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La viticulture dépend des conditions climatiques. Dans le contexte de réchauffement climatique, les changements de la vigne et du raisin sous l’effet des températures élevées vraisemblables pour les prochaines décennies pourraient modifier l’aire de répartition des cépages et même menacer la durabilité de la viticulture des régions chaudes. L’objectif de cette étude était donc d’analyser les effets de l’élévation de température sur la composition du raisin, du transcriptome au métabolome. L’utilisation d’un système « open top » passif au vignoble a permis d’augmenter la température autour des grappes de 0.5-1.6 °C en moyenne, une valeur compatible avec le réchauffement climatique prévisible. Les expérimentations ont été conduites sur Cabernet Sauvignon (CS) et Sauvignon Blanc (SB), de la nouaison à la surmaturité, à Bordeaux (France) et en Barossa Valley (Australie). Les analyses ont ciblé essentiellement les métabolites primaires, les composés phénoliques et aromatiques (l’IBMP, arôme de poivron vert; les précurseurs de 3SH, arôme de pamplemousse et la ß-damascenone, arômes floraux). En complément, des analyses RNA-seq et q-PCR ont été réalisées pour explorer la réponse transcriptomique à cet échauffement modéré en conditions réalistes de vignoble.L’échauffement modéré a peu affecté les concentrations en sucres, acides et total en acides aminés, mais a modifié la distribution des différents acides aminés. La composition en acides aminés s’est principalement différenciée suivant la variété, le stade de développement et le site expérimental.Les concentrations finales en IBMP n’ont pas été affectées par l’échauffement. Cependant, à la fermeture de la grappe, les baies de CS échauffées avaient une moindre concentration en IBMP associée à une sous-expression de VviOMT3. La concentration en IBMP des baies de SB échauffées n’a pas montré de différenciation, bien que les niveaux d’expression de VviOMT3 et VviOMT4 soient diminués. Les effets limités et dépendant du génotype suggèrent qu’une augmentation modérée de la température ne serait pas suffisante pour modifier significativement l’IBMP.Glut-3SH-Al était bien plus concentrée que Glut-3SH et Cys-3SH. le traitement échauffé a fait diminuer la présence de Glut-3SH-Al et Cys-3SH dans les baies de SB, en association avec une sous-expression de VviGST4. Par ailleurs, VIT_08s0007g01420 (GSTU8) a été réprimé par le traitement, et pourrait donc être un gène candidat potentiel impliqué dans la biosynthèse de précurseurs de 3SH.Pour les baies de CS, les concentrations en caroténoïdes totaux et celles des deux caroténoïdes majeurs (la lutéine et le β-carotène) n’ont pas réagi à l’augmentation de température. La zéaxanthine a montré une tendance à la diminution sous l’effet de l’échauffement, jusqu’à une diminution significative. Cette concentration plus faible pourrait limiter la biosynthèse de β-damascenone et expliquer la plus faible teneur en β-damascenone observée dans les baies à sur-maturité en cas de température élevée.Un total de 357 gènes (DEGs) ont été différentiellement exprimés en réponse à l’augmentation de température pour les échantillons de 2015 à Bordeaux. D’après l’analyse d’enrichissement « Gene Ontology », le traitement a principalement régulé quatre catégories en relation avec les microtubules, la paroi cellulaire, l’espace extracellulaire et l’activité des facteurs de transcription. 6 DEGs liés à la biosynthèse des anthocyanes ont été régulés négativement, ce qui pourrait expliquer, au moins en partie, la concentration réduite en anthocyanes totaux observée dans les baies de CS échauffées. En revanche, les tanins n’ont pas été affectés par l’augmentation de la température. / Viticulture depends on climate conditions during the growing season. In the context of global warming, any changes in viticulture caused by the rising temperatures expected for the next decades may alter the geographical distribution of grape varieties and even threaten the sustainability of viticulture in hot areas. The objective of this research was to investigate the effects of moderately elevated temperature on grape composition, both at metabolic and transcriptomic levels. A passive open-top heating system was applied in Cabernet Sauvignon (CS) and Sauvignon Blanc (SB) vines grown with standard practice in Bordeaux, France and the Barossa Valley, Australia (CS only) to increase the bunch zone mean temperature by around 0.5-1.6 °C, which was commensurate with the projected global warming. This moderate heating was applied from fruit-set to two weeks after harvest. Metabolites related to technical, phenolic and aromatic maturities (IBMP, the green pepper aroma, precursors of 3SH, grapefruit aroma, and β-damascenone, floral aroma) were assessed, together with transcriptome analysis via RNA-seq and q-PCR, in order to obtain a comprehensive view of berry responses to this moderately elevated temperature in realistic vineyard conditions.The moderately elevated temperature hardly affected the concentrations of sugars, organic acids and total amino acids, but it altered free amino acid composition depending on varieties, vintages and locations.The final concentrations of IBMP were not affected by warming condition in mature berries. However, the elevated temperature significantly reduced IBMP content and expression level of VviOMT3 (a known key gene of IBMP) in CS berries at bunch closure stage, while it reduced the expression levels of VviOMT3 and VviOMT4 at bunch closure stage without affecting IBMP concentration in SB berries. This limited and genotype-dependent effect of elevated temperature suggests that a moderate temperature elevation may not be sufficient to significantly modify IBMP.Glut-3SH-Al was much more concentrated than Glut-3SH and Cys-3SH. Reduced Glut-3SH-Al and Cys-3SH concentrations were associated with a significantly lower expression level of VviGST4 in heated SB berries. Meanwhile, VIT_08s0007g01420 (GSTU8), was down-regulated by elevated temperature and might be a potential candidate gene involved in the biosynthesis of precursors of 3SH.The concentrations of total carotenoids and two predominant carotenoids (lutein and β-carotene) were not altered by elevated temperature in CS berries, but zeaxanthin was reduced by elevated temperature and was significantly less concentrated at harvest. This lower concentration may limit the biosynthesis of β-damascenone and explain the observed lower β-damascenone concentration in post-ripening berries under elevated temperature.A total of 357 genes were differentially expressed (DEGs) in response to the elevated temperature in Bordeaux samples in 2015. Enrichment analysis of Gene Ontology showed that temperature mainly regulated four GO categories, including microtubule, cell wall, extracellular region, and transcription factor activity. 6 DEGs related to anthocyanins synthesis were down-regulated and it could explain, at least in part, the observed lower total anthocyanins in warmed CS. Conversely, tannins were not affected by elevated temperature.The results provide a better understanding of potential global warming effects on metabolite changes during berry development, along with novel molecular insights into the response of grape berry to moderate heating in vineyard conditions.

The production of Nitric Oxide (NO) under physiological conditions has beneficial roles in acting as a key signaling component of many biological processes as well as having an anti-microbial effect. However its effects following excess production by the inducible NO pathway is potentially detrimental in the pathogenesis of chronic inflammation including sepsis and several other inflammatory diseases. Understanding the mechanisms that regulate the expression of the inducible nitric oxide synthase (iNOS) responsible for producing the excessive amounts of NO in disease states is therefore critical. In this regards, experiments were carried out to identify the signaling pathways that may mediate this process, focusing specifically on the JAK/STAT cascade. The reason for selecting the latter is because our research group, amongst others, has carried out extensive work investigating other signaling pathways, including the mitogen activated kinases (MAPK). Moreover, studies have also been carried out in an attempt to identify the critical role of JAK/STAT signaling for iNOS induction. These studies however failed to conclusively demonstrate whether, as with the MAPKs, the JAK/STATs may also play an essential role. Furthermore there is indeed controversy in the literature with researchers unable to agree whether expression of iNOS does require JAK/STAT activation. Thus, the aim of the project described in this thesis was to establish unequivocally whether activation of the JAK/STATs preceeds induction of iNOS. The studies were extended to L-arginine transport as well because the latter is widely reported to be induced in parallel with iNOS and substrate supply to iNOS may be critical for sustained NO production. Changes in transporter activity as well as their expression profiles were assessed. All experiments were carried out in either rat aortic smooth muscle cells (RASMCs) or in the J774 macrophage cell line. These cell types were selected because RASMCs are one of the prime targets for induced NO production in vascular inflammation and the macrophages are involved in host defence, acting in part through NO production. To establish the role of JAK/STATs, pharmacological and molecular approaches were used. Pharmacologically, two inhibitors were used and these were AG490 and JAK inhibitor I. The former is reported to be a selective JAK2 inhibitor and the other blocks all known JAK proteins. The potential of the GTPases to regulate the induction of iNOS was also examined using selective inhibitor known to regulate these proteins. In addition to these drugs, siRNA targeting JAK2 was also exploited and western blotting was extensively used to detect expression of various proteins including iNOS, native and phosphorylated JAK2 and TYK2. Changes in iNOS activity was monitored by determining nitrite production using the Griess assay and L-arginine transport was monitored using tritiated arginine (L-[3H]arginine). RASMCs were treated with a combination of LPS (100 µg/ml) and IFN- (100 U/ml) and the macrophages with LPS (1 µg/ml) to induce iNOS and transporter activity. Consistent with previous reports, the above treatment of both cell types resulted in the expression of iNOS, production of NO and enhanced transport of L-arginine. These effects were not affected by AG490 but blocked by JAK inhibitor I. Furthermore, although both cell types expressed the key JAKs (JAK2 and TYK2), neither of these proteins were phosphorylated under conditions of induced NO production. Moreover, siRNA experiments showed that JAK2 expression could be abolished without any significant change in NO production, confirming that at least JAK2 may not be required for this process. Whether TYK2 is involved still remains to be resolved as the phosphor-protein could not be detected. However the conclusive siRNA knockdown studies could not be carried out due to time and cost constraints. Apart from iNOS and NO production, changes in induced L-arginine transport were also not significantly affected under the experimental conditions described above suggesting that like with iNOS, induction of L-arginine transport is independent of at least JAK2. Interestingly however, STAT-1 was phosphorylated and this was blocked by JAK inhibitor I but not AG490. Thus, STAT-1 activation may be essential but its activation may be independent of the JAKs. One possible alternate upstream activator of STAT-1 may be the GTPases. Indeed these proteins have been indicated to phosphorylate STAT-1 independent of the JAKs. However, in this project, inhibition of the GTPase pathway enhanced NO production and L-arginine transport suggesting that the GTPases downregulate these processes. In conclusion, the studies carried out in this thesis have shown that induction of iNOS, NO production and L-arginine transport in both RASMCs and J774 macrophages are independent of JAK2 but require STAT-1 activation which may be phosphorylated independently of the JAKs. The role of other JAKs such as TYK2 although unlikely, will need to be resolved using a more specific approach such as siRNA.

Au niveau des fonds océaniques, souvent considérés comme des déserts aux conditions de vie extrêmes, des oasis luxuriants de vie existent pourtant. Les sources hydrothermales et les suintements froids, principalement localisés au niveau des dorsales océaniques et des marges continentales, vont permettre le développement de communautés microbiennes et animales très particulières. Le Bassin de Guaymas, situé dans le Golfe de Californie (Mexique) présente la particularité de regrouper à la fois une zone de sources hydrothermales et une zone de suintements froids, situées à une soixantaine de kilomètres l’une de l’autre, et toutes deux recouvertes par une épaisse couche sédimentaire. Ces deux zones sont également colonisées par des étendues de bivalves, des buissons de vers tubicoles ainsi que de tapis microbiens blancs ou colorés comparables. Afin de mieux comprendre le fonctionnement global de ces deux types d’écosystèmes et notamment le rôle structurant des communautés microbiennes sédimentaires sur la répartition des différents assemblages de surface, les travaux entrepris dans cette thèse se proposaient d’étudier les communautés microbiennes sédimentaires associés à la zone de suintements froids (Marge de Sonora) et la zone de sources hydrothermales (Southern Trough) du Bassin de Guaymas. Pour cela, la diversité des communautés microbiennes (Bacteria et Archaea) de différents habitats caractérisés par une faune et des profils géochimiques particuliers, a été étudiée grâce à l’utilisation d’une technique de séquençage haut-débit, le pyroséquençage par la technique du 454, combinée à d’autres techniques comme le FISH ou la PCR quantitative. Cette étude a permis de déterminer que la structure et la diversité des communautés microbiennes dans ces sédiments étaient très spécifiques de ces environnements. Par ailleurs, les colonisateurs présents en surface des sédiments reflétaient des profils géochimiques et des communautés microbiennes très différentes au sein des sédiments. Dans les sédiments colonisés par les tapis microbiens, riches en méthane, les communautés microbiennes dominantes (ANME, Deltaproteobacteria…) utiliseraient notamment les émissions de méthane des couches sédimentaires profondes et produiraient d'importantes concentrations de sulfures, nécessaires à l’installation des communautés microbiennes thiotrophes de surface formant les tapis. Ces fortes concentrations en sulfure excluraient en revanche les communautés animales de ce périmètre. A contrario, dans les sédiments présentant de faibles concentrations en méthane et en sulfure, permettant l'installation d’assemblages faunistiques variés, les communautés microbiennes méthanotrophes anaérobies et productrices de sulfure étaient minoritaires. L'activité et le métabolisme de ces colonisateurs de surface pourraient par ailleurs permettre le développement des lignées microbiennes détectées dans ces habitats (MBG-D, Chloroflexi…). L'analyse des larges jeux de données obtenus au cours de cette étude nous a donc permis de mettre en évidence un système dynamique complexe fonctionnant en équilibre entre les communautés microbiennes sédimentaires, les organismes colonisant la surface du sédiment et la composition géochimique des eaux interstitielles. / Whereas the deep-sea environment is often considered to be a desert, hydrothermal vents andcold seeps provide “oases” of biological activity on the ocean floor. Vent and seep ecosystems support complex food webs based on microbial chemoautotrophic primary production. These hydrothermal vent and cold seeps ecosystems both release hydrocarbon- and sulfide-rich fluids,fueling various surface assemblages such as mat-forming giant bacteria or symbiont-bearinginvertebrates (e.g. bivalves, tubeworms). In the Guaymas Basin, the nearby presence at a few tens of kilometers of cold seeps and hydrothermal vents coupled with comparable sedimentary settings and depths offer a unique opportunity to assess and compare the microbial community composition of these ecosystems. Tobetter understand their overall functioning, we studied sedimentary microbial communities associated with cold seep and hydrothermal vent areas in the Guaymas Basin. The diversity of microbial communities inhabiting sediments was studied using high throughput sequencing (454pyrosequencing), combined with complementary approaches, such as FISH and quantitative PCR. This study reveals that sediments found in the Guaymas Basin were colonized by microbial communities typically found in these types of ecosystems. Our results revealed a high similarity between microbial communities composition associated with the cold seep and hydrothermal vent areas as a probable consequence of the sedimentary context. Nonetheless, thermophilic and hyperthermophilic lineages (e.g.: Thermodesulfobacteria, Desulfurococcales, etc) were exclusively identified in hydrothermally influenced sediments highlighting the strong influence of temperature gradients and other hydrothermally-related factors on microbial community composition. Furthermore, sediments populated by different surface assemblages show distinct porewater geochemistry features and are associated with distinct microbial communities. Indeed, in the sediments underlying microbial mats characterized by high methane porewater concentrations,microbial communities were dominated by anaerobic methane oxidizers (ANME), known to produce sulfide which provides high fluxes of sulfide to the seafloor. In contrast, sediment associated microbial communities underlying faunal assemblages were characterized by a lower biomass and lower methane porewater concentrations in sediments, limiting porewater sulfide concentrations. Without elevated and toxic sulfide concentrations, faunal assemblages can colonize the surface. Together, geochemical and microbial surveys indicate that porewater methane concentrations play an important role in the microbial community structure and subsequently in the establishment of the surface colonizers. Furthermore, presence and activity of the surface colonizers influence the underlying microbial communities probably because of modification of energy source availabilities. Finally, the existence of similar microbial populations between the two ecosystems also raises the question of their dispersal mechanisms. Our results support the hypothesis of a potential continuity among deep-sea ecosystems. In absence of physical borders, environmental conditions (temperature, specific compounds associated withhydrothermal fluids) might select specific and highly adapted microorganisms from the pool of microorganisms dispersed globally across the seafloor.