AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...

Face au phénomène de désindustrialisation de certains territoires français, accompagné par des mouvements de délocalisation, et dans un contexte de concurrence mondiale exacerbée, la question de l'attractivité des territoires infranationaux se pose avec acuité. Cette thèse porte sur l'attractivité territoriale entendue comme la capacité à retenir des activités économiques étrangères de sorte à favoriser le développement territorial. Ainsi, ces travaux de recherche posent la question du lien théorique et empirique entre action publique locale et ancrage territorial des filiales étrangères / The question of attractiveness of subnational territories is particularly raised due to the fact that some French territories are facing the phenomenon of deindustrialization accompanied with movements of relocation in a context of worldwide competition. This thesis will focus on the territorial attractiveness intended as the capacity to retain foreign economic activities so that favoring local development. Thus, the research works raise the question of the theoretical and empirical link between local public action and the territorial anchoring of foreign subsidiaries. The idea is to understand what this anchoring consists of in terms of local public action, and the way it affects it

Les microARN constituent une classe de petits ARN non codants d’une vingtaine de nucléotides, issus de transcrits cellulaires, qui inhibent l’expression de gènes cibles au niveau post-transcriptionnel. Chez les mammifères, bien qu’ils puissent agir sur une cible parfaitement complémentaire (mode parfait), les microARN ont presqu’exclusivement des cibles partiellement complémentaires (mode imparfait). Puisqu’en mode imparfait une coupure endonucléolytique de la cible est impossible, il est généralement proposé que le mode imparfait soit moins efficace que le mode parfait : conduisant à un silencing moins efficace, nécessitant plus de complexes effecteurs (miRISC) et facilement saturable par une augmentation du nombre de cible. Dans ce travail j’ai développé une approche expérimentale reposant sur l’expression de protéines fluorescentes pour mesurer précisément le silencing au niveau de chaque cellule. J’ai fait trois observations inattendues sur l’efficacité de la régulation par les microARN : i) le silencing en mode parfait et imparfait nécessite des quantités similaires de petit ARN, ii) une augmentation, même très importante, de l’expression du gène cible ne lui permet pas d’échapper à la régulation, iii) le silencing n’est pas intrinsèquement plus faible en mode imparfait (qu’en mode parfait) mais n’est pas actif dans toutes les cellules. Si les deux premiers points sont facilement explicables dans le cadre de l’induction de la dégradation de l’ARNm cible sur un mode catalytique via la déadénylation de l’ARNm, le troisième indique l’existence d’une régulation forte du silencing qui est spécifique du mode imparfait. De plus, comme dans les deux modes le silencing dépend principalement du même partenaire, Ago2, cette régulation intervient après l’assemblage du complexe minimal (Ago2/petit ARN). Ainsi, les différences entre les modes parfait et imparfait ne se situent pas au niveau proposé puisque lorsque la cellule est compétente, leurs efficacités sont comparables. Par contre, mes travaux mettent en évidence l’existence d’un contrôle de la régulation en mode imparfait dont la nature reste à préciser. / MicroRNAs are endogenous small non-coding RNAs about 21 nucleotides in length that inhibit the expression of target genes primarily at the post-transcriptional level. The target recognition of microARN is sequence-specific and requires a partial complementarity between the microRNA and target sequences that are present on the mRNA molecules. microRNAs are part of an effector complex, miRISC, containing multiple proteins which can participate to a repression of translation and/or promotes destabilization of the target mRNA. The mechanism of microRNA silencing is not completely understood to date, but it is assumed that it is globally less potent than that of siRNA acting on perfect targets. By using fluorescent proteins expressing reporter plasmids and flow cytometry, we observed an efficient silencing by microRNA which does not require more active complexes than the perfect target silencing and cannot be easily saturated. This suggests that in cells in culture, microRNA silencing works in a catalytic manner leading to mRNA degradation. In addition, our data also indicates that the efficiency of microRNA silencing is variable among cells and can be almost completely abrogated under some conditions contrary to the siRNA silencing which is active in all cells. As we observed that the protein Ago2 is the only member of Ago family that is implicated in the microRNA silencing, it follows that the regulation of microRNA silencing acts after the formation of the core complex (Ago2/small RNA). So the difference between the silencing in the perfect and imperfect mode are not what is usually proposed, but pertain to a level of cellular control, which remains to be deciphered.

Le réseau de signalisation qui répond à l’insuline et aux nutriments est conservé chez les métazoaires, où il joue un rôle central dans le contrôle du métabolisme et de la croissance. Les nutriments assimilés sont soit directement utilisés pour la croissance tissulaire, soit stockés principalement sous forme de triglycérides. Chez la drosophile, l’activation de ce réseau de signalisation dans le corps gras, un organe qui remplit à la fois les fonctions hépatiques et destockage, induit une augmentation du stockage de lipides sous forme de nombreuses gouttelettes lipidiques (LDs). A l’inverse, la carence alimentaire se traduit par une augmentation de la taille des LDs et une diminution de lipides stockés. La kinase TOR (TargetOf Rapamycine) et son substrat S6 Kinase (S6K) jouent un rôle central dans cette régulation.Chez la drosophile, ces 2 kinases (dTOR et dS6K) contrôlent les aspects autonome-cellulaireset hormonaux de la croissance. En dépit de nombreuses études sur divers organismes modèles,destinées à comprendre les mécanismes régulateurs de S6K, rien n’est connu à ce jour sur lecontrôle de sa dégradation.Nous avons utilisé une banque de lignées exprimant des ARN interférant (RNAi) contre unegrande quantité de gènes de la drosophile, pour réaliser 3 des cribles génétiques destinés à identifier de nouveaux régulateurs du métabolisme et de la croissance. Dans le premier crible,les RNAi ont été induits dans la glande prothoracique, siège de la production de l’hormonestéroïde ecdysone connue pour réguler la croissance et les étapes du développement, souscontrôle de la nutrition et de la signalisation dTOR. Sur 7000 gènes criblés, 620 ont étéidentifiés comme nécessaire à la production d’ecdysone. Dans le second crible, nous avonsexprimé les RNAi de 4000 gènes dans le corps gras pour rechercher ceux qui induisaient uneaugmentation de la taille des LDs. L’objectif était d’identifier des gènes impliqués dans la réponse à la carence alimentaire, et nous avons ainsi retenu 24 candidats intéressants. Le troisième crible représente la majeure partie du travail de thèse, où nous avons criblé les RNAi susceptibles de modifier un phénotype de croissance induit par dS6K. Sur 7000 gènes testés,nous en avons retenu 45 qui ont ensuite été utilisés pour générer un diagramme d’interaction en utilisant les informations disponibles dans les banques de données. Les candidats les plus intéressants ont ensuite été analysés en culture de cellules pour identifier ceux qui régulent l’activité de dS6K et ceux qui régulent sont niveau d’expression. Parmi ces derniers, nousavons identifié le gène codant pour Archipelago (Ago), connue pour contrôler la dégradationrégulée des protéines-cibles au niveau du protéasome. Nous avons réalisé de nombreusesexpériences qui montrent que ago et dS6K interagissent génétiquement. En outre, il est indiquédans les banques de données que ces protéines interagissent entre elles par la technique des 2-hybrides en levure. Tous ces résultats révèlent que Ago régule la dégradation de dS6K, etposent les premières pierres de ce niveau de régulation. / The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.

No description available.

Cette thèse s’intéresse à la dynamique actuelle du développement durable dans l’entreprise et la prise en compte de la responsabilité sociale dans les processus stratégiques. En particulier, elle vise à caractériser les logiques de conception des systèmes de pilotage de la performance globale au sein d’entreprises pionnières au Maroc et la manière dont ces dernières cherchent à articuler exigence de conformation et logique d’innovation en matière de développement durable. La première partie de cette thèse est consacrée à l’élaboration du cadre conceptuel de la recherche, abordant les fondements du concept de la performance globale et analysant la portée et les limites des modèles conçus pour son pilotage. La deuxième partie de la thèse aborde empiriquement la problématique de la recherche. Après une première analyse des démarches des entreprises en matière de développement durable dans le contexte des pays en développement, sont présentés les résultats de deux études de cas multi-sites, réalisées au sein de deux entreprises pionnières au Maroc : Lafarge Maroc et Accor Maroc. Les modalités de déploiement de la démarche de développement durable et les caractéristiques des systèmes de pilotage de la performance globale sont analysées au plan de la stratégie, des structures organisationnelles et de l’instrumentation de gestion. La recherche met en évidence les contraintes spécifiques au contexte marocain qui rendent plus complexe le déploiement stratégique du développement durable. Ces contraintes impliquent un étalement et une gradation des engagements dans le temps permettant à l’encadrement intermédiaire et opérationnel de développer la capacité organisationnelle à porter les projets sociaux et environnementaux du sommet stratégique. On observe par ailleurs un renforcement des structures fonctionnelles et la mise en place de nouvelles démarches qui s’accompagnent d’un décloisonnement organisationnel interne et externe. La mobilisation du cadre d’analyse de la systémique ago-antagoniste a permis de concevoir le pilotage de la performance globale comme un processus d’équilibration sous tension de trois ensembles expansifs de couples ago-antagonistes : performance financière/performance sociale, contrôle/autonomie, routinisation/innovation, relevant respectivement de deux sphères de référence : financière et durable. / This thesis focuses on the current dynamics of sustainable development in the company and the inclusion of social responsibility in strategic processes. In particular, it aims to characterize the logical system design for controlling the overall performance of the pioneers in Morocco and how the latter seek to articulate and logical requirement conformation of innovation in sustainable development. The first part of this thesis is devoted to developing the conceptual framework of research, addressing the foundations of the concept of overall performance and analyzing the scope and limitations of models for its management. The second part of the thesis addresses the problem of empirical research. After an initial analysis of business processes for sustainable development in the context of developing countries, are presented the results of two case studies of multi-site, performed in two pioneering companies in Morocco: Morocco Lafarge and Accor Morocco. The arrangements for the deployment of sustainable development and characteristics of systems for controlling the global performance is analyzed in terms of strategy, organizational structures and management instrumentation.The research highlights the constraints to the Moroccan context that complicate the strategic deployment of sustainable development. These constraints imply a spreading and a gradation of the commitments in time for middle management and operational levels to develop the organizational capacity to carry the social and environmental projects of strategic summit. Moreover, there is a strengthening of the functional and the implementation of new approaches that are accompanied by an opening up our internal and external. The mobilization of the analytical framework of ago-antagonist systems has helped design the global performance management as a process of balancing power of three sets of expansive ago-antagonistic couples: financial performance / social performance, control / autonomy , routinization / innovation, falling within two reference spheres: financial sustainability.

<p>MicroRNAs (miRNAs) are small RNAs that have important roles in post-transcriptional gene regulation in a wide range of species. This regulation is controlled by having miRNAs directly bind to a target messenger RNA (mRNA), causing it to be destabilized and degraded, or translationally repressed. Identifying miRNA targets has been a large area of focus for study; however, a lack of generally high-throughput experiments to validate direct miRNA targeting has been a limiting factor. To overcome these limitations, computational methods have become crucial for understanding and predicting miRNA-gene target interactions.</p><p>While a variety of computational tools exist for predicting miRNA targets, many of them are focused on a similar feature set for their prediction. These commonly used features are complementarity to 5'seed of miRNAs and evolutionary conservation. Unfortunately, not all miRNA target sites are conserved or adhere to canonical seed complementarity. Seeking to address these limitations, several studies have included energy features of mRNA:miRNA duplex formation as alternative features. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement.</p><p>We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide interaction sites to Argonaute (AGO) protein family members, which are essential parts of the miRNA machinery complex. This trend is confirmed on data sets that assay the effect of miRNAs on mRNA and protein expression changes, where a statistically significant change in expression is noted when compared to the control. Furthermore, our method also allows for prediction of strictly imperfect sites, as well as non-conserved targets.</p><p>Recently, new methods for identifying direct miRNA binding have been developed, which provides us with additional sources of information for miRNA target prediction. While some computational target predictions tools have begun to incorporate this information, they still rely on the presence of a seed match in the AGO-bound windows without accounting for the possibility of variations. </p><p>We investigate the usefulness of the site level direct binding evidence in miRNA target identification and propose a model that incorporates multiple different features along with the AGO-interaction data. Our method outperforms both an ad hoc strategy of seed match searches as well as an existing target prediction tool, while still allowing for predictions of sites other than a long perfect seed match. Additionally, we show supporting evidence for a class of non-canonical sites as bound targets. Our model can be extended to predict additional types of imperfect sites, and can also be readily modified to include additional features that may produce additional improvements.</p> / Dissertation

No description available.

• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. • Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages. • This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. • The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.