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Pour s’adapter à leur environnement, les plantes ont acquis la capacité de produire une grande quantité de métabolites spécialisés à partir d’un nombre limité de structures de base. Parmi les modifications apportées à ces structures de base, la réaction de glycosylation permet d’augmenter la solubilité du composé, de réduire sa toxicité et de contribuer à une meilleure stabilité de certaines molécules ayant pour conséquence la modification de leur transport et/ou de leur stockage. Les UDP-glycosyltransférases (UGT) forment une vaste famille de glycosyltransférases chez les plantes. Elles regroupent des enzymes glycosylant principalement des hormones et des phénylpropanoides en utilisant l’UDP-sucre comme donneur de sucre. L’objectif de ce travail consiste à contribuer à la caractérisation fonctionnelle de la famille des UGT72 chez le peuplier afin d’identifier le rôle de ses membres dans les processus développementaux liés aux arbres et dans leurs réponses au stress. Plusieurs membres de cette famille ont déjà été caractérisés chez d’autres espèces comme A. thaliana, M. truncatula et C. sinensis, mettant en évidence la capacité de certaines UGT72s à glycosyler les monolignols, une implication dans le processus de lignification, un rôle dans des processus de défense contre les pathogènes ou encore une fonction de détoxification de certains polluants. Parmi les 8 UGT72s identifiées chez le peuplier, nous avons montré qu’in vitro UGT72AZ2 glycosyle l’acide férulique et l’acide sinapique, UGT72B37 le p-coumaraldéhyde, le coniféraldéhyde, le sinapaldéhyde, le coniferyl alcool et le sinapyl alcool, UGT72B39 le coniféryl alcool et UGT72A2 la naringénine. Tous les membres de la famille UGT72 sont exprimés dans les tissus vasculaires, suggérant un rôle dans le développement vasculaire. La surexpression de UGT72AZ1 ou UGT72AZ2 provoque l’accumulation de glucosides de monolignols (respectivement coniférine et syringine ou coniférine seulement), sans toutefois affecter la quantité totale de lignine. Concernant la localisation subcellulaire, excepté pour UGT72A2, les UGT72s du peuplier sont localisées dans le réticulum endoplasmique et le noyau, suggérant respectivement, un rôle dans la régulation de la voie des phénylpropanoides et dans la maintenance de l’ADN. UGT72A2 se démarque des autres membres de cette famille, car elle est localisée dans les chloroplastes et dans des vésicules associées aux chloroplastes, suggérant un rôle dans la régulation des phénylpropanoides dans le chloroplaste et/ou dans la maintenance du chloroplaste. En appui de ces hypothèses, nous avons constaté que la photosynthèse est affectée dans les lignées sous-exprimant UGT72A2, provoquant un jaunissement des feuilles. De plus, les feuilles de lignées sous-exprimant UGT72A2 développent un stress oxydatif associé à une réduction de l’accumulation des flavonoïdes et de l’activité des enzymes antioxydantes, suggérant un rôle de UGT72A2 dans l’homéostasie des formes réactives de l’oxygène (ROS). / In order to adapt to their environment, plants have developed the capacity to produce a diversified range of specialized metabolites by modifying a core set of molecules. Among those modifications, glycosylation allows to increase the solubility, to reduce the toxicity and to stabilize compounds in order to modify their transport and/or allow their storage. The UDP-glycosyltransferases (UGT) forming the largest glycosyltransferase superfamily in plants, combine enzymes which glycosylate mainly hormones and phenylpropanoids by using UDP-sugar as sugar donor. The purpose of this dissertation is to contribute to the functional characterization of the UGT72 family in poplar to unravel the role of its members in tree developmental processes and in stress response. Members of this family already characterized in other species (e.g. Arabidopsis thaliana, Medicago truncatula and Camellia sinensis) have been found to glycosylate monolignols and some of them have been associated with lignification, defence against pathogens and detoxification of pollutants. Among the 8 UGT72s identified in poplar, we have shown that UGT72AZ2 glycosylates in vitro ferulic acid and sinapic acid, UGT72B37 p-coumaraldehyde, coniferaldehyde, sinapaldehyde, coniferyl alcohol and sinapyl alcohol, UGT72B39 coniferyl alcohol and UGT72A2 naringenin. All the UGT72 members are expressed in vascular tissues suggesting a role in vascular development. The overexpression of UGT72AZ1 or UGT72AZ2 in poplar triggers the accumulation of monolignol glucosides (both coniferin and syringin or only coniferin, respectively) but has no impact on lignin content. With respect to the subcellular localization, except for UGT72A2, poplar UGT72s are localized in the endoplasmic reticulum and in the nucleus suggesting a possible role in the phenylpropanoid pathway regulation and in DNA maintenance, respectively. UGT72A2 stands out from the other poplar UGT72s by being localized in the chloroplast and chloroplast associated bodies, suggesting a role in the phenylpropanoid regulation in chloroplasts and/or in chloroplast maintenance. Moreover, supporting these hypotheses, photosynthesis was affected in lines downregulated for UGT72A2, as shown by a leaf yellowing phenotype and an oxidative stress in these lines as compared to the wild type. The flavonoid biosynthesis and the activity of enzymes involved into the reactive oxygen species (ROS) scavenging seem to be reduced by the downregulation of UGT72A2 suggesting a role of this UGT in the ROS homeostasis. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

L'objectif du groupe Im3i est d'étudier les mécanismes moléculaires impliqués dans la relation symbiotique entre l'hôte et le microbiote intestinal afin de proposer des solutions pour le traitement de certaines pathologies humaines dans le contexte de la nutrition et de la sécurité alimentaire. Nous avons choisi une bactérie à Gram positif, Ruminococcus gnavus E1 , comme modèle d'étude. Cette bactérie, anaérobie stricte, a été isolée à partir du microbiote d'un adulte sain et son génome a été séquencé . L'étude in vivo a été menée sur des groupes de souris mono-colonisées par R. gnavus E1. Nos résultats montrent clairement une surexpression des gènes codant les fucosyltransférases et sialyltransférases intestinales au niveau du côlon. Les observations réalisées en microscopie confocale à l'aide de lectines ont confirmé ces résultats. L'étude in vitro nous a permis d'analyser l'impact des facteurs solubles présents dans le surnageant de R. gnavus E1 sur la glycosylation des cellules épithéliales intestinales. La caractérisation préliminaire de cette fraction soluble indique qu'elle est thermorésistante, de nature non lipidique et qu'elle possède une faible masse moléculaire (< 3 kDa). Nous pouvons conclure que les variations d'expression des ARNm des différentes glycosyltransférases des cellules en gobelet et des entérocytes entrainant un programme de réinitialisation des glycoprotéines du mucus impacté par R. gnavus E1. Cela ouvre des perspectives considérables dans le dialogue moléculaire qui s'établit entre l'hôte et les bactéries endogènes. / The aim of IM3I group is study molecular mechanisms involved in the symbiotic relationship between the host and the intestinal microbiota, accounting for both partners, in order to propose solutions to human health problems, especially in the context of nutrition and food safety. We have chosen a Gram positive bacteria, Ruminococcus gnavus E1, as a model. This bacterium, in strict anaerobia, is isolated from the dominant microbiota of healthy human for which the genome is currently being sequenced. In vivo, using animals that are mono-associates with R. gnavus E1, our RT-qPCR studies clearly show an up regulation of the expression of genes coding for intestinal fucosyltranferases and sialyltransferases, in the colon. This is also confirmed by confocal microscopy using lectins. Thus, R. gnavus E1 is able to reestablish the glycosylation profile. In vitro, we have studied the impact of soluble factors from the R. gnavus E1 supernatant. We found that the soluble supernatant fraction of R. gnavus E1 cultures differentially modulates the intestinal glycosylation process in HT29-MTX, Caco-2, independently or coculture 75% Caco-2 and 25% HT29-MTX cell lines. Preliminary characterization of this soluble fraction indicates that it is heat resistant, is not affected by elimination of lipids, and has a low molecular weight form (< 3 kDa). We conclude that the variation of expression of mRNAs coding for different glycosyltransferases in goblet cells and enterocytes proves the re-initiating program of glycoproteins and mucus protection layer by the dominant bacteria R. gnavus E1 and opens the prospect to a new host-indigenous bacteria molecular crosstalk in the intestine.

Cosmomicina é um antibiótico antitumoral produzido pela bactéria Streptomyces olindensis DAUFPE 5622. Estudos de expressão gênica demonstraram que genes cuja expressão esta relacionada a condições de estresse (dnaJ e 18hsp), assim como genes associados a via biossintética de cosmomicina, são expressos sob condições de produção do antibiótico. Genes que ainda tinham a função desconhecida foram selecionados (cosS e cosY) e foram realizados análises bioinformáticas destes atribuindo-lhes a função de regulador transcricional e ornitina ciclodesaminase, respectivamente. Um cassete para inativação desses genes foi construído visando a futura obtenção de mutantes nulos. Genes de glicosiltransferase (cosK e cosG) também apresentaram diferenças na expressão na presença do antibiótico. Neste trabalho, foi revelada a presença de uma hipotética glicosiltransferase que tem homologia com a B-daunosamine daunomy, glicosiltransferase envolvida na transferência de açúcares na biossíntese do antibiótico daunomicina. / Cosmomycin is an antitumoral antibiotic produced by the soil bacteria Streptomyces olindensis DAUFPE 5622. Gene expression studies established that stress condition genes like dnaJ and 18hsp, and cosmomycin biosynthetic pathways genes are expressed under antibiotic production. Also the genes cosS and cosY (unknowns function), were selected and analyzed by bioinformatics techniques attributing a transcriptional regulator and ornithine cyclodeaminase functions, respectively. A cassette was constructed in order to inactivate these two selected genes and generating void mutants. Another gene cosK, with glycosiltransferase function, also presented differences in its expression when the antibiotic is produced. We described in this work the presence of a hypothetical glycosiltransferase related with B-daunosamine daunomy, which transfers sugar molecules in the biosynthesis of daunomycin antibiotic.

Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria. To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle. In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms. WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>

Carbohydrates are ubiquitous components in nature involved in a range of tasks. They cover every cell and contribute both structural stability as well as identity. Lipopolysaccharides are the outermost exposed part of the bacterial cell wall and the primary target for host-pathogen recognition. Understanding the structure and biosynthesis of these polysaccharides is crucial to combat disease and develop new medicine. Structural determinations can be carried out using NMR spectroscopy, a powerful tool giving information on an atomistic scale. This thesis is focused on method development to study polysaccharide structures as well as application on bacterial lipopolysaccharides. The focus has been to incorporate a bioinformatics approach prior to analysis by NMR spectroscopy, and then computer assisted methods to aid in the subsequent analysis of the spectra. The third chapter deals with the recent developments of ECODAB, a tool that can help predict structural fragments in Escherichia coli O-antigens. It was migrated to a relational database and the aforementioned predictions can now be made automatically by ECODAB. The fourth chapter gives insight into the program CASPER, a computer program that helps with structure determination of oligo- and polysaccharides. An approach to determine substituent positions in polysaccharides was investigated. The underlying database was also expanded and the improved capabilities were demonstrated by determining O-antigenic structures that could not previously be solved. The fifth chapter is an application to O‑antigen structures of E. coli strains. This is done by a combination of NMR spectroscopy and bioinformatics to predict components as well as linkages prior to spectra analysis. In the first case, a full structure elucidation was performed on E. coli serogroup O63, and in the second case a demonstration of the bioinformatics approach is done to E. coli serogroup O93. In the sixth chapter, a new version of the CarbBuilder software is presented. This includes a more robust building algorithm that helps build sterically crowded polysaccharide structures, as well as a general expansion of possible components. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.</p>

La fusariose des épis est l’une des principales maladies des céréales, majoritairement causée par le champignon pathogène et toxinogène, Fusarium graminearum (Fg). Lors son développement in planta, le champignon produit des mycotoxines dommageables pour la santé humaine et animale, dont le déoxynivalénol (DON). De nombreux loci à effet quantitatif sur la résistance à Fg ont été identifiés chez le blé tendre. Certains d’entre eux ont été corrélés à la capacité à détoxifier le DON, en particulier par glucosylation sous l’action d’UDP-glucosyltransférases (UGT). Une UGT d’orge impliquée dans la conjugaison du DON a été identifiée en système hétérologue. Brachypodium distachyon (Bd) a récemment émergé comme modèle d’étude pour les céréales. Ce travail à l’aide d’approches transcriptomique et métabolomique a mis en évidence que lors de l’interaction avec Fg, Bd met en place des réponses macroscopiques, moléculaires et métaboliques similaires à celles connues chez le blé et l’orge. La recherche d’UGTs candidates capables de conjuguer le DON en DON-3-O-glucoside (D3G) chez Bd a permis l’identification d’un candidat. L’analyse fonctionnelle du gène correspondant a été conduite par des approches de mutagenèse et de surexpression. Ceci a montré une sensibilité accrue des lignées mutantes à la toxine et à l’agent pathogène. A l’inverse les lignées surexpresseurs ont montré une tolérance et résistance quantitative à la toxine et l’agent pathogène. Ces résultats ont été corrélés par la détection in planta de DON et D3G, dans des proportions variables selon les lignées. Ces résultats démontrent le rôle majeur que joue la glucosylation du DON dans l’établissement de la résistance observée chez Bd en réponse à Fg. / Fusarium head blight is a major cereal disease, mostly caused by the pathogenic and toxin-producing fungus, Fusarium graminearum (Fg). During its development in planta, the fungus produces mycotoxins harmful to human and animal health, including deoxynivalenol (DON). Many quantitative trait loci exhibiting an effect on resistance to Fg have been identified in wheat. Some of them were correlated with the ability to detoxify DON, particularly by glucosylation by UDP-glycosyltransferases (UGT). A barley UGT involved in the conjugation of DON was identified in a heterologous system. Brachypodium distachyon (Bd) has recently emerged as a model species for cereals. Using transcriptomic and metabolomic approaches, we show that when interacting with Fg, Bd implements macroscopic, molecular and metabolic responses similar to those known in wheat and barley. The search for UGT candidates able to conjugate DON into DON-3-O-glucoside (D3G) in Bd resulted in the identification of the Bradi5g03300 gene. Functional analyses of this gene showed increased sensitivity of the mutant lines to the toxin and to the pathogen. Conversely the overexpressor lines showed a tolerance to the toxin and quantitative resistance to Fg. These results were correlated with the detection of differential amounts of DON and D3G in the different lines. These results demonstrate the important role of DON glucosylation in the resistance establishment of Bd observed in response to Fg.

Cosmomicina é um antibiótico antitumoral produzido pela bactéria Streptomyces olindensis DAUFPE 5622. Estudos de expressão gênica demonstraram que genes cuja expressão esta relacionada a condições de estresse (dnaJ e 18hsp), assim como genes associados a via biossintética de cosmomicina, são expressos sob condições de produção do antibiótico. Genes que ainda tinham a função desconhecida foram selecionados (cosS e cosY) e foram realizados análises bioinformáticas destes atribuindo-lhes a função de regulador transcricional e ornitina ciclodesaminase, respectivamente. Um cassete para inativação desses genes foi construído visando a futura obtenção de mutantes nulos. Genes de glicosiltransferase (cosK e cosG) também apresentaram diferenças na expressão na presença do antibiótico. Neste trabalho, foi revelada a presença de uma hipotética glicosiltransferase que tem homologia com a B-daunosamine daunomy, glicosiltransferase envolvida na transferência de açúcares na biossíntese do antibiótico daunomicina. / Cosmomycin is an antitumoral antibiotic produced by the soil bacteria Streptomyces olindensis DAUFPE 5622. Gene expression studies established that stress condition genes like dnaJ and 18hsp, and cosmomycin biosynthetic pathways genes are expressed under antibiotic production. Also the genes cosS and cosY (unknowns function), were selected and analyzed by bioinformatics techniques attributing a transcriptional regulator and ornithine cyclodeaminase functions, respectively. A cassette was constructed in order to inactivate these two selected genes and generating void mutants. Another gene cosK, with glycosiltransferase function, also presented differences in its expression when the antibiotic is produced. We described in this work the presence of a hypothetical glycosiltransferase related with B-daunosamine daunomy, which transfers sugar molecules in the biosynthesis of daunomycin antibiotic.

No description available.