Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2008. / Titre provenant de l'écran titre. Bibliogr. p. 121-142.

<p> Diabetes mellitus is a major independent risk factor for cardiovascular disease and stroke. However, the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. We have shown that conditions of hyperglycemia are associated with accumulation of intracellular glucosamine, a downstream metabolite of glucose. Our findings indicate that elevated levels of intracellular glucosamine can promote inflammation and lipid accumulation - the hallmark features of atherosclerosis - in vascular cells and HepG2 cells.</p> <p> Here I demonstrate that exposure of HepG2 cells to the branched chain fatty acid, valproic acid, increases cellular resistance to glucosamine-induced lipid accumulation and nuclear factor-KB activation. In vivo I show that hyperglycemic apolipoprotein E-deficient (ApoE-/-) mice fed a diet supplemented with 625 mg/kg valproic acid have significantly reduced lesion volumes relative to non-supplemented controls. Valproate supplementation has no apparent effect on the plasma levels of glucose, or lipids, nor does it affect the expression of ER chaperones. Significant reductions were observed in total hepatic lipids(> 50.4%) and hepatic glycogen synthase kinases (GSK)-3β activity (> 55.8%) in mice fed the valproate supplemented diet.</p> <p> In vitro I demonstrate that valproic acid directly inhibits GSK-3α/β. Also pretreatment with novel GSK-3 inhibitors protects primary mouse hepatocytes from glucosamine-induced unesterified cholesterol accumulation. I further establish the role of GSK-3 by showing that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol after glucosamine treatment. Dietary supplementation with 2-ethylbutyric acid, a novel and potent GSK-3 inhibitor in vitro, did not reduce lesion development in hyperglycemic ApoE-/- mice and significantly increased atherosclerosis in normoglycemic mice. This may be a side effect attributed to multiple cellular pathways controlled by GSK-3 .</p> <p> In conclusion, I have identified a pathway involving glucosamine-induced cellular dysfunction that leads to accelerated hyperglycemia-associated atherosclerosis. This pathway involves GSK-3, which regulates glucosamine-induced unesterified cholesterol accumulation.</p> / Thesis / Doctor of Philosophy (PhD)

La glycochimie est un domaine qui prend de plus en plus d'expansion en chimie bioorganique et médicinale. Les glucides, étant la classe de biomolécules la plus répandue, sont des composants de plusieurs molécules bioactives naturelles et synthétiques. Ils présentent notamment un intérêt dans l'industrie pharmaceutique, puisqu'ils peuvent améliorer différentes propriétés pharmacocinétiques de certains composés. À ce jour, plus de 170 médicaments approuvés par les différentes organisations de la santé possèdent une unité glucidique dans leur structure. Ces molécules actives biologiquement ont une grande variété d'applications comme agents thérapeutiques : elles peuvent notamment être utilisées comme antibiotique ou encore comme médicament contre plusieurs types de cancers. Parmi ceux-ci, l'étoposide est un dérivé glycosylé semi-synthétique de la podophyllotoxine, un composé naturel isolé des espèces Podophyllum, et est employé pour le traitement de divers cancers. L'utilisation de ces deux molécules comme agents thérapeutiques est compromise par différents facteurs comme leur toxicité, les effets secondaires engendrés et leur faible solubilité dans l'eau. C'est pour ces raisons que plusieurs groupes de recherche travaillent sur le développement de nouveaux analogues du produit naturel qui seraient potentiellement moins toxiques, plus hydrophiles et dont l'activité biologique est améliorée, ou, du moins, non affectée. Ce mémoire présente la synthèse d'un dérivé de la glucosamine comportant un acide carboxylique et une amine dans sa structure. À partir de cette molécule, une nouvelle méthodologie synthétique d'oligosaccharides linéaires portant un groupement alcyne a été développée. Ces derniers ont pu être liés à la podophyllotoxine grâce à une réaction dite réaction click afin de générer de nouveaux dérivés glycoconjugués de la toxine. Les divers analogues obtenus diffèrent par le groupement retrouvé à la position C-2 du glucide terminal. Une voie de synthèse d'oligosaccharides utilisant les principes de couplage peptidique a donc été développée afin d'accéder à une librairie de nouveaux composés potentiellement actifs biologiquement. / There is a growing interest for the use of glycochemistry in the bioorganic and medicinal chemistry fields. Being the most abundant class of biomolecules, carbohydrates are components of various natural and synthetic bioactive products. They are of particular interest in the pharmaceutical industry since they can improve various pharmacokinetic properties of certain compounds. Nowadays, more than 170 commercially available drugs contain a carbohydrate unit in their structure. These biologically active molecules present a wide range of applications as therapeutic agents: they can be used as antibiotics or even as anticancer drugs. Among these, etoposide is a semi-synthetic glycosylated derivative of podophyllotoxin, a naturally occurring product in Podophyllum species, used as treatment of various cancers. The use of etoposide and podophyllotoxin as therapeutic agents is compromised by various problematics such as high toxicity, undesirable side effects and low water solubility. For these reasons, research groups are working on the development of new podophyllotoxin analogues that would potentially be less toxic, more hydrophilic and whose biological activity is either improved, or not affected. This thesis presents the synthesis of a glucosamine derivative bearing both a carboxyl and an amine group in its structure. From this molecule, a new synthetic methodology to access linear oligosaccharides carrying an alkyne group has been developed. These glycomolecules could be linked to podophyllotoxin by click reaction to generate new glycoconjugate derivatives of podophyllotoxin. Each analogue differs by the group found at the C-2 position on the terminal carbohydrate. Therefore, a new synthetic approach of oligosaccharides using the principles of peptide coupling has been developed to access a library of new potentially biologically active compounds.

Deux thématiques principales ont été étudiées au cours de cette thèse. La première partie porte sur la synthèse de complexes de manganèse dérivés de porphyrines et de salens. L'activité catalytique de ces complexes a été évaluée dans l'époxydation d'alcènes non-Fonctionnalisés. Suite à cette étude, les propriétés électroniques des ligands ont été étudiées, notamment par le biais de la complexation d'anions. Dans une deuxième étape, nous nous sommes intéressés à la synthèse de phosphines P-Chirogéniques. Afin de trouver un substitut à l'éphédrine, couramment utilisée dans ces synthèses, des dérivés du (1S,2S)-2-Aminocyclohexanol et de la D-Glucosamine ont été synthétisés. L'utilisation de groupements sulfonamides a permis l'obtention aisée d'oxazaphospholidines N-Tosylées. L'un ou l'autre diastéréoisomère de cet hétérocycle peut être obtenu en fonction du degré d'oxydation du réactif phosphoré utilisé. Avec cette stratégie, divers oxydes de phosphines ont été obtenus avec de bons rendements et de bonnes énantiosélectivités / This thesis is divided in two parts. The first part focuses on the synthesis of manganese complexes derived from porphyrins and salens. The catalytic activity of these complexes were evaluated toward the epoxidation of unfunctionalized alkenes. Next, the electronic properties of the ligands were evaluated using their anion binding properties. In a second part, we were interested in the synthesis of P-Chirogenic phosphines. In order to find a surrogate to ephedrine, that is commonly used in those syntheses, derivatives from (1S,2S)-2-Aminocyclohexanol and D-Glucosamine were synthesized. The use of sulfonamides allows the access to N-Tosylated oxazaphospholidines. Both diastereoisomers could be synthesized depending on the oxydation state of the phosphine precursor. Using this strategy, various phosphine oxides were obtained in good yields and enantioselectivities

Heparin glycosaminoglycans mediate a range of biological events, including anticoagulation as well as a diversity of cell proliferation and differentiation processes. Heparin saccharides have been shown to act as inhibitors against angiogenesis and metastasis of tumour cells. This thesis describes work developing chemistry towards varying length oligosaccharide sequences with potential to offer variable sulfation patterns. The main synthetic components to this work were contribution to developing scalable syntheses of an orthogonally protected L-Iduronic acid unit and a differentially protected D-glucosamine unit. The synthetic work also evaluated a recently reported diazo transfer reagent, which allowed for earlier placement of azide protection over that of previously developed routes within the group. This provided a cheaper, more atom efficient route towards protected D-glucosamine building blocks. Glycosylation of the developed D-GlcN donor units with the L-Ido acceptor allowed the production of key disaccharides which facilitated an efficient iterative glycosylation strategy towards longer oligosaccharides, ultimately providing a differentially protected pentasaccharide. The project evaluated methods towards generating various dimeric heparin type systems through forming new O4 ether linkages between GlcN residues across various short linker fragments. The most successful of these dimerisations used a methallyl dichloride core which allowed for further derivatisation towards dihydroxylated species, the analysis of which highlighted some interesting proton NMR data. The final aspect of this project began development of chemistry towards non-reducing end-labelled oligosaccharide sequences by implementation of a masked aldehyde unit on the C4 hydroxyl of GlcN synthesised from the allylated GlcN precursor via dihydroxylation chemistry. Incorporation of this moiety (protected as a 1,2-dibenzyl glycol) within both a trisaccharide and a pentasaccharide was achieved. Further development of this chemistry should allow for late step oxidative cleavage to reveal the reactive aldehyde, potentially allowing for attachment of various amine functionalised fluorophores via reductive amination. Radiolabelling of such a species should also be possible through sodium borotritide reduction for example.

This research focused on the study of the amino sugar recycling and sialic acid degradation pathway as a possible entry point for N-acyl glucosamines for the production of sialic acid analogs. Meeting this objective would allow the development of a bacterial strain capable of producing non-natural nonulosonic acids that could be used in the development of medicines, vaccines or useful compounds for the study of interactions between pathogenic organisms and their host. The first step was to understand how N-acetyl-D-glucosamine-6-phosphate deacetylase reacts to different types of substrates in order to determine its tolerance to the size of acyl groups in acyl amino sugars. This was achieved by studying the enzymatic activity in an in vitro system. We determine that the enzyme has a preference for small and slightly bulky acyl groups. Then, an in silico docking modeling and an in vivo system experiment were carried out. These experiments allowed to confirm the previous results. The second project was carried out due to the uncertainty of whether the kinase involved in the catabolic pathway would be able to phosphorylate the substrates. By quantifying residual ATP, the high specificity of N-acetyl-D-glucosamine kinase could be verified. This result led us to think about the design of an organic synthesis strategy that would allow the phosphorylation of glucosamine in carbon 6. A simple synthetic route was designed based on the protection of the two most reactive moieties of the amino sugars and the reactivity of the hydroxy group on carbon 6. However, we had problems with the purification step of the final product due to its high polarity. The next stage of this investigation was to confirm the transformation of GlcNAc into ManNAc. For this, an NMR analysis was designed that would detect the presence of both sugars in the reaction system. The epimerization of ManNAc to GlcNAc was detected successfully. Notwithstanding, the reverse reaction could not be detected. Based on the results obtained in the previous stage, we realized that an error was made in the epimerization reaction since we placed the wrong kinase because we did not take into account its substrate specificity. Finally, we tried to produce sialic acid analogs in a fermentative system using different genetic variants of Escherichia coli. Two of the expected analogs, Neu5Pr and Neu5nBu, were obtained. In addition, NagA activity towards substrates with small acyl groups was confirmed.

Low molecular weight gelators (LMWGs) have gained great attention over the past two decades. These compounds form self-assembled fibrous networks like micelles, cylindrical, sheets, fibers, layers and so on. The fibrous network entraps the solvent and form gel. LMWGs are interesting compounds with many potential applications in material and biomedical sciences. Many different structures have been found to be good LMWGs. Our interests focus on the carbohydrate based LMWGs. Previously, we have found that several ester derivatives of methyl 4, 6-O-benzylidene-α-D-glucopyranoside are good gelators for organic solvents and aqueous solutions. In this study, in order to understand the structure requirement, we systematically investigated the influence of sugar head groups and the attached hydrophobic tails towards gelation. The design, synthesis and gel properties of esters, amides, ureas, carbamates which derived from sugar head groups show above will be discussed in chapter II, III, IV.

Foram sintetizadas duas novas séries de tensoativos de açúcar derivados da 2-D-glucosamina: os metil 2-acilamido-2-deóxi-6-O-sulfonato-D-glucopiranosídeos de sódio (aniônicos) e os cloretos de metil 2-acilamido-2,6-dideóxi-6-trimetilamônio-D-glucopiranosídeos (catiônicos). Os tensoativos aniônicos foram obtidos pela acilação da 2-D-glucosamina com cloretos de acila (com 8, 12 e 16 carbonos), seguida pela metilação desses derivados com metanol em meio ácido, e posterior sulfatação dos metil glucosídeos com complexo trióxido de enxofre-piridina. Os tensoativos catiônicos foram obtidos pela tosilação dos metil glucosídeos, seguida pela quaternização com trimetilamina e troca do contra-íon tosilato por cloreto com resina de troca-iônica. Esses tensoativos apresentaram c.m.c. similares a de outros tensoativos iônicos de cadeia hidrofóbica de igual comprimento, mas energias livres de transferência do grupo polar para a micela muito mais favoráveis. Esse fato foi atribuído à formação de ligações de hidrogênio entre os grupos polares do tensoativo na micela, e à hidrofobicidade do açúcar. As micelas formadas apresentaram números de agregação maiores que os obtidos para outros tensoativos, provavelmente devido às interações atrativas entre os grupos polares. / Two new sugar-based surfactant series were synthesized from 2-D-glucosamine: sodium methyl 2-acylamido-2-deoxi-6-O-sulfonate-D-glucopyranosides (anionic) and methyl 2-acylamido-2,6-dideoxi-6-trimethylamonium-D-glucopyranoside chlorides (cationic). The anionic surfactants were obtained by the acylation of 2-D-glucosamine with acyl chlorides (with 8, 12 and 16 carbons), followed by the methylation of these derivatives with methanol in acidic media, and the sulfation of the methyl glucosides with sulfur trioxide-pyridine complex. The cationic surfactants were obtained by the tosylation of methyl glucosides followed by the quaternization with trimethylamine and exchange of the tosylate contra-ion with chloride ions on an ion exchange resin. These surfactants showed c.m.c. similar to other ionic surfactants with equal hydrophobic chain lengths, but more favorable free energies of transfer of the polar head to the micelle. This fact is attributed to hydrogen bonding between the head groups of the surfactant in the micelle, and the hydrophobicity of the sugar moiety. The micelles of these surfactants showed aggregation numbers larger than those obtained for other surfactants, problably because of head-group attractive interactions.

Abstract Osteoarthritis (OA) is the most common musculoskeletal disorder affecting Australians and is the leading cause of pain and disability in the country. There is no known cure for OA, but pharmacological and non-pharmacological treatments can relieve symptoms and improve quality of life for OA sufferers: two of these are glucosamine sulphate (GS) and physical activity. Little is known about physical activity behavior and the correlates of physical activity participation among Australians with OA, or about the benefits of combining physical activity and GS for the management of OA. More generally, there is little information on the treatments used by OA sufferers in Australia. The first main aim of this thesis was to describe OA symptoms in people with hip or knee OA; their use of treatments for these symptoms; their health status; their current, past and future participation in physical activities; and their perceptions that could influence physical activity participation (Study 1). The second main aim was to compare the effectiveness of different frequencies and durations of walking, combined with GS intake, for reducing symptoms of OA and improving well-being in people with hip or knee OA (Study 2). Descriptive study (Study 1) A questionnaire was mailed to 2200 members of the Arthritis Queensland Foundation who lived in the Brisbane environs. Of these, 588 participants with hip or knee OA completed the survey. The most common treatments being used to manage osteoarthritic symptoms were weight loss (57%), range of motion exercises (56%), and strengthening exercises (49%). The most popular pharmacological treatments were glucosamine and/or chondroitin (54%) and anti-inflammatory medications (40%). Paracetamol (73%), topical liniment rubs (45%) and anti-inflammatory gels (35%) were the most commonly used ‘as needed’ medications. Most participants had fair to good health (68%). Many reported moderate levels of anxiety and depression as well as other health problems. Hypertension was the most common health problem (43.3%). Twenty-five percent reported that health problems were preventing them from walking, and 33% reported that health problems were preventing them from doing other exercises. Fifty-nine percent were meeting Australian physical activity guidelines (30 minutes of moderate-intensity physical activity most days of the week). Participants reported taking part in a wide range of sports and exercises, walking being the most common. They had moderate levels of self-efficacy for, and perceived many health benefits from being physically active, but perceived a moderate number of barriers to being active. Significant associations between past participation in sports and current OA symptoms were only found for men (p < 0.05), with past participation in Australian Rules football, basketball, soccer, skiing or volleyball associated with more severe symptoms in men. Feasibility study (Study 2) Thirty-six participants were given GS for 18 weeks. Starting in Week 6, they also participated in the 12-week Stepping Out walking program (after being randomly assigned to walk 3 or 5 days each week). Assessments were conducted at baseline, at Weeks 6, 12, and 18 during the intervention, and at a final follow-up during Week 24 of the study. Primary outcome measures were WOMAC pain, stiffness, physical function, and total scores and time to complete a self-paced step test, an objective measure of physical function. Comparisons were made between groups and between assessment weeks. As the data were not normally distributed, non-parametric techniques were used. Given that the study was a feasibility study, data were analysed on a per protocol basis. No significant between-group differences were found at any assessment week for the primary outcome measures. Therefore, changes between assessments were examined for the two groups combined. After 6 weeks of GS intake, WOMAC stiffness (p = 0.01) and physical function scores (p = 0.05) improved, as did physical function, measured objectively (p < .001). Between Weeks 6 and 12, when participants were asked to increase their daily steps over their current steps to an extra 1500-3000 per day, physical activity minutes increased (p = 0.01), and improvements were found for WOMAC pain (p = 0.03), WOMAC physical function (p = 0.03), and objectively-measured physical function (p < .001). Between Weeks 12 and 18, when participants were instructed to increase their daily steps by 6000 from baseline, physical activity minutes increased (p < .001), and further improvements were found for objectively-measured physical function (p < .001). During the follow up between Week 18 and 24, physical activity minutes decreased (p = 0.01) while objectively-measured physical function improved (p < .001). Both studies add to the body of literature on the management of OA of the hip and knee. Study 1 provides information about OA sufferers’ use of over-the-counter medications and their adoption of self-management strategies, information not routinely available from other sources, while Study 2 provides preliminary evidence for the walking ‘dose’ appropriate to relieve OA symptoms and on the effects of difference ‘doses’ on OA symptoms.

Thesis (Ph.D.)--Baylor University, 2006. / Includes bibliographical references (p. 136-146).