit, skin abnormalities, and a predisposition to developing tumors. In addition, patients with Costello syndrome develop hypertrophic cardiomyopathy, hypertension, hypotonia and myopathy of unknown molecular origin. In connection with a patients association and the genetics department of Bordeaux University Hospital, we conducted an exploration of proteomic abnormalities in the tissues of a mouse model of the Costello syndrome as well as in patients’ fibroblasts and cell models expressing mutated forms of HRASG12S and HRASG12A. This global and unbiased analysis revealed alterations in energy metabolism and more particularly in the composition of mitochondria. The functional deficiency of mitochondria, energy plants of the human body, has been characterized by biochemistry, bioenergetics and cell biology approaches. In addition, the 'omic' analysis of Costello syndrome suggested a new pathophysiology hypothesis that considered the involvement of a microRNA, miR-221* in the alteration of oxidative metabolism. Functional genetic analyzes performed on patient cells and cell models demonstrated the inhibition of the expression of the major mitochondrial metabolism regulator AMPK protein by miR-221* under the control of HRASG12S and HRASG12A. These findings led to the development of a preclinical therapeutic strategy to reduce cardiomyopathy in Costello syndrome. Preclinical investigations performed on the cellular models and the murine model made it possible to evaluate the efficacy of a pharmacological stimulation of mitochondrial metabolism. This thesis thus reveals the involvement of mitochondria in Costello syndrome and the molecular analysis carried out makes available a series of 'Omics' data that will allow progress in the understanding of this rare disease.

O controle de qualidade de laranjas desde a colheita até a comercialização é realizado com base em análises físico-químicas. Todavia, estas análises são destrutivas. Neste cenário, sistemas não invasivos para aferir a qualidade, são alternativas promissoras. O objetivo do trabalho foi avaliar os métodos de análises não destrutivas como a Ressonância Magnética em baixo campo (RMN) e espectroscopias de infravermelho médio (MIR) e próximo (NIR), associadas à quimiometria, para analisar parâmetros de qualidade de laranjas de forma não invasiva. O experimento ocorreu na unidade da Embrapa Instrumentação em São Carlos, SP. Foram coletadas 470 laranjas, obtidas em cultivos comerciais no interior do estado de São Paulo. As frutas passaram pelas etapas de seleção, higienização e sanitização. Em seguida, foram submetidas à análise não invasiva pelos equipamentos de RMN, NIR e MIR. Os parâmetros de qualidade avaliados foram, massa fresca, diâmetros longitudinal e transversal do fruto, teor de sólidos solúveis (SST), pH, acidez total titulável (ATT), índice de maturação (ratio) e rendimento de suco. Para os sinais de RMN foi aplicada a suavização de Savitzky-Golay com largura de janela de 21 pontos. Para os sinais de NIR foi aplicado a variação normal padrão (SNV) e para os sinais de MIR foi aplicada a normalização (0-1), seguido da segunda derivada. O modelo de predição foi construído utilizando a regressão por mínimos quadrados parciais (PLS) para cada parâmetro de qualidade. Os modelos desenvolvidos por RMN-PLS validados para predição foram: massa fresca; coeficiente de Pearson da predição (r) = 0,97, erro padrão da predição (SEP) = 13,57. Diâmetro longitudinal; r = 0,91 e SEP = 3,37. Diâmetro transversal; r = 0,92 e SEP = 2,73. SST; r = 0,81 e SEP = 0,88. Rendimento de suco; r = 0,78 e SEP = 3,26 e pH r = 0,74 e SEP = 0,17. Os parâmetros de índice de maturação e ATT não puderam ser validados utilizando RMN-PLS. Os modelos de NIR-PLS validados foram: SST; r = 0,92 e SEP = 0,71. ATT; r = 0,92 e SEP = 0,30. Os demais parâmetros não puderam ser validados por NIR-PLS. Para os modelos de MIR-PLS, o melhor resultado encontrado foi para validação interna do modelo de pH, r Validação = 0,80 e erro padrão da validação (SEV) de 0,16. A classificação desenvolvida utilizando os modelos de parâmetros físicos de RMN-PLS apresentaram acurácia para diâmetro transversal de 80,00%. As classificações por parâmetros químicos, como teor de sólidos solúveis revelou acurácia de 81,10% e para pH de 61,11%. Para as classificações por PLS-NIR para o ratio a acurácia foi igual a 87,95%. Os frutos classificados de forma não invasiva para a análise sensorial no teste de comparação pareada, apresentaram respostas significativas para sucos classificados pelos modelos de RMN-PLS a nível de p=0,05. Para os frutos classificados pelo NIR-PLS de forma não invasiva, a resposta ao segundo teste sensorial foi significativa a nível de p=0,05. Estes resultados comprovam a aplicabilidade destas técnicas como análises não invasivas para mensurar a qualidade de laranjas e classifica-las por parâmetros físico-químicos percebidos por provadores. / The quality control for oranges, after the harvest until commercialization are carried based on physical-chemical parameters. However, these analyzes are invasive. In this scenario, non-invasive systems to measure quality are promising options. Nuclear Magnetic Resonance (NMR) at low field and infrared spectroscopy (mid-infrared - MIR and near-infrared - NIR) were associated with chemometrics to analyze quality parameters in intact oranges. The experiment was carried at Embrapa São Carlos, SP. Four hundred and seventy oranges were obtained from commercial crops in the state of São Paulo. Samples were selected, cleaned and sanitized and then were submitted to non-invasive analysis by NMR, NIR and MIR equipment\'s. The evaluated reference quality parameters were fresh weight, longitudinal and transversal diameter, total soluble solids (TSS), pH, titratable acidity (TA), maturation index (ratio) and juice yield (%). For the non-invasive methods was applied pre-processing techniques on the signal. In NMR signal was applied Savitzky-Golay smoothing with 21 points width of window. For NIR signal was applied standard normal variant (SNV) and on the MIR signal normalization was applied (0-1), followed by the second derivative. The prediction model was constructed using partial least squares regression (PLS) for each quality reference parameter. The models developed by NMR-PLS validated by prediction were: fresh weight; Pearson coefficient prediction (r) = 0.97, standard error of prediction (SEP) = 13.57. Longitudinal diameter; r = 0.91 and SEP = 3.37. Transverse diameter; r = 0.92 and SEP = 2.73. SST; r = 0.81 and SEP = 0.88. Juice yield; r = 0.78 and SEP = 3.26. pH r = 0.74 and SEP = 0.17. The maturation index and titratable acidity parameters could not be validated using PLS-NMR. The validated NIR-PLS models were: SST; r = 0.92 and SEP = 0.71. ATT; r = 0.92 and SEP = 0.30. The others reference quality parameters were not validated by NIR-PLS. For models using MIR-PLS, the best result was found for internal validation of the pH model, r = 0.80 and standard validation error (SEV) 0.16. The classification models developed using NMR-PLS physical parameters showed accuracy in transverse diameter of 80.00%. The classifications by chemical parameters such as soluble solids revealed accuracy of 81.10% and 61.11% for pH. For classifications by PLS-NIR for the accuracy ratio was equal to 87.95%. Fruit classified noninvasively for sensory analysis in paired comparison test, showed significant responses to juices classified by NMR-PLS models at the level of p = 0.05. For fruit classified by NIR-PLS noninvasively, the answer to the second sensory test was significant at the level of p = 0.05. These results demonstrate the applicability of these techniques as non-invasive tests to measure the quality of oranges and sorts them by physicochemical parameters perceived by tasters.

No câncer, alterações em microRNAs (miRNAs), pequenos RNAs que regulam a tradução protéica, exerce efeito oncogênico (oncomiR). Os oncomiRs regulam genes chave para a proliferação celular e apoptose, sendo importantes para a biologia do câncer. O carcinoma papilífero de tiróide apresenta alterações genéticas alinhadas na via MAPK (RET>RAS>BRAF>ERK). Observamos que a indução do oncogene RET/PTC diminui a expressão de let-7 em células foliculares tiroidianas. Na linhagem TPC-1 (com RET/PTC-1), a introdução de let-7 diminui a proliferação celular e a fosforilaçãode ERK, indicando papel de gene supressor tumoral. No carcinoma anaplásico, avaliamos o papel da introdução do cluster miR-17-92 na linhagem ARO. Observamos que in vitro miR-17-92 atua de forma oncogênica aumentando proliferação e viabilidade celular de ARO. No entanto, estas células apresentam diminuição no crescimento em soft-agar. No xenotransplante, os tumores de ARO-miR-17-92 apresentam menor volume e expressam MMP-9 de forma reduzida, indicando também um papel de gene supressor tumoral para o cluster. / In cancer, alteration in microRNA, small RNAs (~22nt) that regulate post-transcriptionally protein levels, exerts oncogenic role (oncomiR). OncomiRs control genes involved in cell proliferation and apoptosis, influencing cancer biology. Papillary thyroid cancer displays activating genetic alterations in MAPK signaling pathway (RET>RAS>BRAF>ERK). Using conditional induction of oncogenes in thyroid cells, we observed that RET/PTC decreases let-7 miRNA expression. In papillary thyroid cancer cell TPC-1 (with RET/PTC-1) we observed that let-7 introduction inhibits cell proliferation and ERK phosphorylation, indicating tumor suppressor role for let-7. In anaplastic thyroid cancer, we evaluate the role of introduction of miR-17-92 cluster in ARO cell line. We observed in vitro that miR-17-92 increases ARO cell proliferation and viability, acting as oncogene. However, these cells show impaired soft agar growth. In xenotransplant, ARO-miR-17-92 tumors are smaller in volume and express reduced levels of MMP-9, indicating a tumor suppressor role for the cluster.

Mehr als 300 Millionen Menschen sind weltweit von Diabetes betroffen, die Mehrheit davon leidet an Typ-2-Diabetes. Typ-2-Diabetes ist durch eine Insulinresistenz charakterisiert, welche meistens durch Übergewicht und Adipositas verursacht wird. Diese Insulinresistenz kann zunächst durch eine erhöhte pankreatische Insulinsekretion kompensiert werden, jedoch können langfristig die pankreatischen beta-Zellen den erhöhten Insulinbedarf nicht mehr decken. Dies verursacht einen starken Anstieg der Blutglucosespiegel und stellt den Beginn der Typ-2-Diabetes Erkrankung dar. Neben genetischen Veränderungen sind Umweltfaktoren, wie erhöhte Nahrungsaufnahme und reduzierte Bewegung, wichtige Faktoren in der Pathogenese des Typ-2-Diabetes. Frühere Forschungsergebnisse zeigten eine wichtige Rolle von microRNA 375 (miR-375) im Wachstum und in der Funktion der Insulin produzierenden beta-Zellen. Die Genexpression von miR-375 ist in diabetischen Nagetieren und Menschen verändert, was auf eine wichtige Rolle dieser microRNA in der Pathogenese des Typ-2-Diabetes hindeutet. Gene, die durch miR-375 reguliert werden, wurden in den pankreatischen beta-Zellen beschrieben, jedoch ist der Mechanismus wie miR-375 das Wachstum und die Funktion der pankreatischen beta-Zellen beeinflusst noch nicht im Detail verstanden. Das Cell Adhesion Molecule 1 (CADM1) ist ein bekanntes Zielgen der miR-375 und vor allem im Gehirn als Regulator von Anzahl und Funktion der Synapsen bekannt. Da es außerdem in den pankreatischen beta Zellen exprimiert ist, könnte es auch dort an der Regulation von beta-Zellwachstum und –funktion beteiligt sein und die Glucose- und Energiehomöostase verändern. Ziel dieser Arbeit war es, in vollständig oder konditionell Cadm1-defizienten Mäusen den Einfluss von CADM1 in pankreatischen beta-Zellen und neuronalem Gewebe an der Regulation von Glucose- und Energiehomöostase zu untersuchen. / More than 300 million people world-wide are affected by diabetes, the majority suffering from type 2 diabetes. Type 2 diabetes is characterized by insulin resistance, usually caused by obesity and overweight. Enhanced pancreatic insulin secretion largely compensates insulin resistance for years. A failure of pancreatic beta-cells to meet increased insulin demands drastically increases blood glucose levels and marks the onset of type 2 diabetes. Besides environmental influences, mainly elevated food intake and reduced physical activity, also genetic mutations are important factors in the pathophysiology of type 2 diabetes. Recent literature highlights the role of microRNA 375 (miR-375) in the growth and function of pancreatic insulin-producing beta-cells. MiR-375 gene expression is regulated in diabetic humans and rodents, suggesting that this microRNA is involved in the pathogenesis of type 2 diabetes. Genes regulated by miR-375 have been described in pancreatic beta-cells. Nevertheless, the exact mechanisms how miR-375 regulates beta-cell growth and insulin secretion have not been understood. Cell adhesion molecule 1 (CADM1) is a known target of miR-375 and has mainly been described as regulator of synapse number and synaptic function in the brain. CADM1 is also expressed in pancreatic beta-cells and might regulate beta-cell growth and function and might be involved in the control of glucose and energy homeostasis. The aim of this work was to investigate whether CADM1 in pancreatic beta-cells or neuronal tissue contributes to the regulation of energy and glucose homeostasis by using total and conditional Cadm1 deficient mice. Total Cadm1 deficient (Cadm1KO) mice showed increased sensitivity to glucose and insulin as well as enhanced glucose-stimulated insulin secretion compared to littermate control mice. Elevated glucose-stimulated insulin secretion after Cadm1 depletion could be confirmed in an in vitro beta-cell model.

Der Cholesterin-Stoffwechsel ist sehr streng auf zellulärer Ebene reguliert und ist essentiell für das Zellwachstum. MicroRNAs (miRNAs), eine Klasse nicht-kodierender RNAs, wurden als kritische Regulatoren der Genexpression identifiziert und entfalten ihre Wirkung vorwiegend auf posttranskriptioneller Ebene. Aktuelle Arbeiten aus der Gruppe um Fernández-Hernando haben gezeigt, dass hsa-miR-33a und hsa-miR-33b, miRNAs die in den Intronsequenzen der Gene für die Sterol-regulatorischen Element- Bindungsproteine (SREBP-2 und SREBP -1) lokalisiert sind, den Cholesterin-Stoffwechsel im Einklang mit ihren Wirtsgenen regulieren. Gleichermaßen inhibiert miR-33 Schlüsselenzyme in der Regulation der Fettsäureoxidation, einschließlich CROT, CPT1A, HADHB, SIRT6, AMPKα, genauso wie IRS2, eine wesentliche Komponente des Insulin-Signalwegs in der Leber. Diese Studie zeigt, dass hsa-miR-33 Familienmitglieder nicht nur Gene in Cholesterin- und Fettsäure-Stoffwechsel sowie Insulin-Signalwege regulieren, sondern zusätzlich die Expression von Genen des Zellzyklus und der Zellproliferation modulieren. miR-33 inhibiert die Expression der CDK6 und CCND1, wodurch sowohol die Zellproliferation als auch die Zellzyklusprogression verringert wird. Die Überexpression von miR-33 induziert einen signifikanten G1 Zellzyklusarrest. Durch eine Inhibierung der miR-33 Expression mittels 2''F/MOE-modifiziert Phosphorothioat-Backbone Antisense-Oligonukleotiden, wird die Leberregeneration nach partieller Hepatektomie (PH) in Mäusen verbessert, was auf eine wichtige Rolle für miR-33 in der Regulation der Hepatozytenproliferation während der Leberregeneration hinweist. Zusammengefasst zeigen diese Daten, dass Srebf/miR-33 Locus kooperieren, um Zellproliferation und Zellzyklusprogression zu regulieren, und könnte somit auch relevant für die menschliche Leberregeneration sein. / Cholesterol metabolism is tightly regulated at the cellular level and is essential for cellular growth. Cellular imbalances of cholesterol and fatty acid metabolism lead to pathological processes, including atherosclerosis and metabolic syndrome. MicroRNAs (miRNAs), a class of noncoding RNAs, have emerged as critical regulators of gene expression acting predominantly at posttranscriptional level. Recent work from Fernández-Hernando´s group and others has shown that hsa-miR-33a and hsa-miR-33b, miRNAs located within intronic sequences of the sterol regulatory element-binding protein (SREBP-2 and SREBP-1) genes, respectively, regulate cholesterol metabolism in concert with their host genes. Similarly, miR-33 targets key enzymes involved in the regulation of fatty acid oxidation including CROT, CPT1A, HADHB, SIRT6 and AMPKα, likewise, IRS2, an essential component of the insulin- signaling pathway in the liver. This study shows that hsa-miR-33 family members not only regulate genes involved in cholesterol and fatty acid metabolism and insulin signaling, but in addition modulate the expression of genes involved in cell cycle regulation and cell proliferation. Thus, miR-33 inhibited the expression of CDK6 and CCND1, thereby reducing cell proliferation and cell cycle progression. Over-expression of miR-33 induced a significant G1 cell cycle arrest and most importantly, inhibition of miR-33 expression using 2’F/MOE-modified phosphorothioate backbone antisense oligonucleotides improved liver regeneration after partial hepatectomy (PH) in mice, suggesting an important role for miR-33 in regulating hepatocyte proliferation during liver regeneration. Altogether, these data establish that Srebf/miR-33 locus may co-operate to regulate cell proliferation, cell cycle progression and may also be relevant to human liver regeneration.

Esta dissertação tem como objetivo central reconstituir a trajetória do poeta brasileiro Nilton Rosa da Silva, enfocando seu período como exilado político no Chile, entre 1971 e 1973. Em Santiago, Nilton da Silva estudou castelhano no Instituto Pedagógico da Universidade do Chile, publicou o livro de poesias Hombre América, e passou a militar na Frente de Estudiantes Revolucionarios (FER), um dos grupos estudantis do Movimiento de Izquierda Revolucionaria (MIR). O jovem brasileiro seria morto aos 24 anos de idade, em junho de 1973 (três meses antes do golpe de Estado liderado por Augusto Pinochet), por membros da Frente Nacionalista Patria y Libertad, milícia de ultradireita que lutava pela derrubada do presidente Salvador Allende. Defendendo a revolução armada para colocar o Chile no caminho do socialismo, mesmo durante o governo democrático da Unidad Popular (UP), o MIR era visto com reservas por setores moderados da esquerda. Ao mesmo tempo, porém, a organização procurava – desde fora – radicalizar os partidos da UP. A partir da análise da vida de Nilton da Silva, e das repercussões da sua morte, é possível discutir as disputas entre as estratégias “rupturista” e “sistêmica” da esquerda chilena durante o governo Allende, as possibilidades de acordo que se desenharam entre esses setores, e as maneiras como o MIR procurou conquistar esferas mais amplas para sua retórica em favor da necessidade de pegar em armas. Os usos políticos do assassinato de Nilton da Silva estão relacionados a essa busca: através da análise das apropriações do episódio, apreende-se o uso imediato que o MIR fez de sua morte, tentando construir o jovem militante como um mártir revolucionário em um período de crescente temor frente a um golpe reacionário. Discute-se, ademais, a forma como a vida e a morte de Nilton da Silva seriam eventualmente ressignificada nas décadas seguintes, passando a incluí-lo em uma narrativa mais ampla, ao lado de outras vítimas das ditaduras brasileira e chilena. / This thesis aims to recreate the trajectory of the Brazilian poet Nilton Rosa da Silva, focusing on his time as a political exile in Chile, from 1971 to 1973. In Santiago, Silva studied Spanish at the Pedagogical Institute of the University of Chile, published his poetry book Hombre América, and became a member of the Revolutionary Students Front (FER), one of Revolutionary Left Movement’s (MIR) groups in the student movement. The young Brazilian was killed at the age of 24 in June 1973 (three months prior to the coup led by Augusto Pinochet), by members of the Fatherland and Liberty Nationalist Front, a far-right militia that fought to overthrow the president, Salvador Allende. Advocating the need of an armed revolution to place Chile in the path of Socialism, even during the Popular Unity’s (UP) democratic administration, MIR was seen with hesitations by the moderate left. At the same time, however, MIR sought to radicalize the UP parties. By analyzing Nilton da Silva’s life and the impact of his death, it is possible to discuss the disputes between the “rupturist” and “systemic” strategies of the Chilean left during the Allende administration, the chances of agreement between these sectors, and the ways in which MIR sought to conquer wider segments to its rhetoric in favor of the need to take up arms. The political uses of Nilton da Silva’s murder are related to this goal: by examining the appropriation of his death, we are able to see the immediate use that MIR did of this episode, trying to construct the young activist as a revolutionary martyr in a period of growing fear towards a reactionary coup. This work discusses, moreover, how the life and death of Nilton da Silva would eventually be re-signified in the following decades, now being included in a broader narrative, along with other victims of the Brazilian and Chilean dictatorships.

En pathologie tumorale, l’étude du micro-environnement tumoral doit prendre en compte différents modes de communication cellulaire : contacts directs entre membranes plasmiques, émission et réception de cytokines et enfin émission et internalisation d’objets biologiques plus complexes comme les microvésicules et les exosomes qui peuvent être assimilés à de véritables organites extra-cellulaires. Le virus d’Epstein-Barr (EBV) participe à l’oncogenèse de plusieurs affections malignes humaines d’origine épithéliale (carcinomes nasopharyngés ou NPC) ou lymphocytaire (lymphomes post-transplantation). Dans ces tumeurs, les cellules malignes qui sont infectées de façon latente par EBV libèrent des exosomes et des microvésicules qui contiennent des protéines et des acides nucléiques d’origine virale. L’étude de ces éléments doit permettre de mieux comprendre les interactions hôte-tumeur et de mettre en évidence de nouveaux biomarqueurs utiles pour le diagnostic précoce et la surveillance de la maladie sous traitement. Le premier objectif de ma thèse consistait à étudier la sécrétion par les cellules malignes d’une famille de microARN viraux appelés miR-BART et leur diffusion dans le sang périphérique chez les sujets porteurs de tumeurs associées à EBV. Pour la première fois j’ai mis en évidence une sécrétion d’exosomes porteurs de miR-BART par les cellules de NPC en culture in vitro. J’ai également montré que les miR-BART, particulièrement miR-BART7, sont détectables dans le plasma de sujets porteurs de NPC. Contrairement à ce qui se passe in vitro les miR-BART plasmatiques ne sont pas transportés par des exosomes. Des données obtenues chez la souris montrent qu’ils peuvent être transportés par des complexes extra-cellulaires que l’on peut précipiter au moyen d’anticorps anti-ago2. Nous cherchons à confirmer ces données sur des échantillons de plasma provenant de patients porteurs de NPC. Ces données pourront guider à l’avenir l’utilisation des miR-BART circulants comme source de biomarqueurs.Le deuxième volet de ma thèse avait pour but d’étudier les modifications du protéome des exosomes induites par une oncoprotéine du virus d’Epstein-Barr appelée LMP1 (latent membrane protein 1). J’ai montré que la LMP1, lorsqu’elle est exprimée dans les cellules lymphocytaires ou épithéliales, infectées ou non par EBV, induit la libération de la protéine PARP1 dans le milieu extra-cellulaire. Cette PARP1 extra-cellulaire n’est pas associée aux exosomes ni aux microvésicules mais à des nano-objets non-vésiculaires contenant notamment des histones et de l’ADN. Nous avons désignés ces objets sous le terme de complexes ADN-protéines extra-cellulaires. Nous ne savons presque rien de la biogenèse de ces complexes ; nous pensons qu’ils ne proviennent pas uniquement de cellules en apoptose. En revanche, des expériences préliminaires suggèrent que la présence de PARP1 dans ces complexes coïncide avec une activation permanente de la PARP1 induite dans les cellules productrices par l’expression de l’oncoprotéine LMP1. Cette hypothèse est en cours de vérification grâce à des expériences menées sur des lignées cellulaires exprimant différentes formes sauvages ou mutées de la LMP1. Ces données sur l’activation de la PARP1 et sur sa sécrétion induite par la LMP1 auront des retombées intéressantes pour notre compréhension des mécanismes d’oncogenèse et d’auto-immunité liés à l’infection par le virus d’Epstein-Barr. / The study of tumoral microenvironment should take into account different modes of intercellular communications: direct contacts between extracellular membranes, secretion and uptake of cytokines and finally emission and uptake of complex biological objects like exosomes and microvesicles.Epstein-Barr virus (EBV) is associated with several human malignancies of epithelial origin (Nasopharyngeal carcinoma or NPC) or of lymphoïd origin (post-transplant lymphoproliferative disorder or PTLD). In these tumors, malignant cells are latently infected by EBV and release exosomes and microvesicles containing viral nucleic acids and proteins. Studying them will enable a better understanding of tumor-host interactions and the discovery of new markers which could be useful for early diagnostic and the follow-up of the disease under treatment.The first aim of this thesis was to study the release by malignant cells of EBV microRNAs belonging to the BART family and their blood diffusion in patients bearing NPC tumors. For the first time, I’ve shown that exosomes released by NPC cells in vitro contain EBV miR-BART microRNAs. Moreover, ebv-miR-BART7 can be detected in the plasma of NPC patients. Unlike what is observed in vitro, circulating BART microRNAs are not carried by exosomes. Recent data from studies in xenografted mice show that they are carried by extra-cellular complexes which can be immunoprecipitated by anti-Ago2 antibodies. We are currently trying to confirm these data in plasma from NPC patients. This work will ease the use of miR-BARTs as potential biomarkers.The second aim was to study the proteome modifications induced by the EBV Latent Membrane Protein 1 protein (LMP1). I’ve shown that LMP1 expression in lymphoid or epithelial cells infected or not by EBV induces the release of PARP1 in the extra-cellular space. This extra-cellular PARP1 is not carried by exosomes or microvesicles but is embedded in non-vesicular nano-objects containing histones and DNA. We have called these objects “DNA-proteins complexes”. We don’t know how they are produced and released by cells. We think that they are not only secreted by apoptotic cells. Recent data show that this release of extra-cellular PARP1 is associated with PARP1 activation by LMP1 oncoprotein expression. We are trying to prove this hypothesis using cell lines expressing wild type or mutated LMP1. The release and the activation of PARP1 induced by LMP1 expression will help to understand the mechanisms of EBV-associated oncogenesis and auto-immunity.

La fibrose radio-induite est une complication sévère et tardive de la radiothérapie. Plusieurs études cliniques ont montré que la combinaison pentoxifylline-vitamine E est un traitement sûr et efficace contre la fibrose. Cependant, les mécanismes moléculaires de son efficacité restent inexplorés. Nous avons montré l’efficacité de la combinaison pentoxifylline-vitamine E dans l’entéropathie radique dans une faisabilité clinique. En parallèle, en utilisant un modèle unique, in vitro, de cellules musculaires lisses intestinales primaires isolées des personnes atteintes de l’entéropathie radique, nous avons montré une synergie entre la pentoxifylline et l’analogue hydrophile de vitamine E (trolox) qui permet à l’association d’inhiber l’expression de TGF-β1 au niveau de l’ARN messager et de la protéine. Cette action inhibitrice intervient au niveau transcriptionnel et conduit à une inhibition conséquente des cibles de la voie de signalisation TGF-β1/Smad (Col Iα1, FN1, PAI-1, CTGF), alors qu’elle semble sans effet sur la voie de signalisation Rho/ROCK. Pour la première fois, dans ces cellules issues de l’entéropathie radique, nous avons montré une surexpression de miR-210 ; microRNA induit par l’hypoxie. L’association pentoxifylline-trolox inverse la surexpression de miR-210 aussi bien dans les conditions normoxique que dans les conditions hypoxiques. L’implication de miR-210 dans l’entéropathie radique n’a pas été préalablement étudiée, néanmoins nous avons montré qu’un inhibiteur de miR-210 diminue l’expression de Col Iα1 dans ce modèle. L’effet anti-fibrosant exercé par l’association pentoxifylline-vitamine E est partiellement induit par l’inhibition de la cascade TGF-β1. L’inhibition de miR-210 est un deuxième mécanisme potentiel nécessitant d’autres investigations. Cette étude renforce les essais clinique antérieurs en montrant in vitro une synergie entre pentoxifylline et vitamine E et permettant de proposer cette association en première ligne thérapeutique dans la fibrose radio-induite. De plus, miR-210 est proposé comme une possible cible thérapeutique pour traiter la fibrose radio-induite. / Radiation-induced fibrosis is a serious late complication of radiotherapy. Pentoxifylline-vitamin E has proven effective and safe in clinical trials as treatment of fibrosis, while the molecular mechanism of its activity is yet unexplored. We showed efficacy of Pentoxifylline-vitamin E combination in radiation-induced enteropathy in a small clinical study. In parallel, using a unique in vitro model of primary smooth muscle cells isolated from intestinal samples isolated from humans with radiation enteropathy we showed that pentoxifylline and the hydrophilic analogous of vitamin E (trolox) synergize to inhibit TGF-β1 protein and mRNA expression. This inhibitory action is mediated at the transcriptional level and leads to subsequent inhibition of TGF-β1/Smad targets (Col Iα1, FN1, PAI-1, CTGF), while it has no effect on the Rho/Rock pathway. We have also demonstrated, for the first time, an overexpression of the hypoxia-induced microRNA miR-210 in the fibrotic cells. Pentoxifylline-trolox combination could reverse this miR-210 overexpression in normoxic and hypoxic conditions. While miR-210 has not been previously shown to be involved in radiation-induced enteropathy, we showed that miR-210 inhibitor could reduce mRNA expression of Col Iα1. The anti-fibrotic effect of combined pentoxifylline-vitamin E is at least in part mediated by inhibition of the TGF-β1 cascade. MiR-210 inhibition is another mechanism which needs further investigations. This study strengthens previous clinical data showing pentoxifylline-vitamin E synergy and supports its use as a first-line treatment of radiation-induced fibrosis. Also, it suggests miR-210 as a new potential therapeutic target for the treatment of this complication.

Avaliamos o efeito da RCIU induzida por glicocorticoides sobre a regulação da expressão de miRNAs no fígado de ratos albinos. Animais expostos intrauterinamente à dexametasona apresentaram menor peso ao nascer, fígados relativamente menores dos que os observados em animais controle e menores concentrações hepáticas de PCNA. Em longo prazo, os animais DEX desenvolveram distúrbios metabólicos caracterizados por intolerância à glicose e maior potencial gliconeogênico no desmame e na vida adulta. Ao avaliarmos o perfil de miRNAs no fígado, detectamos aumento da expressão de todo o cluster do miR-322 no período perinatal, com menor conteúdo de alvos preditos desses transcritos (AKT3, CCND1 e INSR). A superexposição ao miR-322-5P reduz a taxa de proliferação em linhagens celulares de hepatocarcinoma e a expressão dos alvos observados no fígado dos animais estudados. Propomos um link entre a expressão aberrante do miR-322-5P e a reduzida taxa de proliferação detectada no tecido hepático em desenvolvimento, contribuindo para o estabelecimento do fenótipo em longo prazo. / We evaluated the effects of glucocorticoid induced IUGR on the expression of miRNA on Wistar rat livers. Dexamethasone (DEX) treated animals were lighter and had smaller liver weight:body weight ratio when compared to control animals. Furthermore, liver PCNA expression was downregulated, sugesting a reduction on cell proliferation rate. In the long term, DEX animals developed metabolic disturbances such as glucose intolerance e increased gluconeogenesis rate in a fast state. The analysis of miRNA expression profile showed an upregulation of miR-322 cluster on perinatal period, together with a downregulation of three putative targets: Akt3, CCND1 and INSR. Later, we used in vitro studies to prove that overexpression of miR-322-5P arrests cell cycle and impairs proliferation of HEPG2 cells as well as it downregulates the predicted targets. Based on this data, we suggest a link between overexpression of miR-322-5P and impaired proliferation rate on the developing liver, which affects the phenotype in the long term.