La découverte récente d'adipocytes bruns fonctionnels chez les humains adultes a conduit à envisager leur utilisation afin d’augmenter la dépense énergétique dans de potentiels traitements contre l'obésité et les maladies associées. Par ailleurs, des ilots d’adipocytes bruns, appelés adipocytes "brite" (brown in white), émergent dans le tissu adipeux blanc après une exposition au froid ou une stimulation des récepteurs β3-adrénergiques. En utilisant les cellules hMADS, nous avons identifié plusieurs miARNs régulés pendant le « britening ». miR-125b et let-7i ont des niveaux d’expression plus bas dans les adipocytes « brites ». Des analyses fonctionnelles utilisant un « mimic » de miR-125b ou un inhibiteur ont révélé que miR-125b agit comme un frein sur le « brunissage » des cellules hMADS en altérant leur respiration ainsi que leur contenu mitochondrial. In vivo, nous avons montré que miR-125b et let-7i sont moins exprimés dans le tissu adipeux brun par rapport au tissu adipeux blanc. La stimulation des récepteurs β3-adrénergiques ou l'exposition au froid induit une diminution d’expression des miARNs dans les deux tissus et est associée à l'activation du tissu adipeux brun et au recrutement des adipocytes « brites ». Nous avons constaté que l’injection de miR-125b ou let-7i dans le tissu adipeux blanc sous-cutané inhibait l’expression de gènes du « brunissage » induite par la stimulation de la voie β3-adrénergique. En conclusion, nos observations ont montré que miR-125b et let-7i jouaient un rôle important dans la modulation des adipocytes « brites » et des adipocytes « bruns » en ciblant l’expression de gènes mitochondriaux et en diminuant la biogenèse mitochondriale. / The recent discovery of functional brown adipocytes in adult humans has led to the consideration of their use to increase energy expenditure in the treatment of obesity and associated metabolic disorders. Furthermore, in rodents and humans, islands of thermogenic adipocytes, termed “brite” (brown in white) adipocytes, emerge within white adipose tissue after cold exposure or β3-adrenergic receptor stimulation. Using hMADS cells, we identified several miRNAs regulated during “britening” including miR-125b and let-7i which showed lower levels in brite adipocytes. Functional analysis using miR-125b mimic or miR-125b inhibitor transfection revealed that miR-125b-5p acts as a brake of the browning of hMADS cells by impairing respiration rate as well as their mitochondrial content. miR-125b and let-7i levels were lower in brown compared to white adipose tissue. In vivo, we showed that both miRNAs levels were down regulated in mice sub-cutaneous white and brown adipose tissues upon β3-adrenergic receptors stimulation or cold exposure, which is associated with BAT activation and brite adipocyte recruitment. We found that injection of both miRNA mimics in subcutaneous white adipose tissue inhibited β3-adrenergic-induced brown adipocyte markers expression. Altogether, our observations showed that miR-125b and let-7i played an important role in the modulation of brite and brown adipocytes function targeting oxygen consumption and mitochondrial gene expression.

Plus de 90% des érythroleucémies induites par le virus de Friend sont associées à l'activation récurrente de l’un ou l’autre des facteurs de transcription ETS Spi-1 ou Fli-1, ou du cluster miR-17-92. La contribution de ces trois oncogènes à la prolifération des clones érythroleucémiques correspondant a déjà été indépendamment démontrée. De plus, il a été montré dans l’équipe que Spi-1 active de façon directe l’expression de Fli-1 et que les deux facteurs activent des gènes cibles communs. Dans cette thèse, j’ai montré que Spi-1 et Fli-1 activent l’expression du cluster miR-17-92 en se liant sur un motif ETS conservé dans le promoteur de ce cluster. J’ai montré que la réexpression de miR-17 et miR-20a est suffisante pour contrebalancer partiellement la baisse de la prolifération et la survie cellulaire induite par l’inhibition de l’expression de Fli-1. De plus, j’ai identifié Hbp1 comme une cible de ces miARNs dans les cellules érythroleucémiques. Ces résultats montrent que les trois oncogènes activés de façon récurrente par le virus de Friend constituent un seul réseau oncogénique contrôlant la prolifération. Ces résultats suggèrent également un rôle potentiel de réseaux ETS-miR-17-92 dans d’autres contextes normaux ou pathologiques / Clonal erythroleudemia developing in susceptible mice infected by Friend virus complex are associated with higly recurrent orviral insertinons at one of three loci called Spi-1, Fli-1 or Fli-3, leading to deregulated expression of oncogenic Spi-1 or Fli-1 transcription factors or miR-17-92 miRNA cluster, respectively. Deregulated expression of each of these here ocongenes has been independently shown to contribute to cell profileration of erythroleukemic clones. Previous studies showed close relationship between Spi-1 and Fli-1, which belong te the seame ETS family, Spi-1 activating fli-1 gene and both Spi-1 and Fli-1 activating multiple common target genes involved in ribosome biogenesis. In this tehesis, we habe also demonstrated that physiological re-expression of exogenous miR-17 and MiR-20a are able to partially rescue proliferation arrest induced by Fli-1 knock down and we identified Hbp1 as a tarteg of these miRNAs in erythroleukemia cell line.These results establish that three of the most recurrently activated oncogenes in Friend erythroleukemia are arctually involved in the same oncogenic network controlling proliferation . The putative contribution of similar ETS-MiR-17-92 network in other normal or hyper

LRP1 (low-density lipoprotein receptor–related protein 1), un récepteur endocytaire multifonctionnel, a récemment été identifié comme pivot d’un réseau de biomarqueurs pour la prédiction pronostique de plusieurs types de cancers. Son rôle dans le cancer du côlon n'a pas été caractérisé. Notre travail porte sur l’étude de la relation entre expression de LRP1 et cancer du côlon.L'expression de l'ARNm LRP1 a été déterminée dans des échantillons d'adénocarcinome et de muqueuses coliques appariées, ainsi que dans les cellules stromales et tumorales obtenues après microdissection laser. Les associations clinicopathologiques et moléculaires ont été étudiées par immunohistochimie dans une série de cancer colique (n = 307). La présence de méthylation ou mutation du gène LRP1 et l'expression de miR-205 ont été évaluées et comparées aux niveaux d'expression de LRP1.L’ARNm de LRP1 est sous exprimé dans les cellules d'adénocarcinome colique par rapport à la muqueuse colique par rapport aux cellules stromales. La faible expression immunohistochimique de LRP1 dans les adénocarcinomes était associée à un âge plus élevé, à localisation droite, une perte d'expression de CDX2, une expression d'Annexine A10, un statut CIMP-H, MSI-H et BRAFV600E muté. Cette faible expression était associée à un mauvais pronostic, en particulier chez les patients de stade IV. Les mutations du gène LRP1 entrainaient une sous-expression de LRP1. L’expression était peu modifiée par miR-205. Le promoteur de LRP1 n'était jamais méthylé.La perte d'expression de LRP1 est associée à un profil clinico-pathologique et moléculaire particulier et à un un mauvais pronostic dans les cancers du côlon. / LRP1 (low-density lipoprotein receptor–related protein 1), a multifunctional endocytic receptor, has recently been identified as a hub in a biomarker network for multi-cancer clinical outcome prediction. Its role in côlon cancer has not been characterized. Here, we investigate the relationship between LRP1 and colon cancer.LRP1 mRNA expression was determined in colon adenocarcinoma and paired colon mucosa samples, and in stromal and tumoral cells obtained after laser capture microdissection. The clinical potential was further investigated by immunohistochemistry in a population-based colon cancer series (n = 307). LRP1 methylation, mutation and miR-205 expression were evaluated and compared to LRP1 expression levels.LRP1 mRNA levels are significantly decreased in colon adenocarcinoma cells compared to colon mucosa and stromal cells. Low LRP1 immunohistochemical expression in adenocarcinomas was associated with higher age, right-sided tumor, loss of CDX2 expression, Annexin A10 expression, CIMP-H, MSI-H and BRAFV600E mutation. Low LRP1 expression correlates with poor clinical outcome, especially in stage IV patients. LRP1 expression was downregulated by LRP1 mutation. LRP1 expression was slightly modified by miR-205 expression. LRP1 promoter was never methylated.Loss of LRP1 expression is associated with peculiar clinocopathological and molecular characteristics and with worse colon cancer outcomes.

Les dystrophies myotoniques sont les formes les plus communes des dystrophies musculaires chez l’adulte, caractérisées par de nombreux symptômes tels que les défauts de conduction et de rythme cardiaques fatals chez 30% des patients DM, dont les causes moléculaires sont inconnues. Les DM sont des maladies à gain de fonction d’ARN faisant intervenir une séquestration des facteurs d’épissage alternatif MBNL par des ARNs contenant de longues répétitions (C)CUG, conduisant à des altérations de l’épissage alternatif. Par des approches de puces à ADN, nous avons identifié et confirmé la diminution spécifique de miR-1, conduisant à la dérégulation de l’expression de la connexine 43 et du canal à calcium cardiaque Cav1.2 dans le coeur de patients DM. Par séquençage à haut débit d’ARNs de cœur de patients atteints ou non de DM, j’ai montré la dérégulation de plus d’une centaine d’épissages alternatifs dont celui des exons 6A/6B du principal canal à sodium cardiaque, SCN5A. J’ai montré que cet épissage est régulé par MBNL, et j’ai confirmé l’inclusion anormale de l’exon 6A à la place de l’exon 6B dans l’ARNm SCN5A conduisant à une diminution de l’activité du canal SCN5A, pouvant expliquer les défauts cardiaques des patients DM. / The myotonic dystrophies (DM) are the most common forms of muscular dystrophies in adults, characterized by several symptoms such as cardiac conduction defects and arrhythmias, fatals in 30% of DM patients. The molecular causes of DM cardiac defects are unknown. The RNA gain of function involving a sequestration of MBNL, alternatives splicing factors by large RNAs containing large (C)CUG, leading to alternative splicing defects. By microarray analysis, we identified and confirmed the specific decrease of miR-1, leading to misregulation of connexin 43 and cardiac calcium channel Cav1.2 expressions in DM patients’ hearts. By RNA-Sequencing of samples from DM and non-DM patients hearts, we have shown misregulation of more than 100 alternative splicing, such as the most interesting splicing alteration which is that of exons 6A/6B of SCN5A, the maincardiac sodium channel. We have shown this splicing is regulated by MBNL, and we have confirmed the abnormal inclusion of exon 6A instead of exon 6B in SCN5A mRNA in heart of DM patients, resulting in the decrease of SCN5A channel activity. This decrease could explain the cardiac defects of DM patients.

Splicing é o processo de remoção de introns e ligação de exons em eucariotos. É realizado pelo spliceossomo, um complexo macromolecular composto por RNAs e mais de cem proteínas. Alguns introns possuem microRNAs, os quais devem ser processados para gerar moléculas maduras. O cluster intrônico miR-17-92 é composto por sete miRNAs que têm sido associados ao desenvolvimento de diferentes tumores em vários tecidos. Neste trabalho o splicing de dois miRNAs deste cluster foi analisado em células HeLa, BCPAP e TPC-I. Os resultados mostraram que introns com miR19a tem o splicing mais eficiente do que aqueles com miR18a em todas as três células analisadas. Além disso, a composição dos spliceossomos foi analisada por espectrometria de massas. Entre as principais proteínas encontradas, destaca-se a presença das hnRNPs, como hnRNP_A1 e hnRNP_A2/B1. Estes resultados são importantes para entender como esses miRNAs são processados, e quais são os principais componentes recrutados em diferentes tipos celulares. / Pre-mRNA splicing is the process of intron removal and exon ligation in eukaryotes. It is performed by the spliceosome, a multi-megadalton machinery composed of RNAs and more than a hundred proteins. Intronic miRNAs must be processed from the host gene to generate mature molecules. miR-17-92 is an intronic cluster composed of seven miRNAs which have been associated to the development of different tumors, in several different cells. In this work, we analyzed the splicing of two miRNAs belonging to this cluster in HeLa, BCPAP and TPC-I cells. Interestingly, we observed miR19a is more efficiently spliced than miR18a in all three cells. We also searched for specific proteins that can be involved in their respective splicing process. We observed hnRNP proteins are especially concentrated in spliceosomes assembled in introns containing these miRNAs, based on mass spectrometry data. These results are important to understand how these miRNAs are spliced and matured and also can explain their different expression levels in different cells.

Durante a divisão celular as Aurora-quinases (AURKA e AURKB) participam da formação e controle das fibras do fuso mitótico enquanto as isoformas proteicas (DIDO1, DIDO2 e DIDO3), originadas do splicing alternativo do gene DIDO, auxiliam na junção dos microtúbulos aos cinetócoros. Portanto, ambas são relevantes na regulação do ciclo celular. Interessantemente, a superexpressão (ou o ganho de função) das AURKs ou a baixa expressão (ou perda de função) das isoformas de DIDO estão ambos associados com amplificação dos centrossomos e à instabilidade cromossômica (CIN), com consequente aneuploidia. Dentre as doenças hematológicas com registros de CIN, a leucemia linfoide crônica (LLC) pode apresentar amplificação dos centrossomos e alteração nos níveis de expressão das AURKs acarretando aneuplodias. Apesar disso, não existem estudos avaliando a potencial associação destes genes com CIN na LLC. Avaliando seus níveis de expressão gênica em amostras de LLC de pacientes com ou sem aberrações cromossômicas, mostramos que o aumento dos níveis de AURKA e AURKB e, inversamente, a redução dos níveis das variantes de DIDO, são significativamente associados com ganhos cromossômicos e com aumento da contagem de glóbulos brancos (WBC). Claramente, amostras de LLC sem qualquer anormalidade citogenética apresentam níveis de expressão semelhantes às amostras que contêm aberrações não-numéricas. O achado de que níveis de expressão de AURKs e variantes de DIDO são completamente opostos, mostrando um padrão discreto de inter-relação, levou-nos a investigar o potencial mecanismo regulatório por trás disso. Tendo em vista que outros, anteriormente, mostraram que o cluster oncogênico miR-17~92 é significativamente hiper-regulado em células de pacientes com LLC purificadas expressando genes IGHV não mutados (em comparação com células mutadas de pacientes) e, que o miR-17 é expresso em níveis significativamente mais elevados em células IGHV não mutadas ou ZAP-70 positivas (mau prognóstico geralmente associada à CIN), resolvemos investigar o potencial de regulação negativa dos microRNAs deste cluster sobre as variantes de DIDO. Além disso, com base no mecanismo regulatório já descrito pelo qual a superexpressão de AURKA induz a transcrição do cluster miR-17~92, mediada por E2F1 (com uma correlação entre as expressões de ambas as proteínas em diferentes tipos de câncer), decidimos investigar este eixo regulatório em LLC. Notavelmente, todas as variantes de DIDO apresentam-se preditas como fortes alvos de vários microRNAs deste cluster oncogênico. Mostramos, então, que amostras de LLC com baixa expressão de DIDO, além dos já mencionados níveis elevados de AURK, exibiram níveis significativamente mais elevados do fator de transcrição E2F1 e de seu alvo transcricional, o transcrito primário do miR-17~92 (MIR17HG). Além disso, por meio do uso da linhagem de celular NTERA-2, como modelo experimental, mostramos que o siRNA nocaute para AURKA (nos níveis transcricional e proteico, como confirmado por qPCR e western blot) é acompanhada por uma significativa redução de E2F1 e também de MIR17HG. Ainda, a transfecção de células NTERA-2 com sintéticos microRNAs miméticos do cluster miR-17~92 (ou seja, 19a-miR, miR-20a e miR-92a) resultou em uma clara e significativa redução dos níveis de transcrição de todas as variantes de DIDO. Por fim, a inibição do siRNA especifico para a variante DIDO3 (mas não às outras variantes) levou a uma redução significativa dos níveis de transcrição de todas as variantes de DIDO, indicando um mecanismo adicional contribuindo para a downregulação dos transcritos de DIDO. Ao todo, nossos resultados demonstram a existência de um potencial mecanismo regulatório interconectado entre AURK e DIDO, associado à CIN e maior contagem de WBC na LLC. Mais importante, os níveis de expressão elevada de AURKs e os baixos níveis associados das variantes de DIDO são especificamente relacionados com anormalidades citogenéticas apresentando ganhos cromossomais, com destaque para o mecanismo celular específico, subjacente à CIN, observado neste grupo distinto LLC. Dado o papel central da CIN na gênese e progressão do câncer, esses achados provavelmente terão um impacto importante no prognóstico ou tratamento da LLC. / During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17~92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17~92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17~92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-04T18:03:22Z No. of bitstreams: 1 juliatristaodocarmorocha.pdf: 2765026 bytes, checksum: 7a9e6fcc24e70e565d8b382aeff776df (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T13:33:05Z (GMT) No. of bitstreams: 1 juliatristaodocarmorocha.pdf: 2765026 bytes, checksum: 7a9e6fcc24e70e565d8b382aeff776df (MD5) / Made available in DSpace on 2017-05-17T13:33:05Z (GMT). No. of bitstreams: 1 juliatristaodocarmorocha.pdf: 2765026 bytes, checksum: 7a9e6fcc24e70e565d8b382aeff776df (MD5) Previous issue date: 2016-05-25 / Os produtos petrolíferos em geral são altamente complexos e é exigido um esforço considerável para a caracterização de suas propriedades químicas e físicas. Às vezes tem-se urgência no resultado de determinadas análises e isto fica prejudicado pela forma como as análises são feitas. Assim, a quimiometria, associada à espectroscopia molecular (NIR e MIR em particular) vem gerando métodos alternativos para a caracterização e avaliação de propriedades físicas e químicas de petróleos e seus derivados com elevada exatidão, confiabilidade e rapidez. Para melhorar o desempenho previsor têm sido utilizados procedimentos apropriados para a seleção das regiões espectrais associadas com a propriedade de interesse. Desta forma, face às suas aplicabilidades, foi proposto neste trabalho a utilização das ferramentas quimiométricas com seleção de variáveis (método dos mínimos quadrados parciais por intervalos, iPLS, e por sinergismo de intervalos, siPLS; método de eliminação de variáveis não informativas por mínimos quadrados parciais, UVE; e algoritmo genético, GA), associada ao MIR e ao NIR, para a determinação das seguintes propriedades em frações de petróleo: Grau API, Índice de cetano, Índice de refração (a 20°C), Teor de Enxofre (%m/m), Ponto de fuligem (mm), Ponto de anilina (°C), Ponto de congelamento (°C), Ponto de entupimento (°C), Ponto de névoa (°C) e Ponto de fluidez (°C), avaliando, assim, a performance dos modelos obtidos, bem como as técnicas utilizadas na seleção de variáveis. Essa avaliação se deu pela determinação e análise do coeficiente de determinação (R2), de diversos erros calculados para os conjuntos de calibração e previsão. Os modelos foram, ainda, submetidos a testes estatísticos (α=0,05), e tiveram suas figuras de mérito calculadas. Os melhores modelos para a previsão do Grau API e do ponto de névoa foram criados aplicando-se iPLS a dados de MIR, enquanto que para a previsão do teor de enxofre e pontos de refração, de fuligem e de anilina foram criados aplicando-se siPLS também ao MIR. Já para a previsão do índice de cetano e do teor de enxofre e do ponto de entupimento, os melhores modelos foram criados aplicando-se iPLS a dados de NIR. Nesse contexto, o melhor modelo para a predição do ponto de fluidez foi o GA. Finalmente, para a previsão do ponto de congelamento, nenhum método de seleção de variáveis melhorou a capacidade preditiva, quando comparados ao modelo criado aplicando-se PLS a dados de MIR. Dessa forma, conclui-se que houve um melhor desempenho dos modelos criados a partir de dados de MIR. Quanto aos métodos de seleção de variáveis, iPLS e siPLS obtiveram o melhor desempenho. / Petroleum products are, in general, highly complex and a considerable effort is needed to characterize their chemical and physical properties, though sometimes the results of several analyses are urgent and this is compromised by the way the analyses are carried out. Thus, chemometrics associated with molecular spectroscopy (particularly NIR and MIR) has good potential as a tool in analytical chemistry, creating alternative methods to characterize and evaluate physical and chemical properties of petroleum and its derivates with high precision, reliability and rapidity. To improve the predictor performance, appropriate procedures are being used to select spectral regions associated with the property of interest. In face of their applicabilities, this work proposes the use of chemometric tools, with variable selection (Interval Partial Least Square, iPLS and Sinergism Interval Partial Least Square, siPLS; Elimination of Uninformative Variables, UVE and Genetic Algorithm, GA), associated with mid infrared (MIR) and near infrared (NIR) spectroscopies to determine the following properties in petroleum fractions: API gravity, Cetane index, Refractive index (at 20°C), Sulfur content (%m/m), Smoke point (mm), Aniline point (°C), Freezing point (°C), Plugging point (°C), Cloud point (°C) and Pour point (°C), enabling the evaluation of performance of the obtained models, as well as the techniques used in variable selection. This evaluation was performed by determination and analyses of the following requirements: coefficient of determination (R2), several calculated errors for the calibration and prediction set. The models were also subjected to statistical tests (α=0,05), and the figures of merit were calculated. The best models to predict API gravity and cloud point were created by applying iPLS to the MIR data, whereas for prediction of sulfur content, refractive index, and smoke and aniline points the models were created by applying iPLS to NIR data. In this context, the best model to predict the pour point was the GA. Finally, to predict freezing point, none of the variable selection methods improved the predictive capability when comparing to the model created using only PLS in MIR data. Thus, the conclusion is that a better performance was obtained for the models created from MIR data. Regarding efficiency of variable selection methods, the iPLS and siPLS methods resulted in a best performance.

Les microARNs sont de petits ARN non codants régulant négativement l'expression génique au niveau post-transcriptionnel. Ils interviennent dans de nombreux processus biologiques et leur rôle dans la régulation de l'homéostasie cutanée est clairement démontrée. Cependant, leur fonction durant le vieillissement épidermique n'a jamais été étudiée. Nous avons donc réalisé une analyse exhaustive du miRnome épidermique durant son vieillissement afin d'identifier les microARNs différentiellement exprimés avec l'âge dans ce tissu. Plusieurs microARNs significativement modulés dans des kératinocytes âgés, nous ont permis d'établir une signature du vieillissement épidermique. Parmi eux, les deux brins du microARN miR-30a sont induits dans les épidermes âgés. La construction d'un lentivirus permettant la surexpression stable et inductible de ce microARN a facilité son étude fonctionnelle dans un modèle organotypique d'épiderme reconstruit. Nous avons observé que la surexpression de ce microARN dans un modèle de culture tridimensionnelle induit un phénotype épidermique présentant des similitudes avec celui observé durant son vieillissement chronologique et caractérisé par une forte altération de la différenciation des kératinocytes, par une perturbation de sa fonction barrière et par une augmentation de l'abondance des cellules apoptotiques. Ce projet de thèse a permis l'identification de 3 cibles directes de miR-30a dans les kératinocytes. Il s'agit de LOX, codant pour la lysyl oxydase qui intervient dans la balance prolifération/différenciation des kératinocytes, d'AVEN, un inhibiteur de caspase et d'IDH1, codant pour l'isocitrate déshydrogénase, enzyme du métabolisme énergétique. Ainsi, ce projet de thèse a révélé un nouveau microARN acteur du vieillissement épidermique et a permis de de mettre à jour de nouveaux mécanismes moléculaires expliquant certaines altérations phénotypiques observées dans l'épiderme avec l'âge / MicroRNAs are small non-coding RNA that negatively regulate gene expression at the post-transcriptional level. There are involved in many biological processes and play a key role in the regulation of skin homeostasis. However, their function during epidermal aging has never been studied. We performed an exhaustive analysis of the epidermal miRnome during its aging in order to identify microRNAs differentially expressed with age in this tissue. Several microRNAs significantly modulated in elderly keratinocytes, allowed us to establish a signature of epidermal aging. Among them, the two strands of the microRNA miR-30a are induced in aged epidermis. The construction of a lentivirus allowing inducible and stable overexpression of this microRNA facilitated its functional study in an organotypic model of reconstructed epidermis. We observed that the overexpression of this microRNA in a three-dimensional culture model induces an epidermal phenotype similar of those observed during its chronological aging characterized by a strong alteration of keratinocyte differentiation, by a disturbance of its barrier function and by an increase in the abundance of apoptotic cells. This thesis project allowed the identification of three miR-30a targets in keratinocytes : LOX encoding lysyl oxidase, which plays a role in proliferation/differentiation balance of keratinocytes, AVEN encoding a caspase inhibitor and IDH1 encoding isocitrate deshydrogenase, a key enzyme of cellular metabolism.Our work revealed a new miRNA actor and deciphered new molecular mechanisms to explain some alterations observed in epidermis during aging, especially those concerning keratinocytes differentiation and apoptotic death

Toute maladie rénale chronique (MRC) se caractérise par le développement de lésions de fibrose rénale et d’une perte de fonction qui peut, à terme conduire vers l’insuffisance rénale chronique terminale. miR-21-5p est un microARN ubiquitaire impliqué dans le processus de fibrose, notamment rénale. Toutefois, des données expérimentales contradictoires suggèrent que miR-21-5p joue un rôle ambivalent dans la constitution des lésions rénales de fibrose.L’objectif de ce travail est de préciser l’implication de miR-21 au cours des lésions rénales aigües, en s’appuyant sur un modèle murin de toxicité rénale du cisplatine, ou chroniques chez l’homme au cours de la néphropahtie à dépôts mésangiaux d’IgA (maladie de Berger).Dans un premier travail, une cohorte rétrospective de patients porteurs d’une néphropathie à dépôts mésangiaux d’IgA a été caractérisée de façon systématique sur le plan clinico-biologique et anatomo-pathologique (classification d’Oxford). L’expression rénale de trois fibromiR (miR-21-5p, miR-199a-5p et miR-214-3p) est associée aux lésions de fibrose rénale (p ≤ 0,02). Parmi ces microARN, miR-21-5p semble le plus pertinent car il présente des amplitudes de variation d’expression plus importantes selon le niveau de fibrose, il est également associé aux lésions de sclérose glomérulaire (p = 0,001) et son niveau d’expression tissulaire rénale est associée à une moins bonne survie rénale.Un second travail a été mené en utilisant un modèle murin d’insuffisance rénale aiguë secondaire à l’injection intra-péritonéale de cisplatine chez des animaux invalidé pour miR-21a-5p. Deux schémas d’injections ont été réalisés afin explorer le rôle de miR-21a-5p au cours de lésions rénales aiguës (injection d’une dose unique de 10mg/kg de cisplatine) ou subaiguës (injections répétées de 7 mg/kg de cisplatine). Les souris ayant reçu une injection unique de cisplatine ne présentent pas de différence significative d’urée sanguine, de marqueurs de souffrance rénale (NGAL, KIM-1), d’inflammation (TNF-α, IL-6) et de stress oxydant (HO-1, NRF2), ni d’activité apoptotique selon leur statut sauvage ou invalidé pour miR-21a-5p. Le modèle d’injections répétées de cisplatine a, quant à lui, permis de mettre en évidence des lésions plus importantes chez les souris miR-21-/-. En effet, les souris miR-21-/- cisplatine présentent une urée sanguine plus élevée (1,92 g/l ±, 0,72 versus 0,66 g/l ± 0,15 p = 0,014) et une expression rénale de NGAL plus importante (RQ = 118,1 ± 44,8 versus RQ = 45,4 ± 37,7, p= 0,018) que le souris sauvages cisplatine. Enfin les lésions rénales de nécrose tubulaire aiguë observées sont plus sévères chez les souris miR-21-/-.Ainsi ces résultats montrent qu’une forte expression rénale de miR-21-5p est associée à la fibrose et au pronostic rénal chez des patients porteurs d’une néphropathie à dépôts mésangiaux d’IgA. Dans notre modèle expérimental, les souris déficientes pour miR-21a-5p présentent une sensibilité variable au développement de lésions rénales induites par le cisplatine en fonction du type d’administration, aigu ou subaigu. Ces résultats confirment que miR-21-5p joue un rôle ambivalent au cours des lésions rénales, protecteur à la phase précoce d’une agression rénale aiguë et délétère lorsque le processus se prolonge dans le temps. miR-21-5p est présent dans les fluides biologiques, et constitue ainsi un candidat potentiel en tant que biomarqueur de la fibrose rénale. De plus, miR-21-5p constitue une cible thérapeutique innovante, ayant montré son efficacité dans différents modèles murins de fibrose rénale. / Independently of the cause, active CKD leads to the development of fibrotic lesions, responsible for a loss of renal function and ultimately, end-stage renal failure. MiR-21-5p is a ubiquitous microRNA involved in the process of fibrosis, especially renal fibrosis. However, contradictory experimental data suggest that miR-21-5p plays an ambivalent role in the regulation of renal fibrosis.The aim of this work was to investigate the involvement of miR-21 in chronic renal lesions based on human renal samples and in acute lesions by using a murine model of renal toxicity induced by cisplatin.In a first part of the work, a retrospective cohort of patients with IgA nephropathy has been systematically characterized clinically, biologically and pathologically (according to Oxford classification). The renal expression of three FibromiRs (miR-21-5p, miR-199a-5p and miR-214-3p) is associated with renal fibrosis lesions (p ≤ 0.02). Among these microRNAs, miR-21 appears to be the most relevant as it displayed larger amplitudes of variation, it was also associated with glomerular sclerosis (p = 0.001) and its strong expression was associated with lower renal survival.A second part of the work was carried out on a murine model of acute renal failure secondary to the intraperitoneal injection of cisplatin. Two injections schemes were established to investigate the role of miR-21-5p in acute renal lesions (injection of a single dose of 10 mg/kg cisplatin) or subacute (repeated injections of 7 mg/kg cisplatin). After a single injection of cisplatin, no significant difference in blood urea, renal (NGAL, KIM-1), inflammation (TNF-α, IL-6) and oxidative stress (HO-1, NRF2) nor apoptotic activity was observed in miR21-/- mice compared to wild-type mice. In a model of repeated injections of cisplatin, we observed more renal lesions in miR-21-/- mice. Indeed, miR-21-/- mice treated with cisplatin exhibited higher blood urea (1.92 g / l ±, 0.72 versus 0.66 g / l ± 0.15 p = 0.014) and an increased renal expression of NGAL (RQ = 118.1 ± 44.8 versus RQ = 45.4 ± 37.7, p = 0.018) compared to wild-type mice.Thus, these results demonstrate that an increased renal expression of miR-21-5p is associated with fibrosis and renal prognosis in patients with IgA nephropathy. In an experimental model, of cisplatin-induced renal injury, mice deficient for miR-21a-5p exhibit a higher sensitivity when cisplatin was administered several times. These results confirm that miR-21-5p plays an ambivalent role in renal lesions and seems to be protective at an early stage, or deleterious when the process is prolonged over time. As miR-21-5p is present in biological fluids, it might be an efficient biomarker of renal fibrosis. Moreover, miR-21-5p is an innovative therapeutic target validated in several murine models of renal fibrosis.

Le gène Egfl7 codant une protéine majoritairement sécrétée par les cellules endothéliales a été découvert au sein du laboratoire. Ce gène a la particularité d’héberger dans sa séquence intronique deux microARNs complémentaires nommés miR126-3p et miR-126-5p. Les microARNs sont de petites séquences de 20 à 25 nucléotides régulant l’expression de leurs cibles en se fixant sur leurs ARNm pour induire leur dégradation ou l’inhibition de leur traduction. L’expression endothéliale et les fonctions du microARN miR126-3p (microARN principal du duplex miR126-3p/126-5p) ont déjà été très largement abordées alors que celles de miR126-5p (microARN secondaire du duplex) restent inconnues. Les objectifs de notre étude ont donc été d’établir le patron d’expression de miR-126-5p lors du développement vasculaire et de caractériser ses fonctions dans les cellules endothéliales. Par hybridation in situ, miR-126-5p a été détecté dans les vaisseaux sanguins embryonnaires de souris principalement dans les cellules endothéliales. Cette spécificité endothéliale a été retrouvée dans différents organes tels que le cœur et les poumons et est maintenue in vitro. L’inhibition et la surexpression de miR-126-5p dans des cellules endothéliales veineuses (HUVEC) in vitro n’affectent pas les capacités de prolifération, de migration ou d’organisation en pseudocapillaires de ces cellules. En revanche, l’inhibition de miR-126-5p dans les HUVECs entraine une répression de l’adhérence des leucocytes à la surface d’un tapis de cellules endothéliales ainsi qu’une augmentation de la transmigration de monocytes à travers une monocouche endothéliale. A l’inverse, sa surexpression génère des phénotypes opposés. Des analyses in silico de recherche de cibles pour miR-126-5p en lien avec le recrutement leucocytaire ont permis d’identifier une protéine participant à la transmigration des leucocytes in vitro et in vivo nommée ALCAM. A l’aide de test de transactivation, nous avons pu démontrer que miR-126-5p était capable de se fixer au 3’UTR de l’ARNm d’ALCAM afin de réprimer l’expression de la protéine. De plus, l’augmentation de la transmigration induite par la chute d’expression de miR-126-5p dans les cellules endothéliales est inhibée suite au blocage direct de la protéine ALCAM montrant ainsi que l’effet répresseur de miR-126-5p sur ce mécanisme est établi via ALCAM. Une étude par microarray, réalisée sur des HUVECs où miR-126-5p a été inhibé, a permis d’identifier une seconde cible pour miR-126-5p nommée SetD5 pour laquelle aucune fonction n’est connue à ce jour. Des tests de transactivation ont permis de confirmer que SetD5 était une cible de miR-126-5p. De plus, l’effet de miR-126-5p sur l’adhérence des leucocytes aux cellules endothéliales est directement lié à la modulation d’expression de ce gène. Enfin, l’analyse de l’inhibition de miR-126-5p in vivo a permis de montrer que notre microARN d’intérêt contrôle effectivement les expressions d’ALCAM et de SetD5. Cependant, alors que miR-126-5p régule uniquement l’expression d’ALCAM dans les poumons, celle de SetD5 est sous le contrôle de miR-126-5p dans la rétine.Nos travaux ont donc permis de mettre en évidence l’expression endothéliale de miR-126-5p et d’identifier deux de ses cibles lui permettant de jouer un rôle dans le recrutement des leucocytes au niveau de l’endothélium. / The Egfl7 gene which was identified within the laboratory codes for a protein mainly secreted by endothelial cells. This gene harbors in its intronic sequence two complementary microRNAs named miR-126-3p and miR-126-5p. MicroRNAs are 20-25 nucleotides-long non coding RNAs which repress protein expression through binding to a complementary sequence of their target mRNAs, leading to mRNA degradation or translation inhibition. Endothelial expression and functions of miR-126-3p (The main miRNAs of the miR-126-3p/miR-126-5p duplex) was widely described while those of miR-126-5p remain unknown. The goal of our study was to establish the expression of miR-126-5p during the vascular development and to characterize its functions in endothelial cells. By in situ hybridization, miR-126-5p was detected in mouse embryonic vessels mainly in endothelial cells. This miR-126-5p endothelial specific expression was also found in different organs such as in the heart or lungs and is maintained in vitro. The inhibition and overexpression of miR-126-5p in vein endothelial cells (HUVECs) did not affect HUVECs proliferation, neither their migration nor their ability to form pseudocapillaries in vitro. On the other hand, miR-126-5p inhibition in HUVEC led to a repression of leukocyte adhesion onto endothelial cells as well as an increase of leukocyte transmigration across an endothelial cell monolayer. Interestingly, opposite phenotypes were observed after miR-126-5p overexpression. In silico analyses of miR-126-5p targets led to identify ALCAM as a potential mRNA transcript that could be regulated by miR-126-5p and which was already involved in leukocyte transmigration in vitro and in vivo. By transactivation assays, we showed that miR-126-5p was able to bind the 3’UTR of ALCAM mRNA and to repress ALCAM protein expression. Furthermore, endothelial cell treatment with an ALCAM blocking antibody abolished the effect of miR-126-5p inhibition on leukocyte transmigration indicating that miR-126-5p controls this process via ALCAM. A microarray analysis performed on HUVEC after miR-126-5p inhibition allowed the identification of another target for miR-126-5p named SetD5, a gene with unknown function. Transactivation assays confirmed that SetD5 is a target for miR126-5p. Furthermore, we showed that miR-126-5p controls leukocyte adhesion onto endothelial cells by regulating SetD5 expression. Finally, the inhibition of miR-126-5p in vivo demonstrated that miR-126-5p controls ALCAM and SetD5 expression in mouse. However, while miR-126-5p exclusively regulates ALCAM expression in lungs, SetD5 expression is controlled by miR-126-5p in the retina.In this study, we demonstrated that miR-126-5p is a functional microRNA expressed in endothelial cells. We identified two targets for this microRNA indicating that miR-126-5p participates in the control of leukocyte trafficking onto endothelial cells.