Les glycosyltransférases sont responsables de la biosynthèse d’oligosaccharides, de polysaccharides et de glycoconjugués. Etant donné le nombre croissant de processus biologiques reliés aux saccharides, il existe un grand intérêt pour la compréhension des rôles biologiques des ces saccharides et étudier leurs applications thérapeutiques potentielles. Ce projet concerne la conception, la synthèse, l’évaluationbiologique et l’analyse structurale de deux nouveaux types d’inhibiteurs de glycosyltransférases analogues du substrat donneur naturel. L’analogie repose sur l’incorporation d’une unité pyridine ou amino-acide neutre comme mime du motif pyrophosphate afin de fournir des substrats capables de pénétrer les cellules pour des applications potentielles in cellulo ou in vivo. Les synthèses d’inhibiteurs neutres de GTs ont été réalisées en utilisant une combinaison de réactions de conjugaison créant une liaison O-glycosidique, amide ou triazole. Au total, 26 inhibiteurs neutres de GTs ont ainsi été synthétisés. L’évaluation de l’inhibition pour cinq galactosyltransférases et une GlcNAc-transférase(OGT) a révélé des inhibitions modestes de l’ordre du micromolaire. La co-cristallisation des meilleurs inhibiteurs avec l’une de ces galactosyltransférases a démontré que le motif pyridine neutre chélate le cation manganèse impliqué dans le site catalytique de l’enzyme. Cependant, le motif galactose est orienté vers l’extérieur du site catalytique et loin de la position initiale du substart naturel (UDP-Gal) etindique donc un nouveau mode de liaison. Le concept d’inhibiteur neutre a aussi été examiné sur un système modèle de membrane pour leur perméation membranaire. / Glycosyltransferase is an important class of enzyme in living organisms responsible for the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates. As more and more carbohydrate related biological processes are elucidated, there is great interest to define the biological roles of a given carbohydrate and examine its potential therapeutic applications. The present study reports the design, synthesis, biological evaluation and structural analysis of two novel types of glycosyltransferase donor substrate analogues. The design rationale is to make analogues of sugar nucleotide diphosphate substrates, but incorporating a ‘neutral’ pyridine or amino-acid moiety as thepyrophosphate surrogate in order to provide cell permeable substrates for potential in cellulo or in vivo applications. The syntheses of “neutral” GTs inhibitors were performed using a combination of conjugations through O-glycoside bond, amide bond or triazole functionalities. A total number of 26 “neutral” GT inhibitors were prepared. The evaluation of inhibition towards five galactosyltransferasesand one GlcNAc-transferase (OGT) revealed moderate inhibitions in the micromolar range. More interestingly, co-crystallyzation could be achieved for the most potent compounds in complex with a glycosyltransferase. The designed ‘neutral’ pyridine linker could chelate the manganese cation involved in the enzyme catalytic site. Whereas the sugar head-group was oriented away from the position found inthe related complex with natural substrate (UDP-Gal) indicating a new binding mode. The concept of ‘neutral’ inhibitor was examined by an artificial cell membrane penetration test.

Le microbiote désigne l'ensemble des microorganismes (bactéries, virus, champignons) vivant dans un environnement spécifique, en particulier chez un hôte (humain, animal ou végétal). La relation symbiotique existant entre le microbiote et son hôte a été mise en évidence dans de nombreux contextes. Le rôle protecteur du microbiote a été démontré chez l'homme, dans diverses pathologies, dont des infections bactériennes. Le microbiote colonise l'ensemble des muqueuses, dont l'intestin, où il est le plus abondant. Bien que le rôle du microbiote intestinal ait été beaucoup décrit, l'existence de bactéries commensales dans les poumons a été mise en évidence plus récemment. D'abord sujette à controverse, l'existence d'un microbiote pulmonaire, dont la composition est distincte de celle de l'intestin, et qui peut être altérée en conditions pathologiques, est maintenant bien établie. Il est également établi que le microbiote d'un organe peut agir sur la physiologie d'autres organes ; ainsi on parle par exemple d'un axe " intestin-poumons " pour désigner l'action de composés solubles produits par le microbiote intestinal, ainsi que de cellules immunitaires ou cytokines de l'intestin, véhiculés par le sang ou la lymphe, sur la physiologie du poumon. Les poumons sont une cible majeure pour la colonisation par des pathogènes. La tuberculose (TB), une inflammation chronique pulmonaire causée par la bactérie Mycobacterium tuberculosis, est encore aujourd'hui la pathologie respiratoire due à un agent étiologique unique la plus meurtrière. A ce jour la complexité des mécanismes mis en jeu pour expliquer la différence de susceptibilité à la TB entre les individus n'est pas encore complètement comprise. Il est proposé que la balance entre virulence de la souche de M. tuberculosis, et statut immunitaire de l'hôte pourrait expliquer l'inégalité entre les individus face au développement de la maladie. Ici nous avons émis l'hypothèse que le microbiote de l'hôte serait un facteur influençant l'interaction hôte-pathogène dans la TB via i) la modulation de l'immunité antituberculeuse et/ou ii) la physiologie (métabolisme, virulence) du pathogène. Mon travail de thèse a permis de montrer que l'élimination du microbiote par un traitement antibiotique à large spectre conduit à une colonisation plus importante des poumons par M.[...] / The microbiota refers to all microorganisms (bacteria, viruses, fungi) living in a specific environment, especially in a host (human, animal or plant). The symbiotic relationship existing between the microbiota and its host has been demonstrated in many contexts. In particular, it is now well-established that the microbiota plays a protective role during different human pathologies, including bacterial infections. The microbiota colonizes all the mucosal membranes of the body; particularly the intestine where it is more abundant. While role of the gut microbiota has already been widely studied, the existence of bacteria in the lungs has been described more recently. Even if at first controversial, the existence of a pulmonary microbiota, whose composition is distinct from that of the intestine, and which can be altered in pathological conditions, is now well established. It is also well-established that the microbiota of an organ can act on the physiology of other organs; for instance, a "gut-lungs" axis is used to designate the action of soluble compounds produced by the intestinal microbiota, as well as immunes cells or cytokines from the gut, carried by the blood or the lymph, on the physiology of the lung. The lungs are one of the major colonization site for different pathogens. Tuberculosis (TB), a chronic pulmonary inflammation caused by the bacteria Mycobacterium tuberculosis, is still today the most lethal respiratory disease due to a single etiological agent. To date, the complexity of the mechanisms explaining the difference in susceptibility to TB between individuals has not been fully understood yet. It has been suggested that the balance between virulence of the strain of M. [...]

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The Lattrell-Dax method of nitrite-mediated substitution of carbohydrate triflates is an efficient method to generate structures of inverse configuration. In this study it has been demonstrated that a neighboring equatorial ester group plays a highly important role in this carbohydrate epimerization reaction, inducing the formation of inversion compounds in good yields. Based on this effect, efficient synthetic routes to a range of carbohydrate structures, notably β-D-mannosides and β-D-talosides, were designed. By use of the ester activation effect for neighboring groups, a double parallel as well as a double serial inversion strategy was developed. / QC 20101111

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Doctor of Philosophy / Department of Human Nutrition / Mark D. Haub / Maximizing performance and results in competitive events is something that all athletes strive to achieve. Nutritional strategies have been developed to best optimize the likelihood of success in competitive events. While dietary protein was classically believed to be the key macronutrient in exercise performance, overwhelming evidence now supports the role of maximizing carbohydrate intake and availability in endurance performance. The role of carbohydrate intake prior to, during and after endurance exercise has been heavily studied and relevant literature will be discussed herein. This paper consists of three chapters and a summary related to carbohydrate intake and performance outcomes in endurance sports. While nutritional status surrounding the endurance events is discussed, this paper focuses on the ergogenic and metabolic effects of carbohydrates during the endurance bout. Chapter one serves as a literature review of carbohydrate administration during endurance exercise. Types of carbohydrates, their role as substrates in liver and skeletal muscle during exercise, and their effects on endurance performance are discussed. The role of carbohydrate on central factors of fatigue and motor output also are covered. Chapter two addresses the role of multiple carbohydrate supplements on cycling performance. The role of these supplements on blood glucose, insulin, lactate, and IGFBP-1 also are discussed. Chapter three addresses the effect of nutritional status prior to exercise on the ability of a carbohydrate mouth rinse to impart a performance enhancing effect. There were no treatment effects (p>0.05) of the type carbohydrate ingested, compared with placebo, on selected metabolic and performance outcomes. Likewise, there was no ergogenic effect of mouth rinsing, in the fasted or fed state, in moderately trained endurance cyclists.

Streptococcus pneumoniae is a commensal inhabitant of the human nasopharynx that can sometimes become pathogenic and cause diseases such as pneumonia, otitis media and meningitis. Carbohydrate metabolism is a critical component of S. pneumoniae virulence. Among the myriad of carbohydrate-specific pathways involved in the host-pneumococcus interaction, the N-glycan foraging pathway stands out because of its direct implication in numerous aspects of virulence such as fitness, adhesion/invasion and impairment of the host immune response. Much of the literature has been focussed on the importance of step-wise depolymerisation of N-glycans by the enzymes NanA, BgaA and StrH. However, the importance of the liberation of N-glycans from host glycoconjuguates and their intake by the bacterium has yet to be examined. We have identified a Carbohydrate Processing Locus (CPL) that is highly conserved throughout a large number of Firmicutes and whose individual components appear widespread in bacteria that we hypothesize is active on host N-glycans. This locus encodes for two putative α-mannosidases GH92 and GH38, a characterised α-mannosidase GH125, a putative β-hexosaminidase GH20C, a putative α-fucosidase GH29 and a ROK (Repressor, Open reading frame, Kinase) protein. The genomic context of CPL orthologues suggests that an endo-β-N-acetylglucosaminidase (EndoD) and an ABC transporter (ABCN-glycan) are functionally associated with this locus. Based on our bioinformatic analyses and known functions of these proteins we hypothesize that the CPL encodes a concerted pathway responsible for the liberation, transport, and processing of N-glycans. The objective of this research is to characterize the putative components of this pathway and assess their implication in virulence. Specific focus on ABCN-glycan demonstrated its specificity for a range of N-glycans liberated by EndoD, shedding light on a novel import system for branched N-glycans. Furthermore, we provided evidence that GH92 is an α-1,2-mannosidase that likely removes the terminal mannose residues found on high-mannose N-glycans. EndoD and GH92 are shown to participate in virulence in mice; however, their role in virulence has yet to be determined. This work will significantly advance the construction and validation of a model of N-glycan processing by S. pneumoniae. As the components of this model pathway are conserved amongst a wide variety of bacteria, this work is of fundamental relevance to understanding how microbes from various environments degrade and metabolize N-glycans. / Graduate

Lipopolysaccharides (LPS), major virulence determinants in Gram–negative bacteria, are responsible for many pathophysiological responses and can elicit strong immune responses. In order to better understand the role of LPS in host–pathogen interactions and elucidate the immunogenic properties of LPS outer core oligosaccharide, an all α–linked Escherichia coli R3 outer core pentasaccharide was first synthesized with a propyl amino linker at the reducing end. This oligosaccharide was also covalently conjugated to a carrier protein (CRM197) via the reducing end propyl amino linker. An immunological analysis demonstrated that this glycoconjugate can elicit specific anti-pentasaccharide antibodies with in vitro bactericidal activity. These findings will contribute to further exploring this pentasaccharide antigen as a vaccine candidate. Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification and biofunctional studies of HMOs remain a big challenge due to their diversity and complexity. The accessibility of homogenous HMOs library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse HMOs was reported. Based on 3 versatile building blocks and 4 robust glycosyltransferases, a library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications. Glycoproteins are extremely important for all life on the planet. Glycoproteins play important roles in various biological processes. Increasing evidences demonstrate that glycosylation of proteins could improve stability of proteins by stabilizing their tertiary structure and protecting them from proteolysis. Besides, glycosylation of proteins could provide targeting effects through glycan-lectin interaction. Furthermore, carbohydrates play crucial roles in humoral immunity in that many sugar epitopes are identified as antigens for antibodies. Glycoprotein could boost strong T cells mediated intercellular immune responses because homogeneous antigens present on the surface of proteins by multivalent bonds. In this study, the three advantages of glycoproteins, namely stabilizing proteins, targeting effects and eliciting immunological response, were extensively explored by broad collaboration with other groups.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy Stellenbosch University. / ENGLISH ABSTRACT: Phosphorus (P) is essential for the survival of all living organisms and forms part of several key biological molecules and processes. The basic biological function of all cells depends on the availability of P as structural element in phospholipids and nucleic acids. P plays a central role in the energy metabolism of the cell by activating metabolic intermediates of carbohydrate metabolism and by acting as an energy currency in the form of adenosine tri-phosphate (ATP). ATP is produced during photosynthesis from the energy derived from sunlight, probably the most important biological process on earth. The balance of P supply and demand is of critical importance here. Plants assimilate P in the form of orthophosphate (Pi) via its roots and utilises complex mechanisms to redistribute and balance the Pi concentrations throughout the plant. These processes are collectively known as phosphate homeostasis and in this study we utilised molecular techniques to study some key aspects of this complex network of mechanisms in the plant Arabidopsis thaliana. When the role of the PHT1;5 Pi transporter was investigated in photosynthesis under Pi limitation a new mechanism utilised by plants to supply Pi for the production of ATP in the chloroplast was discovered. During periods of adequate Pi supply plants make use of the triose phosphate / phosphate translocator (TPT) to exchange Pi for phosphorylated carbon intermediates. This transporter does, however, not function at the low Pi concentrations present during Pi limitation and the plant therefore express an alternative transporter i.e. PHT1;5. Together with this transporter several genes were identified that was expressed to allow the export of carbon intermediates from the chloroplast via starch turnover. Amongst these, several alternative isoforms of the enzymes responsible for starch turnover are expressed during Pi limiting conditions. It is therefore suggested that the products of starch degradation, e.g. glucose and maltose are the potential candidates for carbon export from chloroplasts under Pi limiting conditions. In an attempt to perturb the Pi concentrations in the Arabidopsis vacuole we expressed the three genes of a newly discovered polyphosphate (PolyP) polymerase from the yeast Sacharomyces cerevisiae in Arabidopsis. This enzyme complex accumulates PolyP in the yeast vacuole and since the plant vacuole is playing a key role in buffering Pi concentrations we anticipated some observable effects that could lead to the elucidation of the mechanisms involved. Production of PolyP was conclusively shown in plant callus, but it was only at very low concentrations with no detectable perturbing effect and undetectable in whole plants. With the aim to apply this technology to the PolyP and PHT1;5 lines developed in the other parts of this study, newly developed fluorescent indicator protein nanosensors (FLIPPi) were evaluated as a method for detecting and monitoring in vivo Pi concentrations in multicellular plant organs. This technique is capable of detecting changes in metabolite concentrations in real-time and it was applied to the roots of Arabidopsis seedlings subjected to Pi limitation. We specifically looked at changes in the cytosol, but our results revealed no detectable changes occurring in the Pi concentrations in this compartment. This was interpreted to indicate lower levels of Pi in this compartment as was previously expected. / AFRIKAANSE OPSOMMING: Fosfaat (P) is essensieël vir die oorlewing van alle organismes en maak deel uit van etlike kern biologiese prosesse en molekules. Die basiese biologiese funksionering van alle selle hang direk af van die beskikbaarheid van P as strukturele element van fosfolipiede en nuklëinsure. Fosfaat speel 'n sentrale rol in die energie metabolisme van 'n sel deur metaboliese intermediante te aktiveer en deur op te tree as die geld eenheid van sellulere energie in die vorm van adenosien tri-fosfaat (ATP). ATP word gegenereer gedurende fotosintese vanaf die energie wat van sonlig vasgevang word, dit is waarskeinlik die belangrikste biologiese proses op aarde. Dit is van kritiese belang dat die fosfaat vraag en aanbod hier fyn gebalanseer word. Plante assimileer P in die vorm van ortofosfaat (Pi) deur hulle wortels en maak gebruik van komplekse meganismes om Pi deur die plant te versprei en konsentrasies te balanseer. Hierdie prosesse staan gesamentlik bekend as fosfaat homeostase en in die huidige studie het ons gebruik gemaak van molekulêre tegnieke om 'n paar belangrike aspekte van hierdie komplekse netwerk van prosesse in die plant Arabidopsis thaliana te bestudeer. Toe die rol van die PHT1;5 Pi transporter in fotosintese onder toestande van Pi tekort bestudeer is, is 'n nuwe meganisme ontdek waarmee plante Pi verskaf aan chloroplaste vir die proses van fotosintese onder toestande van Pi tekort. Gedurende periodes wat die plant genoegsame Pi tot sy beskikking het, word van die triose fosfaat / fosfaat uitruiler (TPT) gebruik gemaak om Pi uit te ruil vir gefosforileerde koolstof metaboliete. Hierdie transporter kan egter nie onder die lae Pi konsentrasies wat voorkom in die sitoplasma onder Pi tekort toestande funksioneer nie, en gevolglik moet die plant van 'n alternatiewe transporter naamlik PHT1;5 uitdruk. Verskeie ander gene is ook geidentifiseer wat saam met hierdie transporter onder toestande van Pi tekort uitgedruk word en die plant toelaat om koolstof tussengangers uit die chloroplaste uit te vervoer via die proses van stysel produksie en afbraak. Onderandere is verskeie alternatiewe isoforme van die gene wat verandwoordelik is vir stysel produksie en afbraak identifiseer wat uitgedruk word onder toestande van Pi tekort. In 'n poging om die Pi konsentrasies in die Arabidopsis vakuool te versteur is drie gene van die nuut ontdekte polifosfaat (PolyP) polimerase kompleks van die gis Sacharomyces cerevisiae in Arabidopsis uitgedruk. Hierdie ensiem kompleks is verandwoordelik vir die akkumulasie van PolyP in die gis vakuool en siende die plant vakuool 'n kern rol speel in die buffering van Pi konsentrasies in die plant, het ons sekere waarneembare gevolge verwag wat kon lei tot die ontrafeling van die meganismes hierby betrokke. Die produksie van PolyP in plant kallus is duidelik gedemonstreer, maar dit was slegs teen baie lae konsentrasies met geen waarneembare versteuringseffek nie, en kon glad nie in heel plante waargeneem word nie. Met die oog daarop om hierdie tegnologie toe te pas op die bestudering van die PolyP en PHT1;5 lyne wat in die ander dele van hierdie studie ontwikkel is, is 'n nuut ontwikkelde fluoresente indikator protein nanosensor (FLIPPi) tegnologie evalueer as 'n metode om Pi konsentrasies in vivo in multisellulere plant organe waar te neem en te monitor. Hierdie tegniek is in staat daartoe om veranderinge in Pi konsentrasies in selle direk te monitor en is gevolglik op die wortels van Arabidopsis saailinge onder Pi tekort toestande toegepas. Daar is spesifiek na veranderinge in die sitosol gekyk, maar ons resultate kon geen waarneembare veranderinge in Pi konsentrasies in hierdie kompartement uitwys nie. Hierdie resultaat beteken waarkeinlik dat die Pi konsentrasies in hierdie kompartement waarskeinlik baie laer is as wat voorheen verwag is.