Common, complex disorders are polygenic and multifactorial traits representing interactions between environmental, genetic and epigenetic risk factors. More often than not, contributions of these risk factors have been studied individually and this is especially true for complex reproductive traits where application of genomic technologies has been challenging and slow to progress. This thesis explores the potential of genetic and epigenetic components contributing to a better understanding of the biological pathways underlying disease risk in two specific female complex reproductive traits - polycystic ovary syndrome (PCOS) and preterm premature rupture of membranes (PPROM). The PCOS projects focus on characterization of a gene, DENND1A, whose association to PCOS has been established by Genome Wide Association Studies (GWAS) and is known to contribute to PCOS steroidogenic phenotype. In addition, differential microRNAs expression contributing to DENND1A expression regulation in PCOS theca cells was identified. The studies on PPROM utilize a Whole Exome Sequencing approach to identify rare variants in fetal genes contributing to extracellular matrix composition and synthesis contributing to PPROM risk. The results suggest that fetal contribution to PPROM is polygenic and is driven by a significant genetic burden of potentially damaging rare variants in genes contributing to fetal membrane strength and integrity. Tissue and location specific expression patterns of the Chromosome 21 miRNA cluster (miR-99a, miR-125b, let-7c) in fetal membranes from term pregnancies with spontaneous rupture were investigated. The results suggest that these miRNAs play potential roles in fetal membrane rupture and fetal membrane defects associated with T21.

Le Glypican-3 (GPC3) est surexprimé dans 72% des carcinomes hépatocellulaire (CHC). C’est un co-récepteur membranaire du récepteur WNT, qui appartient à la famille des protéoglycanes à sulfates d'héparane. L'objectif général de ma thèse vise à étudier les mécanismes de régulation post-transcriptionnelle de l’expression du GPC3 dans le CHC. Pour cela, j’ai développé un test fonctionnel qui m’a permis de cribler une bibliothèque de 876 microARNs humains. Ceci a conduit à l’identification de 5 microARNs régulateurs de l’expression de l’ARNm codant pour le GPC3 via sa région 3’ non traduite (NT). Mon travail de thèse porte plus particulièrement sur le miR-1271 et le miR-1291 car ils sont dérégulés dans le CHC et sont respectivement inhibiteur et inducteur de l’expression du GPC3. Dans un premier projet, j’ai démontré que le miR-1271 cible directement la région 3’NT du GPC3 et diminue la stabilité de son ARNm. Ce microARN est sous-exprimé dans le CHC et son expression corrèle négativement avec celle de l'ARNm du GPC3 dans les CHC associés à une infection par le virus de l’hépatite B. Dans un deuxième projet, j’ai démontré que le miR-1291 régule positivement l’expression du GPC3 en inhibant un facteur intermédiaire. Une analyse in silico a permis d’identifier IRE1α comme candidat. IRE1α est une protéine transmembranaire du réticulum endoplasmique (RE) qui participe à « l’Unfolded Protein Response », une réponse adaptative activée lors de l’accumulation de protéines mal conformées dans le RE. J’ai démontré qu’IRE1α clive l’ARNm codant pour le GPC3 grâce à son activité endoribonucléase. D’autre part, le miR-1291 cible directement l’ARNm codant pour IRE1α dans sa région 5’NT ce qui inhibe son expression et induit une surexpression du GPC3. Le miR-1291 est surexprimé dans le CHC et son expression corrèle positivement avec celle de l’ARNm du GPC3. En conclusion, mon travail de thèse m’a permis de mettre en évidence et de caractériser deux nouveaux microARNs (miR-1271 et miR-1291) contrôlant l’expression du GPC3 par des mécanismes directs ou indirects. La pertinence physiopathologique de ces régulations dans le CHC est en accord avec les niveaux d’expression respectifs de ces microARNs, qui pourraient contribuer à la surexpression du GPC3 dans ces tumeurs. / Glypican-3 (GPC3) is overexpressed in 72% of hepatocellular carcinoma (HCC). It is a co-receptor for WNT receptor and belongs to the heparan sulfate proteoglycans family. The general objective of my PhD thesis was to study the mechanisms by which GPC3 is post-transcriptionnally regulated in HCC. To this end, I developed a functional test that allowed me to screen a library of 876 human microRNAs. This led me to identify 5 microRNAs that regulate the expression of GPC3 mRNA through its 3’Untranslated Region (UTR). The work presented in this thesis particulary focuses on miR-1271 and miR-1291 as both microRNAs present a deregulated expression in HCC and are respectively inhibitor and activator of GPC3 mRNA expression. In a first project, I demonstrated that miR-1271 directly binds to GPC3 mRNA 3’UTR and affects its stability. This microRNA is underexpressed in HCC and its expression negatively correlates with that of GPC3 mRNA in a subgroup of HCC corresponding to those associated with hepatitis B virus infection. In a second project, I demonstrated that miR-1291 postively regulates the expression of GPC3 mRNA by targeting an intermediate factor. An in silico analysis led to the identification of the Inositol Requiring Enzyme 1 alpha (IRE1α) as a potential candidate. IRE1α is an endoplasmic reticulum (ER) resident type I transmembrane protein and contributes to the signaling of the Unfolded Protein Response (UPR). The UPR is an adaptive response activated upon accumulation of improperly folded proteins in the ER. I showed that IRE1α cleaves GPC3 mRNA through its endoribonuclease activity. Moreover I demonstrated that miR-1291 directly targets IRE1α mRNA through its 5’UTR, thereby decreasing its expression and contributing to GPC3 mRNA overexpression. MiR-1291 is overexpressed in HCC and its expression positively correlates with that of GPC3 mRNA. In summary, the work carried out during my PhD allowed the identification and the characterization of two new microRNAs (miR-1271 and miR-1291) that control the expression of GPC3 mRNA through direct or indirect mechanisms. The pathophysiological relevance of these regulatory mechanisms is in agreement with the respective expression levels of these microRNAs in HCC, which could therefore contribute to the overexpression of GPC3 in those tumors.

Acute myeloid leukemia (AML) the most common acute leukemia in adults is characterized by various cytogenetic and molecular abnormalities. However, the genetic etiology of the disease is not yet fully understood. MicroRNAs (miRNAs) are small single- stranded noncoding RNAs that are negative regulators of gene expression. miRNAs influence processes of proliferation, differentiation and apoptosis. Deregulation of miRNAs expression can contribute to human disease. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as breast cancer, colorectal cancer and lung cancer. However, defining a plasma miRNA signature in AML that could serve as a biomarker for diagnosis has been conducted only once. We studied miRNA expression in plasma of 8 AML patients in first detection of the disease and repeatedly after achieving remission using TaqMan miRNA microarray for 750 human miRNA. The plasma expression level of 25 miRNA was down-regulated whilst that of 20 miRNA was up-regulated in the AML group at diagnosis when compared to healthy controls. The plasma expression level of 21 miRNA was down-regulated whilst that of 13 miRNA was up-regulated in the AML group in remission compared to healthy controls. Keywords acute myeloid leukemia (AML), biomarker, microRNA (miRNA), plasma, TaqMan Low...

MicroRNAs (miRNAs) are small non-coding RNAs with a length of 18 to 25 nucleotides playing a pivotal role in post-transcriptional regulation of gene expression. There are miRNAs whose expression is limited to a certain tissue type and diseases which are characterized by a unique miRNA expression profile. I assumed spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (PPROM) would be characterized by a unique miRNA expression profile. I observed the gene expression of 15 placental specific miRNAs (miR-512-5p, miR-515-5p, miR-516b-5p, miR-517-5p, miR-518b, miR-518f-5p, miR-519a-5p, miR-519d-3p, miR-519e- 5p, miR-520a-5p, miR-520h, miR-524-5p, miR-525-5p, miR-526a and miR-526b-5p) in placental tissue of pacients with PTB, PPROM and women with term in labor pregnancies (FG). PTB group consisted of 24 pacients, PPROM group of 75 pacients and FG group of 20 pacients. Quantitative real-time PCR was used to quantify gene expression. In the group of PTB pregnancies I identified 3 significantly upregulated miRNAs (miR-516b-5p, miR-519d-3p and miR-524-5p) and 4 miRNAs (miR-518b, miR-519a-5p, miR-520h and miR-526a) with a trend to upregulation compared to controls (FG). In the group of PPROM pregnancies I identified 3 miRNAs (miR-519d-3p, miR-520h and miR-256b-5p) with a trend to...

Le processus de formation des cils mobiles multiples (multiciliogénèse) est composé de nombreuses étapes. Récemment, nous avons démontré que les microARNs de la famille miR-449 contrôlent plusieurs de ces étapes. Au cours de mon travail, je me suis concentré sur le rôle joué par miR-449 dans deux aspects du développement de l'épithélium multicilié. Dans les cellules multiciliées, un réseau dense d'actine sous-jacent l'aspect apicale de la membrane cellulaire (coiffe d'actine) est nécessaire pour l'ancrage des multiples corps basaux, et donc pour une ciliogenèse approprié. Dans le cadre de mon travail, j'ai participé à l' identification de la petite GTPase R-Ras comme une des véritables cibles de miR-449. J'ai démontré que la réorganisation de la coiffe d'actine et l'ensemble du processus de multiciliogénèse étaient compromis lorsque l'ARN messager de R-Ras se trouve protégé de la liaison avec miR-449. J'ai aussi contribué à identifier une nouvelle cible de miR-449, le gène Steel, qui code pour le ligand du récepteur transmembranaire à tyrosine-kinase KIT. La repression de Steel par miR449 est impliquée dans le processus par lequel les cellules multiciliées atteignent leur position finale dans l'épiderme embryonnaire de Xenopus. STEEL, qui agit probablement comme une molécule de guidage pour les cellules multiciliées qui expriment KIT, doit être réprimé par miR-449 dans ces mêmes cellules en cours de migration pour assurer leur deplacement directionnel approprié. En conclusion, mon travail a contribué à élucider le rôle complexe joué par le miARN miR-449 dans le processus de multiciliogénèse chez les vertébrés. / The process of multiple motile cilia formation (multiciliogenesis) is composed of many different steps. Recently, we demonstrated that microRNAs of the miR-449 family control several of these steps. During my work, I focused on the role played by miR-449 in two aspects of the development of the mucociliary epithelium. In multiciliated cells, a dense actin network underlying the apical aspect of the cell membrane (actin cap) is required for the anchoring of the multiple basal bodies, and therefore for proper ciliogenesis. Small GTPases play important role in the formation of the actin cap. In the course of my work, I took part in the identification of transcripts coding the small GTPase R-Ras as bona fide targets of miR-449. I demonstrated that apical and subapical actin network reorganization and multiciliogenesis were impaired when R-Ras mRNA was protected from miR-449 binding. Moreover, the actin cap formation and multiciliogenesis were rescued when the translation of protected R-Ras transcripts was prevented. I also contributed to the finding that a new miR-449 target, the KIT receptor tyrosin kinase ligand STEEL, is involved in the process through which the multiciliated cells reach their final position within the developing frog epidermis. STEEL, which likely acts as a guidance molecule for the KIT-expressing multiciliated cells, needs to be repressed by miR-449 within the migrating cells to ensure their proper directional migration. Altogether, my work contributed to elucidate the complex role played by the miR-449 miRNA in the process of vertebrate multiciliogenesis.

Le carcinome rénal à cellules claires (cRCC) est le principal type histologique de carcinome rénal et l'une des tumeurs les plus résistantes à la chimio et à la radiothérapie. L'absence de biomarqueurs pour la détection précoce et pour le suivi des patients est responsable d'un mauvais pronostic. Il est nécessaire d'identifier de nouveaux biomarqueurs et des cibles thérapeutiques pour améliorer la prise en charge des patients. Les microARNs, des petits ARN non codants de 22 nucléotides, qui ont été précédemment montrés comme favorisant l'initiation et la progression tumoral, semblent être de bons candidats. Nous avons focalisé notre étude sur (i) miR-21 qui est le principal oncomiR surexprimé dans le cRCC et (ii) le récepteur nucléaire PPARα (Peroxisome Proliferator Activated Receptor), l'une des cibles de miR-21.D'une part, sur une cohorte de 99 échantillons de cRCC primaires, nous avons montré que l'expression de miR-21 était plus élevée dans les tissus cancéreux que dans les tissus non tumoraux adjacents. In vitro, miR-21 est également surexprimé dans les lignées cellulaires de carcinomes rénaux comparées à la lignée cellulaire épithéliale HK-2 provenant de tubes proximaux humains. De plus, nous avons également montré que la surexpression de miR-21 augmente les propriétés de migration et d'invasion des cellules cancéreuses rénales ainsi que les voies de signalisation prolifératives et anti-apoptotiques, alors que des résultats opposés ont été observés en utilisant une stratégie d'inhibition anti-miR-21. Enfin, nous avons évalué le rôle du miR-21 dans la chimiorésistance du cRCC et montré, en outre, que l'inhibition de miR-21 augmentait significativement la chimiosensibilité au paclitaxel, au 5-fluorouracile, à l'oxaliplatine et au dovitinib, diminuait l'expression des transporteurs à efflux MRP1-6/ABCC1-6 et augmentait l'expression des transporteurs à influx SLC22A1/OCT1, SLC22A2/OCT2 et SLC31A1/CTR1. Ces résultats ont permis la publication d'un article dans Tumor Biology se trouvant en annexe.D'autre part, dans les tissus de patients atteints de cRCC, nous avons montré pour la première fois que la surexpression de miR-21 est en corrélation avec une perte d'expression de PPARα. In vitro, nous avons montré que miR-21 cible le 3'-UTR de PPARα et diminue son expression protéique et que la surexpression de miR-21 diminue l'activité transcriptionnelle de PPARα. En outre, la surexpression et l'activation de PPARα diminuent l'expression de miR-21. En effet, PPARα interagit avec les facteurs de transcription AP-1 et NF-κB et empêche ainsi leur liaison au promoteur de miR-21 diminuant ainsi sa transcription.En conclusion, nous avons montré que (i) miR-21 est un acteur clé de la progression du cancer du rein et joue un rôle important dans la résistance aux chimiothérapies et (ii) qu'il existe une boucle de régulation négative entre miR-21 et PPARα dans le cRCC. / Renal clear cell carcinoma (cRCC) is the major histological type of renal carcinoma and one of the most chemo- and radio-resistant tumors. The absence of biomarkers for early detection and for monitoring patients is responsible of a poor prognosis. It is necessary to identify new biomarkers and therapeutic targets to improve patient care. MicroRNAs, small noncoding RNAs of 22 nucleotides, which have been previously shown to promote malignant initiation and progression, appear to be good candidates.We focused our study on (i) miR-21 which is the main overexpressed oncomirs in cRCC and (ii) the nuclear receptor PPARα (Peroxisome Proliferator Activated Receptor), one of miR-21 targets.In one hand, by using a cohort of 99 primary cRCC samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating cRCC chemoresistance and further showed that miR-21 silencing significantly increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin and dovitinib, decreased expression of multi-drug resistance genes and increased SLC22A1/OCT1, SLC22A2/OCT2 and SLC31A1/CTR1 platinum influx transporter expression. These results led to the publication of an article in Tumor Biology in annex.In other hand, in cRCC tissue patients, we showed for the first time that miR-21 overexpression correlates with a loss of expression of PPARα. In vitro, we showed that miR-21 targets PPARα 3'-UTR and decreases its protein expression and miR-21 overexpression decreases the transcriptional activity of PPARα. Furthermore, PPARα overexpression and activation decrease miR-21 expression. In fact, PPARα interacts with AP-1 and NF-kappaB transcription factors and thus prevents their binding to the miR-21 promoter thus decreasing its transcription.In conclusion, we have shown that (i) miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs and (ii) there is a negative regulatory loop between miR-21 and PPARα in cRCC.

O Diabetes Mellitus é uma das maiores causas de morte no mundo. O desenvolvimento do Diabetes Mellitus tipo 2 (DM2) está relacionado com uma série de fatores genéticos e ambientais, culminando com o desenvolvimento do DM2. Já a hiperglicemia, característica marcante da doença, está associada a uma série de complicações metabólicas e comorbidades. No entanto, nào se sabe a influência de um controle apropriado da doença, com menores níveis glicêmicos. No presente trabalho, foi utilizada a técnica de microarrays para comparar os perfis transcricionais (mRNA e microRNA) de células mononucleares de sangue periférico (PBMCs) em três grupos distintos: um grupo de pacientes DM2 descompensados (DM2-D, n=13); um grupo de pacientes DM2 compensados (DM2-C, n=14), e um grupo controle (n=10). Os dados foram analisados por meio de duas linguagens de programação: R e PERL. Após a extração dos dados utilizando-se o software Feature Extraction, versão 10.7 (Agilent Techonologies), foram realizadas correção do background, exclusão dos outliers, normalização dos dados pelo método quantile e, por fim, o ajuste de variações nãobiológicas. Os dados foram então submetidos a análise estatística rank products, sendo identificados 415 mRNAs diferencialmente expressos no grupo DM2-C relativamente aos controles, 285 no grupo DM2-D em comparação aos controles e 478 em pacientes DM2-D comparados aos DM2-C. Posteriormente, os genes diferencialmente expressos foram submetidos à analise de enriquecimento funcional (DAVID). Foram encontrados 22 e 56 termos biológicos enriquecidos (p-corrigido Benjamini-Hochberg < 0,05) para as comparações DM2-C versus controle e pacientes DM2-D versus DM2-C, respectivamente. Em ambas as comparações, um processo biológico foi considerado de interesse para o presente trabalho: resposta inflamatória. Na análise por GSEA e GSA, foram identificados 110 grupos gênicos diferencialmente expressos na comparação DM2-C versus controle. Já para a comparação DM2-D versus controles foram encontrados 297 grupos gênicos diferencialmente expressos, enquanto que na comparação DM2-D versus DM2-C, 161 grupos gênicos diferencialmente expressos. Dentre os grupos gênicos diferencialmente expressos, três merecem destaque: regulação do reparo do DNA (GO: 0006282), resposta ao superóxido (GO: 0000303) e resposta ao estresse do retículo endoplasmático (GO: 0034976). Ainda, 97 microRNAs foram diferencialmente expressos na comparação DM2-C versus controles, 54 na comparação DM2-D versus controles e 101 na comparação DM2-D versus DM2-C. Assim, diferentes grupos gênicos provavelmente foram modulados pela hiperglicemia, além de terem sido descobertos novos microRNAs relacionados a altos níveis de glicose. / Diabetes mellitus is a major cause of death worldwide. The development of type 2 Diabetes Mellitus (T2D) is associated with a number of genetic and environmental factors, culminating in the development of T2D. Hyperglycemia, a hallmark of the disease, is associated with a number of metabolic complications and comorbidities. However, the influence of a proper control of the disease, with lower glucose levels is unknown. In this study, we used the microarrays technique to compare the transcriptional profiles (mRNA and microRNA) of peripheral blood mononuclear cells (PBMCs) in three distinct groups: a group of patients with uncontrolled T2D patients (T2D-U, n = 13) a group of controlled T2D patients (T2D-C, n = 14) and control group (n = 10). Data were analyzed using two programming languages: R and PERL. After extracting the data using the Feature Extraction software, version 10.7 (Agilent Technologies), background correction, outliers exclusion, data normalization by quantile and adjustmesnt of non-biological variations were performed. The data were then statistically analyzed by the rank products test, which identified 415 differentially expressed mRNAs in T2D-C group compared to controls, 285 in group T2D-U in comparison with controls and 478 when T2D-U and T2D-C are compared. Thereafter, the differentially expressed genes were subjected to functional enrichment analysis (DAVID). 22 and 56 biologically enriched terms were found (Benjamini-Hochberg-corrected p value<0.05), when comparing T2D-C with controls and T2D-U with T2D-C, respectively. In both comparisons, inflammatory response was selected as a biological process of interest. The analysis by GSEA and GSA identified 110 differentially expressed gene sets in comparison T2D-C versus control. As for the comparison T2D-U versus control, 297 gene sets were found differentially expressed, whereas in comparison T2D-U versus T2D-C, 161 differentially expressed gene sets were found. Among the differentially expressed gene sets, three stand out: regulation of DNA repair (GO: 0006282), superoxide response (GO: 0000303) and response to endoplasmic reticulum stress (GO: 0034976). Still, 97 microRNAs were differentially expressed in the T2D-C versus controls comparison, 54 when comparing T2D-U versus controls and 101 in the comparison of T2D-U versus T2D-C. Thus, different gene sets were probably modulated by hyperglycemia, and new microRNAs related to high levels of glucose were discovered.

L’insuffisance cardiaque (IC) est un problème majeur de santé publique. La pénurie des greffons cardiaques et la résistance de nombreux patients aux traitements conventionnels ont poussé les chercheurs à développer de nouvelles thérapeutiques dont la thérapie cellulaire. Bien que l’idée initiale de la thérapie cellulaire ait été de repeupler la partie nécrosée du cœur par l’administration de cellules viables et fonctionnelles, leur disparition rapide alors que les bénéfices perdurent dans le temps a conduit à l’hypothèse que les cellules agiraient via un mécanisme paracrine. Les vésicules extracellulaires (VE), incluant les exosomes et les microparticules, seraient principalement impliquées dans ce processus. Elles agiraient ainsi comme de véritables navettes transportant des biomolécules actives permettant d’activer des voies de réparation endogènes dans le tissu traité. Ce projet de thèse a pu mettre en évidence : 1) La non-infériorité des VE issues de progéniteurs cardiovasculaires (Pg) dérivés de cellules souches embryonnaires humaines par rapport à leurs cellules d’origine dans un modèle murin d’IC chronique (ICC). Ces VE activeraient des voies de signalisation endogènes impliquées dans la stimulation de la prolifération cellulaire, la survie cellulaire, la réparation de l’ADN et la diminution de la fibrose. Leur contenu moléculaire spécifique, et notamment les microARN, pourrait être impliqué dans ces phénomènes. 2) L’importance du choix du type cellulaire dans la production de VE efficaces sur le plan thérapeutique puisque ni les VE dérivées de cardiomyocytes matures ni celles de cellules souches mésenchymateuses n’ont eu d’effets bénéfiques sur la fonction cardiaque de souris en ICC. 3) L’implication des VE dans l’effet paracrine des cellules, confirmée par l’amélioration de la fonction cardiaque chez des souris présentant une ICC traitées avec des VE issues de Pg dérivés d’iPS. Des tests fonctionnels in vitro ont montré que les VE auraient un rôle pro-angiogénique, pro-prolifératif et amélioreraient la survie des cellules. Une thérapie a-cellulaire aurait une réelle pertinence clinique en réglant une partie des problèmes techniques, immunologiques et sécuritaires associés aux greffes de cellules. Si cette hypothèse est confirmée, il pourrait en résulter une simplification des problèmes réglementaires, une diminution des coûts de production et de ce fait une plus grande diffusion clinique de la méthode. / Heart failure (HF) is a major public health concern. The lack of donor hearts and the resistance of numerous patients to conventional treatments has led scientists to develop new therapies such as cell therapy. The initial goal of cell therapy was to repopulate the infarcted heart by directly injecting viable and functional cells. However, the rapid disappearance of the transplanted cells contrasts with their long term, ongoing functional benefits, suggesting that cells may act through a paracrine mechanism. Extracellular vesicles (EV), including exosomes and microparticles, may be key to this process, acting as shuttles to transport bioactive macromolecules that stimulate endogenous repair pathways in the host tissue. This PhD project demonstrates: 1) The non-inferiority of EV secreted by cardiovascular progenitors (Pg) derived from human embryonic stem cells as compared to their parent cells in a mouse model of chronic HF (CHF). These EV could act by the activation of endogenous signaling pathways implicated in cell proliferation, survival, DNA repair and decreased fibrosis. Their specific content, such as miRNA, could be involved in these benefits. 2) The importance of the cell type in the production of therapeutically effective EV, since EV derived from mature cardiomyocytes and mesenchymal stem cells did not improve cardiac function in mice with CHF. 3) The importance of EV in paracrine effects of cells, confirmed by the improvement of cardiac function in mice with CHF treated with EV secreted by Pg derived from iPS cells. In vitro data shows that EV might have pro-angiogenic, pro-proliferative and pro-survival effects. An acellular therapy should be clinically relevant by reducing technical, immunological and safety problems associated with cell transplantation. If this hypothesis is confirmed, regulatory concerns would be simplified and production costs reduced, facilitating large-scale production.

Après un infarctus, les acteurs de l’immunité innée et adaptative contrôlent le remodelage et la fonction cardiaque. Dans un premier travail, nous nous sommes intéressés aux mécanismes moléculaires définissant le rôle des monocytes et des macrophages, composants majeurs de l’immunité innée. En particulier, nous avons analysé le rôle de deux récepteurs MertK (myeloid-epithelial-reproductive receptor tyrosine kinase) et Mfge8 (milk fat globule epidermal growth factor-like factor 8), susceptibles d’être impliqués dans la reconnaissance des débris cellulaires peuplant la zone infarcie. L’invalidation de MertK et Mfge8 in vivo chez la souris augmente l’accumulation des cellules apoptotiques, aggrave la fibrose et accroit la taille de l’infarctus par rapport aux souris contrôles. De plus, l’expression du facteur pro-angiogenique VEGF-A, est significativement réduite 3 jours post-infarctus chez les souris chimères Mertk-/-Mfge8-/- par rapport aux animaux contrôles. Enfin, l’invalidation de l’expression de VEGF-A dans les cellules myéloides augmente la fibrose et la taille de l’infarctus et diminue la fonction cardiaque. Ainsi, la reconnaissance des débris cellulaires par Mertk et Mfge8 promeut la libération de VEGF-A par les monocytes et macrophages et participe à la restauration de la densité capillaire et de la fonction cardiaque post-infarctus. Dans un deuxième travail, nous avons abordé les voies de signalisation intracellulaires sous-tendant les effets d’un acteur majeur de l’immunité adaptative, le lymphocyte B. Après un infarctus, les lymphocytes B libèrent la chimiokine Ccl7, qui en se fixant sur son récepteur CCR2, déclenche une mobilisation des monocytes inflammatoires de la moelle osseuse vers le sang puis leur recrutement délétère dans la zone infarcie. Nous avons notamment montré que des activateurs des lymphocytes B, comme des stimuli inflammatoires, stimulent la libération de Ccl7, en partie en initiant des voies de signalisation intracellulaires dépendantes du micro-RNA (miR)21. L’administration exogène de lymphocytes B déficients pour miR-21 améliore d’ailleurs la fonction et le remodelage cardiaque post-infarctus chez des souris immunodéficientes. miR21 cible le gène suppresseur de tumeur PTEN et ainsi augmente l’expression du facteur de transcription HIF1α. De fait, l’analyse par échocardiographie effectuée 14 jours après l’induction de l’infarctus montre une amélioration de la fonction cardiaque ainsi qu’une diminution de la taille de l’infarctus et de la fibrose interstitielle chez les souris invalidées pour HIF1α spécifiquement dans les lymphocytes B par rapport aux contrôles. Les taux de la chimiokine CCL7 dans le plasma de ces souris sont aussi significativement réduits à J1 et J3 post infarctus ; ainsi que la mobilisation et le recrutement monocytaire. Il apparaît donc que la voie de signalisation impliquant miR21/HIF1α conditionne l’effet délétère des lymphocytes B dans un contexte d’infarctus aigu du myocarde. Ces travaux ont permis de révéler des facteurs majeurs de la régulation fine de la réaction inflammatoire post-ischémique, et de souligner l’efficacité potentielle de stratégies thérapeutiques modulant l’activité des cellules immunitaires dans le décours des pathologies cardiaques. / After a myocardial infarction, the innate and adaptive immunity play a role in post ischaemic remodelling and cardiac function. In a first work, we have been interested in the molecular mechanism of the role of monocytes and macrophages, the major innate immunity components. In particular, it has been analysed the function of, the myeloid-epithelial reproductive protein tyrosine kinase (Mertk) and the milk fat globule epidermal growth factor (Mfge8) and their implication in directing cardiac remodelling by skewing the inflammatory response after myocardial infarction. Compared with wild-type, Mertk-deficient (Mertk−/−), or Mfge8- deficient (Mfge8−/−) animals, Mertk−/−/Mfge8−/− mice displayed greater alteration in cardiac function and remodelling. In parallel, Mertk−/−/Mfge8−/− bone marrow chimeras manifested increased accumulation of apoptotic cells, enhanced fibrotic area, and larger infarct size, as well as reduced angiogenesis and VEFA expression. Combined Mertk and Mfge8 deficiency in myeloid cells either obtained from in vitro differentiation of bone marrow cells or isolated from infarcted hearts altered their capacity of efferocytosis and subsequently blunted vascular endothelial growth factor A (VEGFA) release. On the contrary, the recognition of necrotic cells by Mertk and Mfge8 promote VEGFA liberation improving cardiac function and angiogenesis. In the second work, we have focused on intracellular signalling pathway underlying the effects of another important actor of adaptive immunity, the B lymphocytes. After acute myocardial infarction multiple subtypes of inflammatory cells are known to orchestrate post-ischemic cardiac remodelling. In particular, Mature B lymphocytes selectively produce Ccl7 chimiokine and fixed on its CCR2 receptor it is able to induce monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Many B lymphocytes activators, such as inflammatory stimuli, may promote CCL7 release partially involving an intracellular signalling pathway depending on the micro-RNA miR21. We speculate that endogenous activation of the miR21/HIFα-related pathways balances the effect of B lymphocytes on post-ischemic cardiac remodelling. The treatment with TLR ligands resulted in induction of the microRNA miR-21, which targeted PTEN, leading to subsequent up regulation of HIF1α and HIF2α levels. In Rag1-/- immunodeficient mice with acute MI, we showed that re-supplementation with miR21-/- B lymphocytes restored cardiac repair and function when compared to injection of wild-type B cells. These effects were associated with a reduction in Ly6Chigh monocyte infiltration in the ischemic myocardium as well as with a decrease in infarct size and interstitial fibrosis. This work reveals several factors implicated in regulation of post-ischaemic inflammatory reaction, and underline the potential efficacity of a therapeutic strategy to module the activity of immune cells alongside the cardiovascular diseases.

A fibrilação atrial (FA) é a arritmia mais comum no pós-operatório de cirurgia cardíaca. Apesar de estar relacionada a alterações estruturais, alguns pacientes, mesmo que sem tais condições, ainda assim, cursam com fibrilação atrial no pós-operatório (FAPO) causando aumento no tempo de internação e custos. Estudos recentes vem ampliando o conhecimento sobre pequenos fragmentos de RNA, chamados de microRNAs (miRNAs) que podem interferir diretamente no aparecimento de algumas doenças na área cardiovascular. O objetivo do presente estudo é: 1) comparar a expressão dos miRNAs 1, 23 e 26 entre pacientes com e sem FAPO; 2) comparar nos grupos a expressão destes miRNAs entre os período pré e pós-cirúrgico; 3)comparar a expressão dos genes GJA1, KCNJ2, CACNB1, CACNA1C e KCNN3 entre os tempos pré e pós-cirúrgico no grupo FAPO; 4) comparar estes últimos genes no tecido atrial; 5) comparar os genes relacionados à produção de interleucinas (IL)-1, 6 e fator de necrose tumoral alfa (TNF?) entre os grupos e entre os tempos pré e pós-cirúrgico; 6)avaliar as características clínicas e evolutivas da população estudada. Pacientes submetidos à cirurgia de revascularização miocárdica foram submetidos à coleta de 20ml de sangue pré e pós-cirurgia bem como fragmento de tecido atrial. Um total de 143 pacientes compuseram os grupos: FAPO (24 pacientes), controle genético (24 pacientes) e controle total (97 pacientes + 24 grupo controle genético). Do ponto de vista clínico observou-se maior idade, tempo de anóxia, tempo de internação em terapia intensiva e hospitalar no grupo FAPO. A análise genética revelou menor expressão do miRNA-23 no grupo FAPO (p=0,02). A comparação entre os períodos pré e pós-cirúrgico revelou redução dos três miRNAs no tempo pós-cirúrgico (p<0,05) e dos genes relacionados às proteínas de canal (p<0,05). A comparação no tecido não evidenciou alterações entre os grupos. Os genes relacionados ás citocinas revelaram redução no período pós-cirúrgico (p<0,05) em ambos os grupos. Concluiu-se que o miRNA-23 pode ter implicação no surgimento da FAPO e outros miRNAs não estudados devem estar envolvidos neste processo uma vez que houve redução de outros genes de canais relacionados ao aparecimento de FAPO. / Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. AF is related to cardiac structural changes although a group of patients still remains developing post-operative atrial fibrillation (FAPO) even without those changes, leading to more days in the hospital and costs. Recent studies showed that short fragments of RNA, called microRNA (miRNA) can contribute to the development of several diseases in the cardiovascular area. The aim of this study is to 1) compare the expression of miRNA-1, 23 and 26 between the group with and without FAPO; 2) compare, in the FAPO group, the expression of these miRNAs in the pre and post-surgery periods; 3) compare the expression of GJA1, KCNJ2, CACNB1, CACNA1C e KCNN3 genes between the pre and post-surgery periods; 4) compare this genes in atrial tissue; 5) compare the genes related to inflammation cytokines as interleukin(IL)-1, 6 and alpha tumoral necrosis factor between the groups in the pre and post-surgery periods; 6) evaluate clinical and evaluative patterns of the study population. Twenty milliliters of blood samples in the pre and post-operative periods and an atrial fragment were extracted from patients submitted to coronary artery bypass graft surgery. A total of 143 patients were divided in the FAPO group (24 patients), genetic control group (24 patients) and a total control (97 + 24 genetic control patients). The clinical analysis showed bigger age and clamp-time, more days in the intensive care unit and hospital in the FAPO group. The genetic analysis revealed less expression of miRNA-23 in the FAPO group (p=0.02). The comparison between the pre and post-surgery periods showed reduction in the three studied miRNAs (p<0.05) and reduction in the genes related to the production of the membrane protein channel sites. The comparisons in the atrial tissue didn´t show any difference in the study groups. The cytokines showed post-surgery reduction (p<0.05) in both groups. The conclusion is that miRNA-23 can be implicated in FAPO as others miRNAs not studied can also be, once there was a significative reduction in the genes related to FAPO development.