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The aim of the work described in this thesis was to explore the synthesis of glycosidase inhibitors and to evaluate their potential as anti-cancer agents. Glycosidases catalyze the fission of glycosidic bonds and are involved in vital biological functions. With regard to their potential for anti-cancer therapy, two glycosidases were identified: α-N-acetyl-galactosaminidase and β-N-acetyl-hexosaminidase. The former has been implicated in causing immunosuppression in advanced cancer patients by negating the effect of the macrophage activating factor (MAF), while the latter is secreted by invading cancer cells and hence associated with metastasis formation. The synthetic focus was on generating piperidine and azetidine iminosugars, carbohydrate mimetics with their endocylic oxygen replaced by nitrogen. Their structural similarity to carbohydrates make iminosugars excellent inhibitors of glycosidases. Following synthesis of a pipecolic amide, its previously reported potent β-N-acetyl-hexosaminidase inhibition was confirmed. This data, along with inhibition profiles of several pyrrolidines, allowed the generation of a molecular model for predicting activity of β-N-acetyl-hexosaminidase inhibitors. The model was used to select azetidines in the D/L-ribo and D-lyxo configuration as suitable candidates to be explored in novel chemical space, leading to the first synthesis of a fully unprotected 3-hydroxy-2-carboxy-azetidine. The potent α-N-acetyl-galactosamindase inhibitor 2-acetamido-1,2-dideoxy-D-galacto-nojirimycin (DGJNAc) was successfully derivatised via N-alkylation. Important structural discoveries with regard to glycosylation of vitamin D₃-binding protein, the precursor of MAF, were made using MALDI mass-spectrometry. By comparing the enzymatic and cellular inhibition of N-alkylated derivatives of DGJNAc and a pyrrolidine the following generalization on iminosugar biodistribution was found: N-butylation promotes uptake into the cell/organelles, while hydrophilic side-chains restrict cellular access. An in vitro assay evaluating cancer cell invasion was devised and β-N-acetyl-hexoamindase inhibitors were shown to retard cell migration, including with the highly metastatic breast cancer cell line MDA-MB-231. Additive effects where found when the iminosugar was combined with a protease inhibitor, suggesting potential for future combination therapy.

L’objectif de ces travaux de thèse était de rechercher un potentiel effet inhibiteur de l’acide acétique endogène sur le métabolisme oxydo réductif de Saccharomyces cerevisiae, afin d’évaluer la pertinence d’une stratégie d’amélioration des capacités de production d’éthanol par la modulation de la synthèse de cet acide. Ces travaux devaient également permettre d’approfondir la compréhension des principaux facteurs commandant la synthèse de l’acide acétique et plus largement des acides organiques. La stratégie de modulation de la synthèse d’acide acétique mise en place reposait sur des approches microbiologique et génétique, consistant en l’ajout d’acide oléique et / ou de carnitine dans le milieu de culture ainsi que la surexpression du gène CIT2 ou la suppression du gène ALD6.Cette démarche a permis de montrer que, contrairement à la version exogène, l’acide acétique endogène ne présentait pas d’effet inhibiteur du métabolisme oxydo réductif de Saccharomyces cerevisiae ou qu’il était négligeable par rapport au stress éthanol. En outre, la modulation de la production de cet acide ne semble pas être une stratégie envisageable en vue de l’amélioration des capacités de production d’éthanol de cette levure, bien qu’une corrélation ait été observée entre les titres finaux de ces deux molécules.En outre, il a été montré que l’isoforme 6 de l’acétaldéhyde déshydrogénase (Ald6p) était essentiel pour assurer la croissance cellulaire normale ainsi que les mécanismes de résistance au stress éthanol dans ces conditions de culture. Plus largement, l’interrelation entre les différents isoformes ne paraissait pas aussi flexible qu’en anaérobiose. Saccharomyces cerevisiae semblait également présenter un métabolisme flexible en réponse à une modulation de la synthèse d’acide acétique. La voie des pentoses phosphates serait ainsi capable de prendre le relais de l’Ald6p pour assurer la régénération du NADPH cytosolique, bien que le flux à travers cette voie semble avoir été limité par le ratio NADP+ / NADPH. Enfin, les cellules paraissaient capables de réguler la synthèse de l’acétyl coA à partir d’acide acétique en réaction à une évolution des besoins anaboliques lors de la fin de la phase de croissance. Elles seraient toutefois incapables de pallier le manque d’acétyl coA suite à la suppression du gène ALD6. La modulation de la synthèse des acides pyruvique et succinique a également fait l’objet de discussions. / The aim of this work was to investigate a potential inhibitory effect of endogenous acetic acid on the oxido-reductive metabolism of Saccharomyces cerevisiae, to assess the relevance of a strategy based of the modulation of the synthesis of this acid, to improve ethanol production capacities. This work should also help to broaden the understanding of the main factors controlling the synthesis of acetic acid, and more generally organic acids. The strategy to modulate the synthesis of acetic acid was based on microbiological and genetic approaches, consisting in the addition of oleic acid and / or carnitine in the medium as well as the overexpression of the gene CIT2 or the deletion of the gene ALD6.This approach has shown that, contrary to exogenous version, endogenous acetic acid did not induce inhibitory effects on the oxido-reductive metabolism of Saccharomyces cerevisiae, or was negligible compared to stress caused by ethanol. Moreover, the modulation of the synthesis of this acid appear to be not an attractive strategy to improve ethanol production capacities of the yeast, although a correlation was observed between the end-culture titer of these two molecules.In addition, it has been shown that the isoform 6 of acetaldehyde dehydrogenase (Ald6p) was essential to ensure regular growth and mechanisms of ethanol stress resistance under these conditions of culture. More broadly, the interrelation between the different isoforms did not seem as flexible as under anaerobic conditions. Saccharomyces cerevisiae also seemed to have a flexible metabolism in response to a modulation of the synthesis of acetic acid. The pentose-phosphate way would be able to take over from Ald6p for regeneration of cytosolic NADPH, although the ratio NADP+ / NADPH seemed to lessen the flux through this pathway. Finally, the cells appeared to be able to regulate the synthesis of acetyl-CoA from acetic acid in response to changing in anabolic needs at the end of the growth phase. However, yeasts would be unable to overcome the lack of acetyl-CoA following the suppression of the gene ALD6. The modulation of the synthesis of pyruvic and succinic acids has also been discussed.

Peripheral nerve trauma is a common cause of considerable functional morbidity, and healthcare expenditure. Particularly in the ~15% of injuries unsuitable for primary repair, standard clinical management results in inadequate sensory restitution in the majority of cases, despite the rigorous application of complex microsurgical techniques. This can largely be explained by the failure of surgical management to adequately address the neurobiological hurdles to optimal regeneration. Most significant of these is the extensive sensory neuronal death that follows injury, and which is accompanied by a reduction in the regenerative potential of axotomised neurons, and in the supportive capacity of the Schwann cell population if nerve repair is delayed. The present study aimed to accurately delineate the timecourse of neuronal death, in order to identify a therapeutic window during which clinically applicable neuroprotective strategies might be adopted. It then proceeded to investigate means to increase the regenerative capacity of chronically axotomised neurons, and to augment the Schwann cells’ ability to promote that regenerative effort. Unilateral sciatic nerve transection in the rat was the model used, initially assessing neuronal death within the L4&5 dorsal root ganglia by a combination of morphology, TdT uptake nick-end labelling (TUNEL), and statistically unbiased estimation of neuronal loss using the stereological optical disector technique. Having identified 2 weeks, and 2 months post-axotomy as the most biologically relevant timepoints to study, the effect upon neuronal death of systemic treatment with acetyl-L-carnitine (ALCAR 10, or 50mg/kg/day) or N-acetyl-cysteine (NAC 30, or 150mg/kg/day) was determined. A model of secondary nerve repair was then adopted; either 2 or 4 months after unilateral sciatic nerve division, 1cm gap repairs were performed using either reversed isografts, or poly-3-hydroxybutyrate (PHB) conduits containing an alginate-fibronectin hydrogel. Six weeks later nerve regeneration and the Schwann cell population were quantified by digital image analysis of frozen section immunohistochemistry. Sensory neuronal death begins within 24 hours of injury, but takes 1 week to translate into significant neuronal loss. The rate of neuronal death peaks 2 weeks after injury, and neuronal loss is essentially complete by 2 months post-axotomy. Nerve repair is incompletely neuroprotective, but the earlier it is performed the greater the benefit. Two clinically safe pharmaceutical agents, ALCAR & NAC, were found to virtually eliminate sensory neuronal death after peripheral nerve transection. ALCAR also enhanced nerve regeneration independently of its neuroprotective role. Plain PHB conduits were found to be technically simple to use, and supported some regeneration, but were not adequate in themselves. Leukaemia inhibitory factor enhanced nerve regeneration, though cultured autologous Schwann cells (SC’s) were somewhat more effective. Both were relatively more efficacious after a 4 month delay in nerve repair. The most profuse regeneration was found with recombinant glial growth factor (rhGGF-2) in repairs performed 2 months after axotomy, with results that were arguably better than were obtained with nerve grafts. A similar conclusion can be drawn from the result found using both rhGGF-2 and SC’s in PHB conduits 4 months after axotomy. In summary, these findings reinforce the significance of sensory neuronal death in peripheral nerve trauma, and the possibility of its` limitation by early nerve repair. Two agents for the adjuvant therapy of such injuries were identified, that can virtually eliminate neuronal death, and enhance regeneration. Elements in the creation of a bioartificial nerve conduit to replace, or surpass autologous nerve graft for secondary nerve repair are presented.

Made available in DSpace on 2014-12-17T14:03:43Z (GMT). No. of bitstreams: 1 AdilaLML.pdf: 743306 bytes, checksum: 8c2b4b2827dafcc2f2edd7664a266e10 (MD5) Previous issue date: 2006-11-30 / Two b-N-acetylhexosaminidases (F11 e F15) were purified from Echinometra lucunter gonads extracts. The purified enzymes were obtained using ammonium sulfate fractionation, followed by gel filtration chromatographies (Sephacryl S-200, Sephadex G-75 and Sephacryl S-200). The F11 fraction was purified 192.47 -fold with a 28.5% yield, and F15 fraction 85.41 -fold with a 32.3% yield. The molecular weights of the fractions were 116 kDa for F11 and 42 kDa for F15 using SDS-PAGE. In Sephacryl S-200, F15 was 84 kDa, indicating that it is a dimeric protein. When p-nitrophenyl-?-D-glycosaminide was used as substrate, we determined an apparent Km of 0.257 mM and Vmax of 0.704 for F11 and for F15 the Km was 0.235 mM and Vmax of 0.9 mM of product liberated by hour. Both enzymes have optimum pH and temperature respectively at 5.0 and 45 ?C. The enzymes showed inhibition by silver nitrate, while the glucuronic acid was a potent activator. The high inhibition of F15 by N-etylmaleimide indicates that sulphydril groups are involved in the catalysis of synthetic substrate / Neste trabalho foram purificadas e caracterizadas parcialmente duas N-acetil-b- hexosaminidases (F11 e F15) extra?das de g?nadas do equinoderma marinho Echinometra lucunter. As enzimas foram purificadas com protocolo seq?encial por precipita??o com sulfato de am?nio e cromatografias de exclus?o molecular (Sephacryl S-200, Sephadex G-75 e Sephacryl S-200). A fra??o F11 foi purificada 192,47 vezes com recupera??o de 28,5% e F15 85,41 vezes com recupera??o de 32,3%. Suas massas moleculares, determinadas por eletroforese em gel de poliacrilamida com SDS, foram respectivamente 116 e 42 kDa. Em Sephacryl S-200 F15 apresentou massa molecular de 84 KDa, sugerindo que esta enzima possui forma dim?rica. Utilizando-se p-nitrofenil N-acetil-b-glicosamin?deo como substrato obtivemos Km aparente de 0,257 mM e Vmax de 0,704 unidades de absorb?ncia a 405 nm / h para a fra??o 11, e 0,235 mM e Vmax de 0,9 unidades de absorb?ncia a 405 nm / h para F15. Ambas fra??es apresentaram pH e a temperatura ?tima de cat?lise 5,0 e 45 ?C, respectivamente. A atividade N-acetil-b-glicosaminid?sica foi potencialmente inibida por prata, iodoacetamida, N-etilmaleimida e PMSF. A forte inibi??o de F15 por N-etilmaleimida indica o envolvimento de radicais sulfidrila na hidr?lise do substrato sint?tico, caracterizando tamb?m ser uma enzima altamente sensitiva a este sal

(2-Acetyl­ferrocen-1-yl)boronic acid, [Fe(C5H5)(C7H8BO3)] or 2-C(O)CH3-1-B(OH)2–Fc [Fc = Fe(η5-C5H3)(η5-C5H5)], crystallizes in the centrosymmetric space group P21/n. The boronic acid functionality inter­acts via intra­molecular hydrogen bonds with the acetyl group and with the –B(OH)2 functionality of an adjacent mol­ecule. The resulting centrosymmetric dimer exhibits an anti-positioning of the ferrocenyl moieties towards the central B2O4 plane. Consequently, an (Rp,Sp)-, i.e. a meso configuration is present for this dimer. In the crystal, weak C—H⋯O hydrogen bonds consolidate the mol­ecular packing.

В настоящей работе для электрохимического определения глюкозы предложены электрохимические катализаторы на основе органических комплексов никеля (II) и рутения (III) в присутствии карбоксилизированных многостенных углеродных нанотрубок и полиэтилеимина. Исследована каталитическая активность комплексов рутения (III) и никеля (II) при их раздельном и совместном присутствии на рабочем электроде в электрохимическом окислении глюкозы. Описан алгоритм проведения процедуры электрокаталитического определения глюкозы с использованием модифицированных электродов. Рассчитаны аналитические характеристики модифицированных электродов для электрохимического определения глюкозы. Выбран модификатор с оптимальными характеристиками, с наивысшей чувствительностью. / In this work, for the electrochemical determination of glucose, we propose electrochemical catalysts based on organic complexes of nickel (II) and ruthenium (III) in the presence of carboxylated multi-walled carbon nanotubes and polyethyleneimine. The catalytic activity of the complexes of ruthenium (III) and nickel (II) was investigated in the case of their separate and joint presence on the working electrode in the electrochemical oxidation of glucose. An algorithm for carrying out the procedure for electrocatalytic determination of glucose using modified electrodes is described. The analytical characteristics of the modified electrodes for the electrochemical determination of glucose are calculated. The selected modifier with optimal characteristics, with the highest sensitivity.

Lignocellulosic biomass is believed to serve a prominent role in tomorrow’s sustainable energy and material development. Among the polysaccharide fractions of lignocellulosic biomass, the potential of hemicelluloses as a valuable material resource is increasingly recognized. Thanks to their hydrophilic structure, hemicelluloses are suitable substrates for hydrogel design. The work summarized in this thesis aims to develop feasible strategies for the conversion of O-acetyl galactoglucomannan (AcGGM), an ample hemicellulose in softwood, into hydrogels. Within this framework, four synthetic pathways targeting the formation of crosslinked hydrogel networks from pure or unrefined AcGGM fractions were developed.   Aqueous AcGGM-rich and lignin-containing side-stream process liquors of forest industry, known as softwood hydrolysates (SWHs) were formulated into highly swellable hydrogels by: i) allyl-functionalization of AcGGM chains of crude SWH to obtain a viable precursor for hydrogel synthesis via free-radical crosslinking, ii) directly incorporating unmodified SWH fractions into semi-interpenetrating polymer networks (semi-IPNs). SWH hydrogels and semi-IPNs were characterized with appreciable maximum swelling ratios of Qeq = 170 and Qeq = 225, respectively.   Rapid crosslinking of AcGGM through thiol-click chemistry was addressed by first imparting thiol functionality onto pure AcGGM chains in a one-pot procedure. The thiolated AcGGM proved to be a suitable substrate for the synthesis of hemicellulose hydrogels via thiol-ene and thiol Michael addition reactions. Finally, sequential full IPNs were developed by subjecting single network hydrogels of pure AcGGM to a second network formation. IPNs obtained through either free radical crosslinking or thiol-ene crosslinking exhibited higher shear storage moduli than their single network counterparts. / <p>QC 20161102</p>