Les fucosyltransférases sont les enzymes responsables du transfert d'un groupement fucose à partir du GDP-fucose sur des accepteurs variés (oligosaccharides, protéines,...). Chez l'homme, les rôles de ces glycosyltransférases sont importants dans un grand nombre de processus biologiques et pathologiques. De nombreuses fucosyltransférases existent chez les végétaux. Notamment FuT1, qui transfert un fucose en 1,2 sur un résidu galactose du xyloglucane : l'une des hémicelluloses majeures de la paroi des dicotylédones. Ce polysaccharide ramifié est très étudié en raison de ses applications actuelles et potentielles dans différents secteurs de l'industrie : textile, alimentation, pharmaceutique, etc. Les objectifs de ce doctorat ont été d'obtenir des informations biochimiques et structurales sur la fucosyltransférase AtFuT1 de la plante modèle Arabidopsis thaliana. Pour cela, une forme recombinante soluble de l'enzyme a été produite avec le système baculovirus /cellules d'insectes. De manière à obtenir suffisamment de protéines pour les études structurales, une méthode de culture en suspension des cellules a été mise en place au laboratoire. Un protocole de purification en deux étapes, impliquant une chromatographie par affinité puis par exclusion de taille, a permis d'obtenir à partir d'un litre de culture cellulaire entre un à deux mg de protéine pure et homogène. Ce résultat a permis de mieux comprendre le comportement de l'enzyme vis-à-vis de ses substrats (GDP-fucose et xyloglucane), d'obtenir des cristaux de la protéine et de résoudre la structure 3D d'AtFuT1 en complexe avec le GDP et un oligosaccharide dérivé du xyloglucane, à une résolution de 2,2 Å. La forme AtFut1 produite se comporte en solution et dans le cristal sous forme d'un dimère non covalent. La protéine adopte un repliement qui est un variant du type GT-B classique. Parallèlement à ces travaux, des tests d'activité glycosyltransférase ont été mis au point permettant le criblage de nombreuses conditions de réactions. L'ensemble des méthodes et techniques développées constitue une base utile, devant faciliter la caractérisation d'autres glycosyltransférases. / Fucosyltransferases are enzymes that transfer a fucose residue from GDP-fucose on varied acceptors (oligosaccharides, proteins). In Human, these glycosyltransferases are involved in many biological and pathological processes. Numerous fucosyltransferase exist in the plant kingdom. Among them, FuT1 transfers a fucose linked in 1,2 onto a galactose of xyloglucan: a major hemicellulose of dicots cell wall. This branched polysaccharide is intensively studied because of its current and potential industrial applications in textile, food, pharmaceuticals, etc. The main objective of this PhD program was to obtain biochemical and structural information on the fucosyltransferase AtFuT1 from the model plant Arabidopsis thaliana. A recombinant form of this protein has therefore been produced, using the baculovirus/insect cell system. In order to get sufficient amount of protein for structural studies, a suspension cell culture method has been set-up in the lab. A two-step purification protocol, involving affinity and size exclusion chromatography was established. The active, and highly pure recovered protein was used to determine the biochemical properties of the protein towards its substrates (GDP-fucose and xyloglucan), to get protein crystals and hence to solve its 3D structure in complex with GDP and a xyloglucan derived oligosaccharide (2.2 Å resolution). AtFuT1 behaves in solution and in crystallo as a non-covalent dimer. The protein adopts a variant of the classical GTB fold. In addition, novel glycosyltransferase assay have been designed allowing the screening of numerous reaction conditions. Methods and techniques that were developed during this study should be a useful base for the characterization of other glycosyltransferase.

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan β1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor, of RA receptor α, implicating RA receptor α in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.

No description available.

The research presented in this dissertation consists of two primary parts. The initial focus centered on understanding the distribution of Golgi resident glycosyltransferases between the ER and Golgi at steady-state. Retrograde trafficking of these Golgi proteins has been demonstrated experimentally mandating the existence of a dynamic equilibrium between the Golgi apparatus and ER. Our published studies also included the development of a quantitative method for analysis of data collected using fluorescent microscopy. The second part of this dissertation presents results pertaining to the quantification of a unique Golgi resident protein that cycles in the late endosome bypass pathway. Using the published method of analysis and techniques developed during the initial project, the anterograde and retrograde transport kinetics of this Golgi protein were determined and used to develop a compartmental model for pH sensitive trafficking in the bypass pathway. The spatial Golgi distribution of the protein during retrograde transport to the Golgi following endosomal exit was also investigated. This research lies at the interface of experimental cell biology and quantitative computational analysis. These experiments combined more traditional experimental biological approaches with more recent computational approaches to understanding cellular mechanisms. Additionally, development of a quantitative method of analysis validated the use of fluorescent microscopy as a quantitative tool for studying intracellular proteins. / Ph. D.

Les chaînes de glycosaminoglycanes (GAGs) des protéoglycanes (PGs) jouent un rôle majeur dans la régulation de multiples événements cellulaires et le maintien de l'architecture des tissus. Des perturbations de la synthèse des GAGs sont impliquées dans des pathologies d'origine dégénérative, tumorale et génétique, tel que le syndrome progéroïde d'Ehlers-Danlos (ED). Ce déficit résulte de mutations de la [bêta]1,4-galactosyltransférase 7 ([bêta]4GalT7) humaine associées à des atteintes sévères du système musculo-squelettique. En effet, cette enzyme catalyse une étape essentielle de l?initiation de la synthèse des GAGs à partir de la protéine "core" des PGs et de xylosides exogènes. Notre travail a porté sur l'étude structure-fonction de la [bêta]4GalT7 recombinante humaine. Nous avons associé des approches in vitro et ex vivo afin d?explorer le rôle des acides aminés des motifs 163DVD165, 221FWGWGREDDD230 et 257HLH259, strictement conservés au sein des [bêta]4GalTs. L'étude des conséquences de mutations systématiques sur les propriétés cinétiques et fonctionnelles de la [bêta]4GalT7 recombinante a permis d'identifier des acides aminés essentiels du site actif. Nous avons montré que les résidus D165 et H257 forment des liaisons de coordination avec le cation Mn2+ et proposé le rôle du résidu D228 dans la catalyse. Nous avons mis en évidence un rôle central du résidu W224 dans les interactions avec les substrats donneur et accepteur. Nous avons également établi les bases moléculaires des mutations de la [bêta]4GalT7 associées au syndrome ED. Enfin, l'étude de mécanismes de régulation épigénétique des voies de biosynthèse des GAGs dans les cellules H-EMC-SS de chondrosarcome humain a mis en évidence une hyperméthylation spécifique des gènes de la famille des 3-O-sulfotransférases, associée à un phénotype invasif. L'ensemble de ce travail ouvre des perspectives vers de nouvelles stratégies thérapeutiques dans le traitement des arthropathies / Proteoglycans (PGs) and their glycosaminoglycan chains (GAGs), play a major role in the architecture of extracellular matrices and are implicated in numerous cell events. The impairment of GAG synthesis and sulfation is involved in degenerative, tumor and genetic diseases, such as the progeroid form of Ehlers-Danlos (ED) syndrome. This inherited disorder is due to mutations of human [bêta]4GalT7 ([bêta]4GalT7) causing a defect in GAG synthesis, associated with severe musculo-skeletal alterations. Indeed, this enzyme catalyzes a key step in GAG synthesis linked to the core protein of PGs and from exogenous xylosides. Our work has been focused on the structural and functional characterization of human recombinant [bêta]4GalT7 enzyme. We combined in vitro and ex vivo approaches to explore the role of amino acids located in 163DVD165, 221FWGWGREDDD230 and 257HLH259 motifs, which are highly conserved within [bêta]4GalTs. The study of the consequences of site-directed mutations on kinetic and functional properties of the [bêta]4GalT7 enzyme allowed us to identify key active site amino acids. Our results indicate that D165 and H257 residues form coordination bonds with Mn2+ divalent cations. Furthermore, we suggested a catalytic role for D228 residue and highlighted a central role of W224 residue via interactions with the donor and acceptor substrates. We also determined the molecular basis of [bêta]4GalT7 mutations associated with ED syndrome. Finally, the study of epigenetic regulation mechanisms by DNA methylation of GAG biosynthesis in human chondrosarcoma cells (H-EMC-SS) revealed the specific hypermethylation of the 3-O-sulfotransferase gene family, associated with the invasive phenotype of these cells. Together, this work paves the way towards innovative strategies in the treatment of arthropathies

Les glycosyltransférases (GTs) sont une famille importante d’enzymes responsable de la biosynthèse des chaînes de glycosaminoglycane (GAG) des protéoglycanes, composants clés de la matrice extracellulaire et de la membrane plasmique cellulaire impliqués dans la communication, l'adhésion, la migration et la prolifération cellulaires. Les GTs jouent donc un rôle central dans de nombreux processus physiologiques et physiopathologiques tels que les cancers ou encore les maladies dégénératives et génétiques. Parmi ces GTs, la ß1,4-galactosyltransférase 7 (ß4GalT7) est une cible thérapeutique potentielle puisqu'elle : i) catalyse une étape précoce et majeure de la biosynthèse des chaînes de GAG, ii) est impliquée dans une forme rare de maladie génétique des tissus conjonctifs, le syndrome d’Ehlers-Danlos, iii) prend en charge des xylosides exogènes modulant son activité in vitro et in vivo. Ce travail de thèse s'organise sur une étude structure/fonction de cette enzyme afin de cerner les résidus d'acides aminés clés dans l'interaction de l'enzyme avec un substrat modèle, le 4-methylombelliferyl-ß-D-xylose (4-MOX). Les résidus Y194, Y196 et Y199 ont ainsi été identifiés comme clés dans l'architecture du site de fixation du substrat accepteur et dans l'interaction de l'enzyme avec le xyloside. Au contraire, les résidus H195, R226 et le résidu R270, muté dans la forme progéroïde du syndrome d'Ehlers-Danlos, apparaissent comme des résidus « modulant » l'activité de l'enzyme, notamment du fait d'interactions moléculaires impliquant leur squelette peptidique pour H195 et R226 et une boucle flexible pour R270. Ces travaux ont permis de guider la synthèse d’analogues xylosidiques visant à inhiber l'activité de la ß4GalT7 humaine. Parmi les propositions, un dérivé fluoré du 4-MOX apparaît comme un inhibiteur efficace de la ß4GalT7 in vitro et in cellulo. Faiblement cytotoxique, ce dérivé réduit la prolifération des cellules de lignées cancéreuses SW1353 et MB MDA 231. Ces résultats ouvrent la perspective de nouvelles stratégies thérapeutiques utilisant les xylosides comme agents potentiels dans le traitement de cancers ou encore des maladies génétiques des tissus conjonctifs / Glycosyltransferases (GTs) are an important family of enzymes involved in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans which are key components of cell plasma membranes and of the extracellular matrix, and are thus implicated in cell communication, adhesion, migration and proliferation. GTs are thus key players in numerous pathophysiological processes such as cancers, degenerative and genetic diseases. Among these GTs, ß1,4-galactosyltransférase 7 (ß4GalT7) is a potential therapeutic target since : i) it catalyzes a rate-limiting step in the early phase of the GAG chains biosynthesis, ii) it is implicated in a rare genetic connective tissue disorder (Ehlers-Danlos Syndrome), iii) its in vitro and in vivo activity can be modulated by exogenous xyloside molecules. This PhD work is focused on a structure/function study of the enzyme aiming to identify key amino acid residues that interact with 4-methylumbelliferyl-ß-D-xylose (4-MUX), taken as reference substrate. Y194, Y196 and Y199 have been identified as key residues for the architecture of the acceptor substrate binding site and establish interactions with 4-MUX. By contrast, H195, R226 and R270, a residue mutated in the progeroid form of Ehlers-Danlos Syndrome, should rather be considered as “modulating” residues towards the ß4GalT7 activity, H195 and R226 interacting with 4-MUX with their polypeptide backbone, and R270 via a flexible loop. This work guided the design of xylosidic compounds that would potentially inhibit the ß4GalT7 activity. Thus, a fluorinated derivative of 4-MUX appeared as an efficient in vitro and in cellulo inhibitor of the enzyme. Poorly cytotoxic, this compound also reduced the proliferation rate of cancer cells SW1353 and MB MDA 231. Altogether, these results offer new therapeutic strategies using xylosides as potential therapeutic agents in the treatment of cancer or rare genetic disorders

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2018-01-09T11:15:21Z No. of bitstreams: 1 marcospaulomiola_dissert.pdf: 10829870 bytes, checksum: 279acd690e09b25b71e67463ae29eb6b (MD5) / Made available in DSpace on 2018-01-09T11:15:21Z (GMT). No. of bitstreams: 1 marcospaulomiola_dissert.pdf: 10829870 bytes, checksum: 279acd690e09b25b71e67463ae29eb6b (MD5) Previous issue date: 2017-03-13 / Introduction. ABO histo-blood group system is the most important transfusional system and the identification of its phenotypes is often performed by means of direct and reverse typing, which must always present concordant results. However, some genetic factors such as natural chimerisms and point mutations in the ABO gene may affect the expression of the antigens and antibodies of this system, contributing to the discrepancy in the phenotyping, requiring investigations to define the correct phenotype of receptors and blood donors. Objectives. The main objective of this study was to investigate the variations in the expression of the antigens of the ABO histo-blood group system. Its specific objectives were: 1. Selection of recipients and blood donors that presented discrepancies between the results of the direct and reverse phenotyping of the ABO histo-blood group system; 2. Investigation, using serological and molecular methods, of the causes of phenotypic changes and discrepancies between the results of direct and reverse phenotypes in the ABO histo-blood group system in the recipients and donors of blood. Material and Methods. Samples of recipients (n = 2) and blood donors (n = 7) presenting discrepancies between the direct and reverse phenotyping were selected. Phenotyping were performed using conventional and modified hemagglutination methods in tubes and gel columns with commercial antisera and lectins. Molecular investigations were performed using PCR-RFLP method and sequencing of exons 6 and 7 of the ABO gene and exon 2 of the FUT2 gene. Results. Four cases with poor expression of antigen A and absence of expected antibody, observed in hemagglutination, were identified as A2B, Ael and Aw. Four cases without antigenic alteration but carrying an irregular antibody anti-A1 or absence of expected antibody were characterized as AB, A1 and O and presented common ABO alleles. A case of non-dizygotic twins, phenotyped as AB and with double red blood cell population was characterized as hematopoietic chimera after extensive family analysis. The DNA extracted from buccal swab revealed the ABO (A101/B101) and FUT2 (SE*25.01.01/SE*25.01.01) genotypes in the male twin and the ABO (O01/O02) and FUT2 (SE*01.04.01/SE*01.06.03) genotypes in the female twin. Sequences of two new ABO (ABO*Aw.38; KT906366.1) and FUT2 (SE*01.06.03; KX550421) allele sequences were deposited on GenBank. Conclusions. Our results demonstrate that the use of serum and salivary serological assays combined with molecular methods are good tools to solving discrepancies between the direct and reverse phenotyping of the ABO histo-blood group system as well as elucidate cases of twin chimerism in humans, with a double population of red blood cells. In addition, they contribute to the identification of new alleles of the ABO and FUT2 genes. / Introdução. O sistema histo-sanguíneo ABO é o de maior importância transfusional e a identificação de seus fenótipos é frequentemente realizada por meios das tipagens direta e reversa as quais sempre devem apresentar resultados concordantes. Entretanto, alguns fatores genéticos como quimerismos naturais e mutações pontuais no gene ABO, podem afetar a expressão dos antígenos e anticorpos deste sistema, contribuindo com a discrepância nas fenotipagens, requerendo investigações para se definir o correto fenótipo de receptores e doadores de sangue. Objetivos. O objetivo geral deste estudo foi investigar as variações na expressão dos antígenos do sistema histo-sanguíneo ABO. Seus objetivos específicos compreenderam: 1. Seleção de receptores e doadores de sangue que apresentaram discrepâncias entre os resultados das fenotipagens direta e reversa do sistema histo-sanguíneo ABO; 2. Investigação, com o uso de métodos sorológicos e moleculares, das causas das alterações fenotípicas e discrepâncias entre os resultados das fenotipagens direta e reversa no sistema histo-sanguíneo ABO nos receptores e doadores de sangue. Material e Método. Foram selecionadas amostras de receptores (n=2) e doadores (n=7) de sangue com discrepâncias entre as fenotipagens direta e reversa. As fenotipagens foram realizadas com o uso dos métodos de hemaglutinação convencional e modificada, em tubos e colunas de gel, com antissoros comerciais e lectinas. As investigações moleculares foram realizadas com o uso dos métodos PCR-RFLP e sequenciamento dos exons 6 e 7 do gene ABO e do exon 2 do gene FUT2. Resultados: Quatro casos com fraca expressão do antígeno A e ausência do anticorpo esperado, observados na hemaglutinação, foram identificados como A2B, Ael e Aw. Quatro casos sem alteração antigênica, mas com presença de anticorpo irregular ou ausência do anticorpo esperado, foram caracterizados como AB, A1 e O e apresentaram alelos comuns. Um caso de gêmeos não dizigóticos, fenotipados como AB e com dupla população de hemácias foi caracterizado como quimera hematopoiética, após extensa análise familiar. O DNA extraído de swab bucal revelou os genótipos ABO (A101/B101) e FUT2 (SE*25.01.01/SE*25.01.01) no gêmeo masculino e os genótipos ABO (O01/O02) e FUT2 (SE*01.04.01/SE*01.06.03) no gêmeo feminino. As sequências de dois novos alelos dos genes ABO (ABO*Aw.38; KT906366.1) e FUT2 (SE*01.06.03; KX550421) foram depositadas no GenBank. Conclusões: Nossos resultados demonstram que o uso de análises sorológicas eritrocitárias e salivares combinadas a métodos moleculares são fundamentais na resolução de discrepâncias entre as fenotipagens direta e reversa do sistema histo-sanguíneo ABO bem como no esclarecimento de casos de quimerismo gemelar em humanos, contendo dupla população de hemácias. Além disso, contribuem para a identificação de novos alelos dos genes ABO e FUT2.

La glycosylation des protéines est une modification co/post-traductionnelle, localiséeprincipalement dans l’appareil de golgi, impliquée dans divers processus physiologiques. Àl’inverse de la synthèse des protéines et des nucléotides, celle des sucres est plus complexe,en partie, à cause des nombreux branchements structuraux et de la diversité stéréochimiquedes glycannes. Les glycosyltransférases sont les enzymes responsables de la biosynthèse desoligo- et polysaccharides. Cependant, il existe différents procédés permettant de réaliser lasynthèse in vitro d’oligosaccharides tels que des procédés chimiques, enzymatiques ouchimio-enzymatiques. La Core 2 β(1,6)-N-acétylglucosaminyltransférase I (C2GnT-I) est uneglycosyltransférase (GT) transmembranaire de type II qui crée une liaison β1,6 entre une Nacétylglucosamine(GlcNAc) et la N-acétylgalactosamine (GalNAc) d’un noyau Core 1,formant ainsi une structure branchée de type Core 2. Une fois le branchement Core 2 initié,au moins trois réactions successives de transfert peuvent avoir lieu, impliquant les β(1,4)-galactosyltransférases, les α(2,3)-sialyltransférases et les α(1,3)-fucosyltransférases. Il estadmis que les GTs sont réparties séquentiellement dans les compartiments cis, médian ettrans de l’appareil de Golgi selon leur ordre d’intervention et le type cellulaire. Nous avonsmis au point un procédé de reconstitution membranaire, comportant des glycoprotéinesgolgiennes, dont les GTs, par l’intermédiaire d’une lectine, la wheat germ agglutinin (WGA).Il est admis que la WGA interagit avec les composants de la membrane plasmique et ceux del’appareil de Golgi. L’étude de notre système membranaire reconstitué a mis en évidence uneactivité élevée de la C2GnT-1 in vitro, et son efficacité à synthétiser des oligosaccharidesbranchés Core 2, par glycosylation séquentielle. / Protein glycosylation is a co/posttranslational modification, localized in the Golgi apparatus,involved in various physiological processes. Sugar synthesis is more complex than that ofproteins and nucleic acids, in part because of the glycosidic bond and glycan stereochemistrydiversity. Glycosyltransferases are enzymes responsible of oligo- and polysaccharidesbiosynthesis. Various methods are used for the synthesis of oligosaccharides, in vitro, suchas chemical, enzymatic or chemo-enzymatic. Core 2 β(1,6)-N-acetylglucosaminyltransferase I(C2GnT-I) is a type-II transmembrane glycosyltransferase (GT). This enzyme create a β1,6bond between N-acetylglucosamine (GlcNAc) and Core 1 N-acetylgalactosamine (GalNAc),forming a Core 2 branched structure. Once the Core 2 branch is initiated, at least threesuccessive transfer reactions can take place, involving β(1,4)-galactosyltransferases, α(2,3)-sialyltransferases and α(1,3)-fucosyltransferases. It is known that GT are distributedsequentially in the cis, medial and trans Golgi apparatus in order of intervention andaccording to cell type. We have developed a membrane reconstitution process comprisinggolgi glycoproteins, including GT, by the use of a lectin, the wheat germ agglutinin (WGA). Itis known that WGA interacts with plasma membrane and Golgi apparatus components. Ourreconstituted membrane system showed a high C2GnT-1 activity in vitro and its effectivenessin synthesizing Core2 branched oligosaccharides by sequential glycosylation.

L'induction du métabolisme secondaire fait partie du système de défense des plantes lors d’une attaque par un microorganisme pathogène. Les propriétés antimicrobiennes ou de signalisation des métabolites secondaires (MS) peuvent être régulées par un processus majeur et efficace, la glycosylation. Cette réaction est catalysée par les glycosyltransférases (UGTs) qui assurent le transfert d’une molécule de sucre sur un MS. Mon travail de thèse a porté sur l’étude de trois UGTs d’Arabidopsis thaliana (Arabidopsis), UGT76E12 d’une part, et UGT73B3 et UGT73B5 d’autre part, et a montré que 1) UGT76E12 est impliquée dans la mise en place de la résistance basale à la souche virulente de la bactérie Pseudomonas syringae pv. tomato, Pst DC3000, et participe au dialogue entre les voies de signalisation hormonale de l’acide jasmonique (JA) et de l’acide salicylique (SA). Des analyses par clustering hiérarchique ont révélé une co-expression forte entre le gène UGT76E12, et deux gènes codant une terpène synthase (TPS4) et un cytochrome P450 (CYP82G1), impliqués dans la biosynthèse d’un diterpène, le TMTT ((E,E)-4,8,12-triméthyletridéca-1,3,7,11-tetraène), suggérant un rôle pour UGT76E12 dans la régulation de la voie de biosynthèse des terpènes ; 2) UGT73B3 et UGT73B5 sont impliquées dans la mise en place de la résistance spécifique d’Arabidopsis à la souche avirulente Pst DC3000-AvrRpm1, notamment par la régulation de MS intervenant dans le maintien de l’état redox cellulaire au cours de la réaction d’hypersensibilité (HR). Ces deux gènes appartiennent à la classe des « SA-early induced-genes » et présentent un profil co-expression avec le gène TOLB-related et deux gènes codant des glutathion S-transférases (GSTU7 et GSTU24) impliqués dans la réponse au stress oxydant et la détoxication des MS. Nos résultats suggèrent un rôle pour UGT73B3 et UGT73B5 dans la glycosylation de métabolites oxydants associés à la mise en place de la HR. / Plant secondary metabolism induction is part of an integrated defense system after pathogen infection. Signalling and antimicrobial properties of secondary metabolites (SM) can be regulated by an efficient process named, glycosylation. This reaction is ensured by glycosyltransferases (UGTs) which catalyze the transfer of a sugar moiety on a SM. My PhD work was divided in two parts, the first on UGT76E12 and the second part on UGT73B3 and UGT73B5, three UGTs of Arabidopsis thaliana (Arabidopsis) and it demonstrated that 1) UGT76E12 is involved in basal resistance establishment during plant challenge with the virulent strain of Pseudomonas syringae pv. tomato (Pst DC3000), and participates to the hormonal crosstalk between jasmonic acid (JA) and salicylic acid (SA). Clustering analyses revealed a strong co-expression between UGT76E12 and two genes encoding a terpene synthase (TPS4) and a cytochrome P450 (CYP82G1), both involved in the biosynthesis of a diterpene named TMTT ((E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene), which suggests a role for UGT76E12 in terpene biosynthesis pathway regulation. 2) UGT73B5 and UGT73B3 are involved in the establishment of Arabidopsis specific resistance against the avirulent strain of Pst DC3000-AvrRpm1, especially in the regulation of MS playing a role in redox homeostasis status during the hypersensitive response (HR). These two genes are part of SA-early induced-genes and showed co-expression patterns with the TOLB-related gene and two genes encoding glutathione S-transferases (GSTU7 and GSTU24) involved in oxidative stress responses and in MS detoxification. Our results suggest a role for UGT73B3 and UGT73B5 in the glycosylation of oxidant MS associated with HR establishment.

Carbohydrates are abundant in nature and have functions ranging from energy storage to acting as structural components. Analysis of carbohydrate structures is important and can be used for, for instance, clinical diagnosis of diseases as well as in bacterial studies. The complexity of glycans makes it difficult to determine their structures. NMR spectroscopy is an advanced method that can be used to examine carbohydrates at the atomic level, but full assignments of the signals require much work. Reliable automation of this process would be of great help. Herein studies of Escherichia coli O-antigen polysaccharides are presented, both a structure determination by NMR and also research on glycosyltransferases which assemble the polysaccharides. The computer program CASPER has been improved to assist in carbohydrate studies and in the long run make it possible to automatically determine structures based only on NMR data. Detailed computer studies of glycans can shed light on their interactions with proteins and help find inhibitors to prevent unwanted binding. The WaaG glycosyltransferase is important for the formation of E. coli lipopolysaccharides. Molecular docking analyses of structures confirmed to bind this enzyme have provided information on how inhibitors could be composed. Noroviruses cause gastroenteritis, such as the winter vomiting disease, after binding human histo-blood group antigens. In one of the projects, fragment-based docking, followed by molecular dynamics simulations and binding free energy calculations, was used to find competitive binders to the P domain of the capsid of the norovirus VA387. These novel structures have high affinity and are a very good starting point for developing drugs against noroviruses. The protein targets in these two projects are carbohydrate binding, but the techniques are general and can be applied to other research projects. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript. Paper 6. Manuscript.