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<p>Streptomycetes biology and genetics encompasses a variety of interesting features including multicellular growth, rich secondary metabolite production, and extensive environmental sensory and response systems. The characteristically large genomes of streptomycetes makes studying the diverse external stimuli intricate and internal regulation of these gene systems a challenge. Currently, there does not exist an efficient, cost-effective method of high throughput gene expression analysis in streptomycetes. Luciferase reporters have been used successfully in <em>Streptomyces coelicolor</em> to measure select promoter activity, however, they have demonstrated limited success in other strains and are not favourable to gene expression studies on a larger scale. Here, I present pLHR, a novel luciferase-based reporter tool designed specifically for high throughput gene expression studies in streptomycetes as well as the preliminary results which support the further development of this tool for gene expression profiling in <em>S. coelicolor</em>. Once developed, pLHR may be used to generate libraries of <em>Streptomyces</em> reporter stains to measure promoter activity repeatedly under variable conditions for the duration of the organism’s complex lifecycle.</p> / Master of Science (MSc)

Le 29 juillet 1881, la loi sur la liberté de la presse marque le point de départ d’un développement sans précédent de la presse écrite en France. Un développement qui amène cette presse à jouer un rôle désormais essentiel dans la vie politique, sociale et culturelle de tous les français. Très rapidement, la presse du Puy-de-Dôme profite de ce bouleversement institutionnel pour se développer à son tour. Parallèlement au succès des romans feuilletons, l’actualité criminelle devient alors un des atouts commerciaux majeurs de cette presse écrite devenue populaire, et ce depuis le début des années 1860 et la naissance du Petit Journal. Tous les moyens sont bons pour faire voir à son lectorat. Les dépêches ne suffisent plus, il faut désormais se déplacer, enquêter, s’interroger et révéler, avec ou sans la collaboration des autorités judiciaires. En effet, la criminalité a depuis toujours suscité au sein de la population un éventail d’émotions aussi diverses que la peur, le dégoût, la curiosité, la réprobation et la fascination. En réponse à ces émotions, les révélations et les jugements d’actes criminels sont l’occasion pour la presse de multiplier les éloges ou d’émettre des critiques vis-à-vis du système politique et judiciaire, de s’inquiéter de la déchéance des valeurs morales, de s’effrayer des menaces anarchistes et des monstres tapis dans les ruelles et les champs. Plus que de simples outils d’information et de politisation, les journaux deviennent alors par le biais de l’actualité criminelle le relais des interrogations et des convictions de toute une société. Le but de cette réflexion est de mettre en avant les spécificités de cette représentation médiatique de la criminalité en insistant sur sa dimension provinciale et sur son évolution, des premières années du Second Empire aux dernières heures de la Belle Époque. / On July 29th 1881, the law on the freedom of the press marked the starting point of an unprecedented development in the French written press which led to its playing what then became an essential role in the social, political and cultural life of all French people. Before long, the press in Puy-de-Dôme benefited from this development, to evolve in the same way. In parallel with the success crime reports brought to serialised fiction, these crime reports became one of the major commercial assets of this newly popular written press, and had done so in particular from the beginning of the 1860s and the founding of the Petit Journal. Newspapers would stop at nothing to show the readership what was involved; dispatches no longer sufficed. From then on it became necessary to go and see, investigate, reason things out and disclose information, with or without the collaboration of the judicial authorities. Indeed, crime has always aroused a wide range of emotions in people such as fear, disgust, curiosity, reprobation and fascination. In response to these emotions, the revelations of and verdicts given for criminal acts provided the press with an opportunity to endlessly praise or else to express criticism of the political and legal system, to show concern about the decline in moral values and to be scared of anarchistic threats and of monsters hiding in alleyways and fields. More than being simple informational or politicizing tools, newspapers, through crime reports, then became the indispensable relay of the questionings and convictions of society as a whole. The aim of this reflection is to highlight the specificities of this media representation of crime while insisting on the form it took in the provinces and on its evolution in the first few years of the Second Empire and the final hours of the Belle Époque.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

Rendue possible par les NTIC, une nouvelle forme de journalisme numérique privilégiant la parole publique a pris son essor. Les amateurs se retrouvent désormais au coeur de l’actualité en tant que source ou témoin. Ce phénomène est présent partout dans le monde, mais la Corée du Sud est considérée comme le pays où la participation des amateurs est la plus active. Dans ce pays, les expériences du journalisme « citoyen » sont liées à la logique d’une alternative aux médias de masse. Le succès du site OhmyNews, considéré comme précurseur du journalisme participatif, est dû à la volonté des Coréens de se saisir d'une liberté de parole dont ils ont été privés jusqu'à la fin des années 80. Initiés par ce succès, de multiples pure players et d’innombrable plateformes participatives sont apparus. Ce phénomène a favorisé le développement du journalisme « amateur » et a engendré la fin de l’ère du monopole des médias traditionnels. Le journalisme amateur à l'heure d'internet est-il et a-t-il les moyens d'être une véritable voix d'expression populaire ? Cette recherche montre que la pratique des amateurs permet une nouvelle forme de l'expression médiatique, mais produit désormais une surabondance de l’expression privée. En particulier, les sites-portails génèrent des recettes publicitaires en exploitant le contenu apporté par les utilisateurs en privilégiant de l’information divertissante. Leur domination sur le marché des médias en ligne est le facteur le plus inquiétant non seulement pour les médias traditionnels mais également pour l’avenir de l’information. En parallèle, nous avons observé l’évolution de la culture de la participation politique depuis l’explosion d’Internet. Les internautes informés par diverses plateformes ont participé activement dans les lieux de discussion proposés par la plupart des sites d’actualités. Ces espaces numériques, en favorisant l’apparition d’une nouvelle scène de débat public, ont accru les capacités de mobilisation des citoyens. Mais, cela a finalement provoqué un contrôle trop sévère d’Internet par l’Etat. Dans ce contexte, le journalisme amateur nous semble instrumentalisé par la logique du marché, tandis que l’espace de la parole citoyenne est de plus en plus réduit dans le champ politique. / Thanks to the NTIC, a new form of digital journalism has arisen, which favours public speech. Amateur journalists now find themselves at the heart of the news, as a source or as a witness. This phenomenon is observed everywhere in the world, but South Korea is thought to be the country where amateur journalists are the most involved. In this country, experiments in "citizen" journalism are linked to the logic of an alternative to mass media. The success of the website Ohmynews, considered as the forerunner of participative journalism, is due to the Koreans' desire to grasp the freedom of speech they have been deprived of until the end of the 80s’. Following this success, numerous pure players and countless participative platforms have appeared. This phenomenon has fostered the development of "amateur" journalism and triggered the end of an era of traditional media monopoly. Still one may asks whether, in the internet era, amateur journalism has the means to be a real voice for popular expression. This research shows that the practice of journalism by amateurs allows a new form of media expression, but at the same time produces a superabundance of private expression. In particular, portal sites generate advertising revenues and highlights entertainment news among the content brought by the users. Their domination over the market of online media is the most worrying factor not only for traditional media but also for the future of information as a whole. At the same time, we have observed, in the internet population, an evolution in the culture of political involvement. The net surfers, who are informed through various platforms, have actively taken part in the spaces of discussion available on most news information websites. These digital spaces have encouraged the emergence of a new scene of public debate which, have increased the means of mobilization of digital citizens. But in the end, it has provoked very severe measures of State control. In this context, it appears that amateur journalism is undermined due to the prevailing rules of marketing, and that the space for citizen speech is more and more reduced.

Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...

Mitochondrial processing peptidase (MPP) is a heterodimeric enzyme which belongs to M16B subfamily of metaloendopeptidases. A universal function of this enzyme is in recognition and cleavage of great number of mitochondrial preprotein presequences, which differ in length and amino acid sequence. MPP consists of catalytical β-MPP and probably recognizing α-MPP. The most conservative region in α-MPP is GRL - glycine-rich loop. Its function is supposed in primary interaction with preprotein presequence. It is possible to study conformational change of GRL after binding the substrate by fluorescence experiments. In this diploma thesis the constructs coding the α-MPP with the single reporter tryptophan residue in the position 289 or 299 were prepared using site-directed mutagenesis. These forms of α-MPP were produced in E. coli BL21(DE3)+pGroESL. Activities of MPP dimer containing α-MPP with the single tryptophan residue in the reporter position were compared with MPP from wild type of S. cerevisiae. Used substrate was yeast malate dehydrogenase precursor with fused presequence (pMDH) from three organisms (yeast, mouse and melon). These presequences differ in their length. Activities of MPP dimer containing α-MPP with the single reporter tryptophan residue in the position 289 were about 70 % while...