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.

L'anévrysme de l'aorte abdominale (AAA) est un problème de Santé Publique qui touche principalement les hommes de plus de 65 ans. L'AAA souvent asymptomatique évolue vers la rupture associée à un taux de mortalité élevé. Parmi les acides ribonucléiques (ARNs) non codants, les microARNs (miARNs), stables dans le tissu et les biofluides, sont des candidats intéressant dans la recherche de biomarqueurs. L'inflammation, la dégradation de la matrice extra-cellulaire (MEC) et la raréfaction de la média participent à l'AAA. De nombreuses cellules inflammatoires sont impliquées dans l'AAA. La raréfaction des cellules musculaires lisses (CML) est secondaire à l'anoïkis, apoptose par détachement cellulaire de la MEC. Une analyse protéomique différentielle de CML en culture, issues de patients porteurs d'AAA, réalisée au laboratoire a montré que la désintégrine et metalloprotéinase avec un motif de thrombospondine 5 (ADAMTS 5) est surexprimée dans les CML de patients présentant un AAA. L'isolement des cellules par la microdissection laser permet de conserver le phénotype des cellules isolées et de mettre en évidence des marqueurs potentiels de l'AAA masqués par l'analyse du tissu global. Mon travail de thèse a consisté à partir des cellules isolées de la paroi anévrysmale de l'aorte abdominale sous-rénale chez l'Homme: à effectuer une analyse globale des miARNS et une analyse ciblée de l'ADAMTS 5, métalloprotéase qui a une action enzymatique sur les protéines de la MEC. Les objectifs de ce travail sont une meilleure compréhension de l'AAA et l'identification de nouveaux biomarqueurs.La distribution des cellules dans la paroi anévrysmale est étudiée par immunohistochimie sur des biopsies anévrysmales et d'aortes saine obtenues chez l'Homme. Les cellules localisées sont isolées par microdissection laser. L'analyse par criblage de l'expression des miARNs des cellules isolées de l'AAA et des CML issues d'aorte saine est réalisée sur puce. L'expression différentielle de miARNs sélectionnés est analysée par PCR quantitative dans des cellules isolées de l'AAA et dans du tissu global. L'expression des miARNs sélectionnés est ensuite comparée dans le plasma des patients présentant un AAA et de patients athérosclérotiques sans AAA par PCR pour identifier de potentiels biomarqueurs. Dans l'AAA, les macrophages M1 proinflammatoires sont retrouvés dans l'adventice et les macrophages M2 anti inflammatoires dans le thrombus intraluminal, les lymphocytes de type B sont retrouvés organisé en organe lymphoïde tertiaire adventitielle ou ATLOs dans 11 échantillons sur 20 analysés. Les CML sont rares et strictement localises au niveau de la média. Sur 850 miARNs testés dans la puce, 205 miARNs sont exprimés dans les cellules isolées. Les miR-29b et let-7f sont augmentés dans le plasma de patients porteurs d'AAA et représentent de potentiel biomarqueurs.L'expression d'ADAMTS 5 dans les CML de la paroi anévrysmale est évaluée par immunohistochimie dans la paroi aortique saine et anévrysmale et quantifiée par Western-blot dans les CML isolées de la paroi aortique saine et anévrysmale.Deux morphotypes de CML anévrysmales ont été identifiés: un morphotype arrondi positif au marqueur de l'apoptose, caspase 3 et un morphotype allongé, similaire aux CML de l'aorte saine. Le profil d'expression des sous-unités d'ADAMTS 5 est diffèrent dans les CML arrondies et les CML allongées anévrysmales et saines. La mise en apoptose des CML a été mise au point in vitro pour étudier les mécanismes impliqués dans les modifications d’ expression d'ADAMTS 5 dans l'AAA et les conséquences sur son action enzymatique.L'approche systématique de l'expression transcriptomique des cellules anévrysmales isolées a identifié des marqueurs potentiels de l'AAA, les miR-29b et let-7f et l'analyse ciblée suggère l'implication d'ADAMTS 5 dans le profil évolutif des CML vers l'anoïkis dans l'AAA. Des études complémentaires permettront une meilleure compréhension de l'AAA. / Abdominal aortic aneurysm (AAA) is a public health problem, which mainly affects men older than 65 year. AAA are usually asymptomatic with a natural evolution towards rupture associated with a high mortality rate. Among non coding ribonucleic acids (RNAs), microRNAs are stable in tissue and biofluids and are interesting candidates for the search of biomarkers. Inflammation, extracellular matrix (ECM) degradation and media rarefaction are involved in AAA. Many inflammatory cells are involved in AAA. Anoikis is an apoptosis secondary to a cell detachment from ECM and is responsible for rarefaction of smooth muscle cells (SMC). Differential proteomic analysis of cultured SMC from AAA patients was performed in the laboratory and highlighted the overexpression of a disintegrin and metalloproteinase with thrombospondin motif of type 5 (ADAMTS 5) in SMC of AAA patients. Isolation of cells with laser microdissection allows to keep their phenotype and to find potential markers that may be masked by global tissue analysis.The aim of my PhD work was to perform a global miRNA screening of cells isolated from human aneurysmal wall and an analysis targeted on ADAMTS 5, a metalloprotease with an enzymatic activity on ECM proteins. The main objectives were a better understanding of AAA and the identification of new biomarkers.The distribution of cells in the aneurysmal wall was studied by immunohistochemistry in human aneurysmal and healthy aortic samples. Once located, the cells were isolated by laser microdissection and screened for miRNAs by microarrays. Differential expression of selected miRNAs was quantified by PCR in the cells isolated by laser microdissection and in whole aortas. They were then compared in plasma of AAA patients and atherosclerotic patients without AAA by quantitative PCR to identify potential biomarkers.In AAA, the M1 proinflammatory macrophages were located in the adventitia and the M2 antiinflammatory macrophages in the intraluminal thrombus; the type B lymphocytes were organized in tertiary lymphoid organs (ATLOs) in 11/20 of analysed samples. SMC were rare and restricted to the media. Among the 850 miRNAs tested on microarray, 205 miRNAs were detected in isolated cells. MiR-29b and let-7f were upregulated in plasma of AAA patients, and thus are potential biomarkers.The expression of ADAMTS 5 in aneurysmal SMC was evaluated by immunohistochemistry of healthy and aneurysmal aortic wall and quantified by Western blot in isolated SMC from healthy and aneurysmal wall.Two aneurysmal SMC morphotypes were identified: a rounded morphotype positive for caspase 3, an apoptotic marker, and a spindle-shaped morphotype similar to the healthy aortic SMC. The expression profile of ADAMTS 5 subunits was different in rounded SMC compared to aneurysmal and healthy spindle-shaped SMC. In vitro induction of apoptosis of SMC was established in order to study the mechanisms involved in ADAMTS 5 expression in AAA and their consequences on enzymatic actions.The global transcriptomic screening of aneurysmal cells isolated by laser microdissection has identified potential markers of AAA, miR-29b and let-7f. The targeted analysis suggested that ADAMTS 5 is involved in the evolution profile of SMC towards anoikis in AAA. Further investigations will allow a better understanding of AAA pathophysiology.

Le tissu adipeux brun (TABr) est devenu ces dix dernières années le centre d’intérêt de nombreux laboratoires en raison de sa capacité à dissiper l’énergie apportée par les substrats sous forme de chaleur. Chez les mammifères, le développement du TABr intervient à la fin de la gestation et devient fonctionnel à la naissance. Sa capacité thermogénique permet aux nouveau-nés de s’adapter face à l'environnement extra-utérin, puis son activité régresse avec l’âge. Ceci suggère que l’environnement intra-utérin joue un rôle important dans la programmation de la physiologie et du métabolisme du TABr. Dans un modèle bien décrit de retard de croissance intra-utérin, qu’est la carence protéique maternelle (CP), la jeune progéniture CP est normoglycémique malgré un défaut de sécrétion de l'insuline mais développe avec l'âge une résistance à l'insuline et une hyperglycémie. Lors de mon arrivée au laboratoire, des résultats suggéraient un rôle du TABr dans les changements dynamiques du profil métabolique de la progéniture CP en fonction de l'âge. En effet, le TABr des rats CP est hyperactif à 3 mois par rapport aux animaux contrôles alors qu’il revient au niveau des contrôles chez la progéniture CP âgée de 18 mois, ce qui corrèle avec l’apparition des troubles métaboliques caractéristiques du diabète de type 2. Durant ma thèse, mon premier objectif a été de démontrer le rôle causal du TABr dans le maintien de l’homéostasie glucidique chez les jeunes animaux CP. Pour ce faire, nous avons exposé au froid de jeunes rats CP pour solliciter leur TABr et nous avons procédé à l’ablation chirurgicale de ce tissu. Nos résultats montrent que la jeune progéniture CP est mieux protégée que les contrôles à une exposition au froid grâce à l’activité thermogénique accrue de leur TABr. / Brown adipose tissue (BAT) has grown over the last ten years into the center of interest for many laboratories due to its capacity to burn energy derived from metabolic substrates into heat. Indeed, in mammals, the development of BAT occurs at the end of gestation to become fully functional at birth. Its thermogenic capacity allows newborns to face extrauterine environment, and thereafter its activity declines with age. This suggests that the intrauterine environment plays an important role in the programming of BAT physiology and metabolism. In a well-known model of intrauterine growth retardation (IUGR), the maternal protein restriction model (called LP for low protein), the young LP progeny is normoglycemic despite an insulin secretion defect but develops insulin resistance and hyperglycemia with age. When I started my thesis work, available results in the laboratory suggested a role of BAT in the dynamic changes of the LP progeny metabolic profile according to the age. Indeed, BAT of young LP rats is hyperactive at 3 months compared to controls while this activity drops back to control levels in old 18-months LP progeny, consistent with the appearance of a type 2 diabetic phenotype. During my thesis, the first objective was to search for the causal role of BAT in the maintenance of glucose homeostasis in young LP progeny. Using a first strategy, we exposed young LP progeny to a cold challenge to activate their BAT. In a second approach, we performed surgical ablation of their BAT. Our results show that young LP progeny is more protected against a cold challenge than controls, due to the high thermogenic capacity of their BAT. However, BAT ablation induces hyperglycemia in young LP animals showing that this tissue is required to maintain their normoglycemia. This work, published in Diabetes in March 2017, suggests that a deleterious fetal environment could reprogram BAT metabolism. The second objective of my thesis was to identify the molecular mechanisms allowing the maintenance of active BAT in young LP progeny. To do so, we compared two models of BAT activation, ie our LP model and a well-known model of BAT activation with an agonist of β-3 adrenergic receptors. In both cases, when BAT is active, we observed a global increase in microRNA (miRNA) expression associated to augmented miRNA machinery expression, and in particular AGO2 expression. Interestingly, when BAT is inactive in old LP animals, miRNA expression and miRNA machinery expression return to control levels. While activation of mature brown adipocytes in vitro leads to an increase in AGO2 protein expression, partial deletion of this protein is sufficient to decrease the thermogenic activity of these cells. Collectively our data suggest that AGO2 and increased miRNA expression contribute to BAT activation. The manuscript concerning this research is in the review process at Molecular Metabolism. In the third part of my PhD research efforts, I have found that in the BAT of young LP progeny several miRNAs are robustly downregulated. We have focused on let-7cp and miR-22-3p, which have the most severe decrease in expression. Our key finding is that these two miRNAs act synergistically to hinder mature brown adipocyte thermogenic activity. This work is in the process of being finalized for publication. In conclusion, during my PhD training I have revealed several novel findings, which lead to a better understanding of BAT physiology and its dysregulation in situations eventuating in perturbed glucose homeostasis. While additional efforts are certainly needed, these contributions advance our vision to leverage BAT as a promising target for the prevention and/or treatment of metabolic perturbations associated to obesity and type 2 diabetes.

L’arthrite juvénile idiopathique (AJI) est un groupe hétérogène de rhumatismes inflammatoires chroniques affectant les enfants de moins de 16 ans. Cette atteinte inflammatoire d’origine inconnue est caractérisée par une arthrite persistant plus de 6 semaines en l’absence de traitements.Afin de mettre en évidence des mécanismes impliqués dans la physiopathologie de l’AJI, une inclusion de patients atteints d’une autre forme d’arthrite juvénile, à savoir l’arthrite septique, a été effectuée. En parallèle, des études comparatives entre le sang périphérique (SP) et le liquide synovial (LS) des patients atteints d’AJI ont été réalisées afin de rechercher des mécanismes spécifiques aux perturbations articulaires. Un intérêt particulier a été porté sur les sous-populations de monocytes et de cellules dendritiques (DCs) ainsi que les profils d’expression des microARNs (miARNs) dans le sang périphérique et le LS des patients. Ces différents marqueurs biologiques ont été choisis car ils jouent un rôle majeur à la fois dans la régulation de l’inflammation et la pathogénèse des maladies inflammatoires.L’analyse de l’expression des miARNs par une approche de séquençage à haut débit suivie d’une validation par RT-qPCR a mis en évidence des miARNs dérégulés de façon spécifique dans l’AJI par rapport à l’AS. De plus, la caractérisation phénotypique des sous-populations de cellyles myéloïdes a montré une accumulation et une activation cellulaire propre à l’AJI. Dans l’ensemble, ce projet m’a permis d’identifier différents acteurs cellulaires et moléculaires pouvant être impliqués dans la physiopathologie de l’AJI. / Juvenile idiopathic arthritis (JIA) is a heterogeneous group of chronic inflammatory rheumatism affecting children under 16 years of age. This inflammatory disorder of unknown origin is characterized by arthritis lasting more than 6 weeks in the absence of treatments.In order to highlight mechanisms involved in the pathophysiology of JIA, an inclusion of patients suffering from septic arthritis, another form of juvenile arthritis, was performed. In parallel, comparative studies between the peripheral blood (PB) and the synovial fluid (SF) of patients with JIA were carried out in order to search for mechanisms specific of joint disturbances.We focused on monocytes and dendritic cells (DCs) subsets as well as the expression patterns of microRNAs (miRNAs) in the PB and SF of patients. These different biological markers are known to play a major role both in the regulation of inflammation and the pathogenesis of inflammatory diseases.Analysis of miRNA expression by a high-throughput sequencing approach followed by RT-qPCR validation revealed specifically deregulated miRNAs in JIA compared to AS. In addition, the phenotypic characterization of myeloid cell subpopulations showed an accumulation and activation profile specific of JIA cells. Overall, this project allowed me to identify different cellular and molecular actors that might be involved in the pathophysiology of JIA.

A detailed annotation of non-protein coding RNAs is typically missing in initial releases of newly sequenced genomes. Here we report on a comprehensive ncRNA annotation of the genome of Trichoplax adhaerens, the presumably most basal metazoan whose genome has been published to-date. Since blast identified only a small fraction of the best-conserved ncRNAs—in particular rRNAs, tRNAs and some snRNAs—we developed a semi-global dynamic programming tool, GotohScan, to increase the sensitivity of the homology search. It successfully identified the full complement of major and minor spliceosomal snRNAs, the genes for RNase P and MRP RNAs, the SRP RNA, as well as several small nucleolar RNAs. We did not find any microRNA candidates homologous to known eumetazoan sequences. Interestingly, most ncRNAs, including the pol-III transcripts, appear as single-copy genes or with very small copy numbers in the Trichoplax genome.

No description available.

Robustness is a ubiquitous property of biological systems, however, underlying mechanisms that help reinforce the optimal phenotypes despite environmental or physiological perturbations are poorly understood. C. elegans development consists of four larval stages (L1-L4) and well-characterized invariant cell lineages, within which the heterochronic pathway controls the order and timing of cell-fates. Environmental or physiological stress signals can slow or temporarily halt larval stage progression; remarkably, however, temporal cell-fate progression remains unaffected. We show that two widely conserved signaling pathways, insulin and TGF- β, that regulate C. elegans larval stage progression in response to starvation and crowding, respectively, also regulate a rewiring of the heterochronic pathway so that cell-fates remain temporally anchored to appropriate larval stages. This rewiring is mediated by the nuclear hormone receptor DAF-12, and it involves a shift from the reliance on let-7-family microRNAs to the reliance on LIN-46 for proper downregulation of the transcription factor, Hunchback-like-1 (HBL-1), which promotes L2 cell-fates and opposes L3 cell-fates. LIN-46 (which is a homolog of bacterial molybdopterin molybdenum transferase (moeA) and human gephyrin) post-translationally inhibits HBL-1 activity. LIN-46 expression is repressed by the RNA-binding protein LIN-28 at the early stages to permit HBL-1 activity and hence the proper execution of L2 cell-fates. Our results indicate that robustness mechanisms of temporal cell-fate progression in C. elegans involves 1) coordinated regulation of temporal cell-fates and larval stage progression and 2) collaboration between translational regulation exerted by microRNAs and post-translational regulation exerted by LIN-46 to coordinate HBL-1 downregulation with stage progression.

Background To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice. Methods Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters. Results We distinguished numerous, unique plasma miRNAs both up- and down-regulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and down-regulated miRNAs, included, but were not limited to, 574-5p, 466g, 466f-3p, 375, 29c, and 148a. There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point ( P < 0.001). There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point ( P = 0.011). Conclusion We identified unique plasma miRNAs both up- and down-regulated in lethally dosed APAP poisoned mice.

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that posttranscriptionally regulate gene expression primarily through binding to the 3’ untranslated region (3’UTR) of target mRNAs, and are known to play important roles in various developmental and physiological processes. The work presented in this thesis was centered on understanding how Caenorhabditis elegans miRNAs are modulated by genetic, environmental, or physiological factors and how these small RNAs function to maintain the robustness of developmental processes under stressful conditions. To identify modulators of the miRNA pathway, I developed sensitized genetic backgrounds that consist of a panel of miRNA gene mutants and miRNA biogenesis factor mutants with partially penetrant phenotypes. First, I found that upon infection of Caenorhabditis elegans with Pseudomonas aeruginosa, an opportunistic pathogen of diverse plants and animals, let-7 family miRNAs are engaged in reciprocal regulatory interactions with the p38 MAPK innate immune pathway to maintain robust developmental timing despite the stress of pathogen infection. These let-7 family miRNAs, along with other developmental timing regulators, are also integrated into innate immune regulatory networks to modulate immune responses. Next, I demonstrated that loss-of-function mutations of Staufen (stau-1), a double-stranded RNA-binding protein, increase miRNA activity for several miRNA families, and this negative modulation of Staufen on miRNA activity acts downstream of miRNA biogenesis, possibly by competing with miRNAs for binding to target mRNA 3’UTRs. In summary, these studies provide a better understanding on how miRNAs are modulated by various environmental and cellular components, and further support the role of the miRNA pathway in conferring robustness to developmental processes under these perturbations.