The hormonal control of tomato (Solanum lycopersicum) fruit development have been extensively studied, mainly during ripening. Notwithstanding, considerable gaps still exist in our understanding of the function and spatial-temporal distribution of hormones during fruit development. Here, we performed histochemical analysis of tomato cv. Micro-Tom transgenic lines harboring the gene reporter GUS fused to five different promoter responsive to the hormones, auxin, cytokinins (CKs), gibberellins (GAs), abscisic acid (ABA) and ethylene, in order to redefine their spatial-temporal distribution during tomato fruit development, from early pre-anthesis stages to red ripe (RR) stage. CKs levels were high during pre-anthesis, initially in placental tissues and subsequently in ovule, indicating a major function of this hormone during pre-fertilized ovary growth. At early post-fertilization stages, CKs contents were high in seeds, and afterward in the outer and inner epidermical and subepidermical layer of pericarp. High auxin content was observed, during all pre-anthesis and fruit development stages, in pedicel, suggesting a basipetal transport to the mother plant. Ethylene contents increased during pre-anthesis ovary growth. Interestingly, ethylene contents did not decrease, immediately, post-fertilization. The content of GAs and ABA was low at pre-anthesis and early post-fertilization stages, specific in placental tissues and pericarp. Conversely, these hormones accumulated mainly during cell expansion phase of fruit growth. Pollen grains that reached the stigma, post anther dehiscence, showed high content of auxin and ABA. In addition, we reported an antagonism between ethylene and GAs contents during tomato fruit development, where the level of ethylene started to decrease during the cell expansion phase of the fruit growth, moment when the level of GAs started to increase in placental tissues and pericarp. / O controle hormonal do desenvolvimento do fruto do tomateiro (Solanum lycopersicum cv. Micro-Tom) já foi extensamente estudado, principalmente no amadurecimento. No entanto, ainda existem consideráveis lacunas em nosso conhecimento da função e distribuição espaço-temporal dos hormônios durante o desenvolvimento do fruto. Foram realizados ensaios histoquímicos com cinco linhas transgênicas de tomate cv. Micro-Tom que carregavam o gene repórter GUS fusionado a cinco diferentes promotores responsivos aos hormônios, auxina, citocinina (CKs), giberelina (GAs), ácido abscísico (ABA) e etileno, com o fim de redefinir a distribuição espaço-temporal deles ao longo do desenvolvimento do fruto, desde estágios iniciais pre-anteses até o estágio red ripe (RR). Os níveis de CKs foram altos na fase pre-anteses, inicialmente na placenta e posteriormente nos óvulos, indicando um papel importante deste hormônio no crescimento do ovário, pre- fertilização. Nos estágios inicias, post-fertilização, o conteúdo de CKs foi alto nas sementes e depois nas camadas epidérmicas e subepidérmicas internas e externas do pericarpo. Alto conteúdo de auxina foi observado, durante todos o desenvolvimento do fruto, no pedicelo, sugerindo o transporte basípeto de auxina para a planta mãe. Os níveis de etileno aumentaram durante o crescimento do ovário antes da anteses. Interessantemente, o nível de etileno não diminuiu imediatamente post-fertilização. Os conteúdos de GA e ABA foram baixos prévios a anteses e nos estágios inicias pre-fertilização, especificamente na placenta e pericarpo. Inversamente, o conteúdo desses hormônios aumentou na fase de expansão celular do crescimento do fruto. Os grãos de pólen que chegaram ao stigma, post deiscência das anteras, apresentaram altos níveis de ABA e auxina. Além disso, reportamos um antagonismo entre conteúdo de etileno e giberelina durante o desenvolvimento do fruto, onde o nível de etileno começou a decrescer durante a fase de expansão celular do crescimento do fruto, momento em que o nível de giberelina começou a aumentar na placenta e pericarpo.

The bacteroides group is a subdivision in the <I>Cytophaga-Flavobacterium-Bacteroides</I> phylum. This group is as phylogenetically distinct from other Gram-negative enterics, including <I>Escherichia coli</I>, as they are from Gram-positive organisms. Furthermore, there is no cross expression between genes of <I>E. coli</I> and <I>Bacteroides</I> species. It is thought that this difference in gene expression lies in part at the level of transcription initiation and is due to the sequences within the promoter region itself. A putative consensus sequence for <I>Bacteroides</I> promoters has been published by C. Jeff Smith&#146;s research group based on alignments of the sequences upstream of certain regulated genes. However, this consensus has not been found within all putative <I>Bacteroides</I> promoters. In this study, the promoter structure and function of a strong housekeeping <I>B. thetaiotaomicron</I> 16S rRNA promoter was examined and compared to an <I>E. coli</I> 16S rRNA promoter. Our hypothesis is that there are significant differences between the promoters of these two organisms. Analysis of <I>B. thetaiotaomicron</I> sequence upstream of the 16S rRNA gene has revealed the same overall structure known for <I>E. coli</I> 16S rRNA promoters in that there are two putative promoters separated by approximately 150 bp. However, the <I>B. thetaiotaomicron</I> 16S rRNA promoter contains the proposed <I>Bacteroides</I> &#151;7 and &#151;33 consensus sequences instead of the well known <I>E. coli</I> &#151;10 and &#151;35 consensus sequences. The biological activity of the<I> B. thetaiotaomicron</I> 16S rRNA full-length promoter was confirmed using a <I>Bacteroides lux</I> reporter system. A newly designed <I>Bacteroides lux</I> reporter was used to analyze specific regions of the <I>B. thetaiotaomicron</I> 16S rRNA promoter. In addition, by pairing the <I>B. thetaiotaomicron</I> 16S rRNA promoter with an <I>E. coli</I> ribosomal binding site, and vice-versa, the improved <I>lux</I> reporter was used to further confirm that the difference in gene expression between the two species lies at the level of transcription in <I>E. coli</I>. In <I>Bacteroides</I>, however, transcription and translation may work together to create a barrier to efficient gene expression of foreign genes. </P> / Master of Science

Avec de nombreuses qualités exceptionnelles (biocompatible et ostéogénique), la nacre représente un biomatériau naturel comme substitut osseux. Mais les composés ostéogéniques dans la nacre ne sont pas encore connus. Nos travaux visent à l’identification des composés ostéogéniques de la nacre. L’ESM (éthanol soluble matrix) est un extrait de la nacre qui est démontré ostéogénique. A partir d’ESM, nous avons essayé plusieurs approches pour cibler et identifier ces composés. Grâce au couplage des cellules MC3T3-E1 et d’ostéoblastes humains arthrosiques, nous avons démontré que la partie cationique d’ESM est ostéogénique, sans interaction avec la partie anionique. Le calcium joue un rôle dans l’activité ostéogénique d’ESM. Ensuite, nous avons créé une lignée cellulaire exprimant de manière stable un plasmide contenant un gène rapporteur ostéogénique (ATDC5 pMetLuc2 ColX promoteur). Grâce à cette lignée, nous avons découvert que les lipides et les sucres présents dans l’ESM ont un effet ostéogénique. Les peptides précipités par TCA sont aussi démontrés ostéogéniques, et ont conduit à leur identification partielle par LC-MS. Ces résultats nous permettent d’avancer plus loin et plus rapidement vers l’identification des composés ostéogéniques de la nacre et vers les applications de la nacre en orthopédie clinique / With many exceptional qualities (biocompatible and osteogenic), nacre represents a natural biomaterial as a bone substitute. However, the osteogenic compounds in nacre are not yet known. Our work aims at the identification of the osteogenic compounds in nacre. The ESM (soluble ethanol matrix) is an extract of nacre that is shown to be osteogenic. From the ESM, we have tried several approaches to target and identify these compounds. Thanks to the coupling of MC3T3-E1 cells and the human osteoarthritis osteoblasts, we demonstrated that the cationic part of the ESM is osteogenic, without interaction with the anionic part. Calcium plays a role in the osteogenic activity of the ESM. Then, we created a cell line stably expressing a plasmid containing an osteogenic reporter gene (ATDC5 pMetLuc2 ColX promoter). Thanks to this cell line, we found out that the lipids and sugars in the ESM have an osteogenic effect. The peptides precipitated by TCA are also demonstrated to be osteogenic, which have led to their partial identification by LC-MS. These results allow us to move farther and faster towards the identification of osteogenic compounds in nacre and the applications of nacre in clinical orthopaedics

La régulation des gènes est à l’origine de la diversité cellulaire en permettant aux cellules de se différencier et de se spécialiser. La régulation génique repose largement sur l’existence de séquences d’ADN non codantes dans le génome, appelées "éléments cis-régulateurs", qui vont permettre de recruter de nombreux facteurs de transcription afin de former d’importants complexes (nucléo)protéiques qui vont agir sur le niveau de transcription des gènes. Ce recrutement est notamment contrôlé par des modifications épigénétiques. Le développement des techniques de séquençage et des méthodes d’analyse bioinformatiques permettent d’intégrer de grandes quantités de données pour étudier le fonctionnement des éléments régulateurs. Dans un premier temps, l’intégration de l’ensemble des données ChIP-seq disponibles dans les bases de données nous a permis de créer un catalogue d’éléments régulateurs putatifs chez l’Homme. L’analyse de ce catalogue nous a alors mené à caractériser ces éléments et à mettre en évidence la complexité combinatoire des facteurs de transcription. Dans un deuxième temps, nous avons réalisé une étude basée sur l’analyse des éléments régulateurs impliqués dans la différenciation précoce des lymphocytes T chez la souris. Cette étude a permis de mettre en évidence deux niveaux de complexité impliqués dans la régulation des gènes : le premier est basé sur la combinatoire des facteurs de transcription au sein des éléments régulateurs et le second repose sur la combinatoire des éléments eux-mêmes. Finalement, nous avons développé une nouvelle technique d’analyse quantitative et à haut débit de l’activité régulatrice de régions génomiques chez les mammifères. / Gene regulation is responsible for cell diversity by allowing cell differentiation and specialisation. Gene expression regulation relies mainly on the existence of non-coding DNA sequences in the genome, called "cis-regulatory elements", which recruit numerous transcription factors to form (nucleo)protein complexes which act on the gene transcription level. This recruitment is controlled in particular by epigenetic modifications. The rapid development of sequencing technologies and bioinformatics methods makes possible the integration of large amounts of data to study regulatory elements. First, the integration of ChIP-seq data for all transcription factors available in public databases has allowed us to create an extensive catalogue of putative regulatory elements in the human genome. The overall analysis of this catalogue led us to further characterize these elements and to highlight the high level of combinatorial complexity of transcription factors in the genome. Secondly, we conducted a more specific study based on the analysis of the regulatory elements involved in the early differentiation of T-cells in mice. This study provided an opportunity to highlight two levels of complexity based on regulatory elements and involved in gene regulation: the first rests on the transcription factor combinatorial in regulatory elements and the second is based on the combinatorial of elements themselves within loci. Finally, to validate experimentally the regulatory elements, we have developed a new quantitative and high-throughput technique to assess the regulatory activity of genomic regions in mammals.

NUR77 est un récepteur nucléaire (RN) orphelin impliqué dans la régulation de processus biologiques dont la mort cellulaire, notamment dans la maladie de Parkinson (MP), découlant de la perte de neurones dopaminergiques, et dans le cancer du sein, résultant de la prolifération de cellules mammaires. NUR77 est impliqué dans le déclenchement et la protection de la mort cellulaire et son activité serait indépendante de la liaison d’un ligand. Nous avons émis l’hypothèse que l’activité de NUR77 est influencée par ses modifications post-traductionnelles (MPTs) et ses partenaires d’interactions. L’objectif général de cette thèse était de caractériser les MPTs et les partenaires d’interaction modulant l’activité de NUR77, dans des modèles de cellules en culture, afin de mieux comprendre ses fonctions biologiques - notamment dans la mort cellulaire. Le premier objectif de ce doctorat était de caractériser le rôle de la SUMOylation, une modification modulant l’activité des RN, chez NUR77, par des essais rapporteurs dans les cellules Human Embryonic Kidney 293 (HEK293). La surexpression de la E3 SUMO ligase PIASγ et/ou de l’isoforme 2 de la SUMO, protéines importantes dans la régulation de la SUMOylation chez les RN, a engendré un effet répresseur sur l’activité transcriptionnelle de NUR77. L’effet de PIASγ sur l’activité de NUR77 est modulé par la Sentrin SUMO-specific protease 1, qui hydrolyse la liaison des SUMO. Les mutations des résidus lysine dans des sites consensus de SUMOylation, de NUR77 (K102 et K577), empêchant cette MPT, ont causé des effets opposés sur son activité transcriptionnelle, suggérant le recrutement différent de corégulateurs de la transcription. Ces résultats combinés indiquent que la SUMOylation et les PIASγ et SUMO2 sont, respectivement, une MPT et des corégulateurs importants dans l’activité de NUR77. Le deuxième objectif de cette thèse était de caractériser l’interactome de NUR77 dans des HEK293 vivantes afin d’identifier les interacteurs pouvant moduler son activité, à l’aide d’une méthode de marquage des protéines proximales avec la biotine basée sur la peroxydase APEX2, combinée à la spectrométrie de masse. Ce procédé a identifié 336 potentiels interacteurs de NUR77, dont plusieurs connus. Des essais de coimmunoprécipitation et de coimmunofluorescence menés dans les HEK293 et dans les cellules du cancer du sein MCF-7 ont montré, respectivement, que la protéine régulatrice de l’apoptose Apoptosis Inhibitor 5 (API5), interagissait et colocalisait avec NUR77. La privation de sérum dans le milieu de culture des cellules et la diminution de l’expression de API5 a conduit à une augmentation des niveaux protéiques et de l’activité de NUR77 et à une diminution de la survie cellulaire. Ces données suggèrent que API5 constitue un régulateur de NUR77 dans les voies de signalisation associées à la mort cellulaire et que cette interaction pourrait constituer une cible pour moduler l’apoptose. Elles valident également l’approche d’identification d’interacteurs de NUR77. Les travaux de cette thèse ont donc permis de générer des outils pour caractériser l’activité de NUR77 et ont révélé des corégulateurs de cette activité. La poursuite de ces projets pourrait révéler le caractère opportun de cibler NUR77 comme modulateur de la mort cellulaire, notamment dans la MP et le cancer du sein. / NUR77 is an orphan nuclear receptor (NR) involved in the regulation of multiple cell biology processes including cell death, in particular in Parkinson's disease (PD), which results of the loss of dopaminergic neurons, and in breast cancer (BC), which is caused by the proliferation of mammary epithelial cells. NUR77 is involved in triggering and inhibiting cell death and its activity is believed to be independent of a ligand binding. We hypothesized that the regulation of NUR77 activity does not occur through a ligand, but through the influence of its post-translational modifications (PTMs) and its interaction partners. The general objective of this PhD project was to characterize the PTMs and the interacting partners that modulate the activity of NUR77 in cultured cell models, to better understand its physiological roles, in particular in the regulation of cell death. The first objective of this thesis was to characterize the role of SUMOylation, a modification that regulates NR activity, in regulating NUR77 transcriptional activity in reporter assays in Human Embryonic Kidney (HEK293) cells. Overexpression of the E3 SUMO ligase PIASγ or/and the isoform 2 of SUMO, both important regulators in SUMOylation of the NUR77 homolog NURR1, produced a repressive effect on the transcriptional activity of NUR77. The effect of PIASγ on the activity of NUR77 was shown to be modulated by the Sentrin SUMO-specific protease 1 protein, which removes SUMO tags on target proteins. In addition, mutations of lysine residues in SUMO consensus sites in NUR77 (K102 and K577) had opposite effects on its transcriptional activity, suggesting different recruitment of coregulators of transcription in the regions. The combination of these results indicates that SUMOylation is an important PTM for the regulation of NUR77 activity and that PIASγ and SUMO2 proteins are important transcriptional coregulators of NUR77. The second objective of this thesis was to evaluate NUR77 interactome in HEK293 living cells to identify the interactors that can modulate its activity, using a biotin-labelling method for proximal proteins based on the APEX2 peroxidase combined with mass spectrometry. This approach identified 336 potential interactors of NUR77, some that are consistent with the literature. Coimmunoprecipitation and coimmunofluorescence assays carried out in HEK293 cells and in MCF-7 breast cancer cell line have shown that the regulator of apoptosis Apoptosis Inhibitor 5 vi (API5), interacted and colocalized with NUR77. By depriving cells of serum and decreasing API5 expression, increased protein levels and activity of NUR77 was observed, as well as a decrease in cell viability. These data support that API5 is a regulator of NUR77 in its involvement in signalling pathways associated with cell death and that this interaction could be a target for modulating apoptosis. More generally, they validate the APEX2 tool which can be used to identify novel NUR77 interactors. In conclusion, the work of this thesis resulted in the generation of tools to better understand the activity of NUR77 and revealed important coregulators in this activity. The continued characterization of these interactors may provide opportunities to target NUR77 as a regulator of cell death, particularly in PD and in breast cancer.