Une consommation de fibres alimentaires adéquate semble protéger du risque de développer un diabète de type 2 dont a contrario la prévalence ne cesse de progresser à travers le monde. Parmi les mécanismes d'action pouvant expliquer comment les fibres contribuent à l'homéostasie du glucose et à la sensibilité à l'insuline, l'implication du microbiote intestinal semble aujourd'hui très probable. Cependant il n'est pas toujours aisé de différencier ses effets de ceux pouvant découler d'une réduction de l'indice glycémique voir de ceux induits par des micronutriments parfois associés aux fibres. Les travaux de cette thèse ont permis de confirmer que les fructo-oligosaccharides (FOS), fibres prébiotiques peu visqueuses, peuvent améliorer la sensibilité à l'insuline de différents modèles animaux d'obésité induite par le régime alimentaire. La réduction de la sensibilité à l'insuline induite par le surpoids n'est pas complètement contrebalancée par les FOS mais leurs effets sont visibles même sans perte de poids associée. D'autre part une corrélation directe entre les modifications de la composition et des activités du microbiote intestinale induites par les FOS et le métabolisme de l'hôte a pu être mise en évidence. Au-delà d'une augmentation des bifidobactéries déjà bien documentée, les travaux de cette thèse ont révélé la modulation par les FOS d'autres clusters bactériens plus prépondérants au sein de l'écosystème digestif comme ceux des Clostridium leptum et Clostridium coccoides – Eubacterium rectale. En modulant ces populations, les FOS influencent le métabolisme des acides biliaires et le niveau d'hydroxylation d'acides gras monoinsaturés. / An adequate intake of dietary has proven to protect against the risk to develop type 2 diabetes, for which the prevalence is currently increasing all around the world. Among the different mechanisms that could explain how dietary fibres can contribute to insulin sensitivity and glucose homeostasis, involvement of the gut microbiota seems more than likely. However it is not always possible to differentiate the effects that linked to the microbiota from those induced a reduction of the glycaemic index or those induced by some micronutrients sometimes closely associated to dietary fibres. The experiments conducted during this PhD validate that fructo-oligosaccharides (FOS), poorly viscous prebiotic fibres, can enhance insulin sensitivity if different animal models of diet-induced obesity. Reduction of insulin sensitivity resulting from overweight status is not completely solved by FOS but their effect is not linked to weight loss. Moreover, a direct correlation between the modifications of the composition and activities of the intestinal microbiota triggered by FOS and metabolism of the host has been highlighted. This work reveals that more than a single increase of Bifidobacteria already well documented, FOS can alter other bacterial clusters more predominant within the digestive ecosystem like Clostridium leptum and Clostridium coccoides – Eubacterium rectale. By modulating these populations, FOS will alter the metabolism of biliary acids and the level of monounsaturated fatty acids hydroxylation.

Aspergillus fumigatus é um fungo filamentoso e saprofítico encontrado em todas as regiões do mundo. A principal forma de infecção ocorre através da inalação de conídios do fungo com predominância de infecções no trato respiratório, principalmente em pacientes imunocomprometidos. Foi caracterizada a função mitocondrial de A. fumigatus e sugeriu-se a presença de vias alternativas, dentre elas, a presença da NADH desidrogenase alternativa. A fim de colaborar com estudos para elucidação do papel desta enzima, foi realizada a clonagem dos genes das NADH desidrogenase alternativa interna e externa de A. fumigatus. A análise da seqüência de aminoácidos revelou um perfil de hidropaticidade com a presença de quatro regiões hidrofóbicas, semelhante às outras seqüências já descritas. Além disso, foram identificados dois motivos (GXGXXG) altamente conservados para ligações a nucleotídeos, dentro de um domínio os quais estão relacionados com a estrutura e atividade da enzima. A seqüência de cDNA do gene ndhiAf foi clonada em plasmídeo pYES2 e expressa em S. cerevisiae cepa CEN.PK873-2B; a expressão da NDHIAf foi verificada por técnica de Western-blot. A proteína foi expressa de forma ativa, conferindo à levedura uma respiração e a formação de um potencial de membrana resultantes da oxidação de substratos clássicos do complexo I, sugerindo sua localização na membrana mitocondrial interna. Além disso, as cepas expressando essa proteína foram capazes de crescer em meio contendo apenas lactato como fonte de carbono, uma característica atribuída à presença da NADH desidrogenase alternativa interna (NDHi). Considerando que a atividade das NDHIAf e NDHEAf estão sob controle metabólico, nos avaliamos o efeito de diferentes concentrações de glucose como única fonte de carbono no meio de cultura. Durante a germinação conidial, não foi observada expressão da NDHIAf e NDHEAf na presença de baixas concentrações de glucose. Entretanto, durante a fase de hifas foi observado um aumento na expressão de NDHIAf e NDHEAf. Por outro lado, em alta concentrações de glucose, durante a germinação conidial, foi observado expressão da NDHIAf mas nenhuma expressão da NDHEAf. Na fase de hifas observou-se um aumento da expressão da NDHIAf e uma diminuição da NDHEAf. / Aspergillus fumigatus is a filamentous and saprophytic fungus found in all regions of the world. The main form of infection occurs through inhalation of fungi conidial, with predominance of infections in the respiratory treat, mainly in immunocompromised host. It was characterized the mitochondrial function of A. fumigatus and suggested the presence of alternative pathways, including the alternative NADH dehydrogenase. In order to elucidate its role, genes of the external and internal enzymes were cloned. Analysis of the amino acid sequence and hydropathy plot revealed a profile with four hydrophobic regions, similar to other already described sequences. Moreover, two highly conserved motifs (GXGXXG) for the nucleotides interaction, related with the structure and activity of the enzyme, were identified inside the a domain. The sequence of DNA of the ndhiAf gene was cloned in an expression plasmid pYES2 and expressed in CEN.PK873-2B S. cerevisiae strain; the expression was confirmed by Western-blot analysis. The protein was expressed in an active form, providing to the yeast an oxygen consumption and a potential membrane formation due to oxidation of the complex I substract, suggesting its localization in the inner mitochondrial membrane. Moreover, strains expressing this protein were capable of growing in medium with only lactate as a carbon source, a characteristic associated with the presence of internal alternative NADH dehydrogenase (NDHI). Considering that NDHI and NDHE activities are under metabolic control, we evaluated the effect of different concentrations of glucose as the only carbon source in the medium of culture. During conidia germination, it was not observed neither an expression of NDHI nor NDHE in the presence of low concentrations of glucose. However, in hyphae phase was observed an increase an expression of NDHI and NDHE. On the other hand, in high concentration of glucose, during conidia germination was observed an expression of NDHI and not expression of NDHE. In hyphae phase was observed a decrease an expression of NDHI and an increase of NDHE.

Les enregistrements életrophysiologiques ont apporté des informations considérables sur le fonctionnement et le dysfonctionnement du cerveau. Améliorer les dispositifs d'enregistrement permettrait d'approfondir les connaissances au niveau de la science fondamentale et serait bénéfique pour les patients. Les principales limitations des électrodes en contact direct avec le cerveau comprennent leur invasivité, leur biocompatibilité et leur SNR. Il serait aussi souhaitable de mesurer simultanément les signaux électriques et moléculaires. Le couplage entre l'activité électrique et métabolic est encore mal comprise. Le but de ce travail était de fournir des solutions technologiques à ces défis dans le contexte de l’épilepsie.Nous avons développé des grilles flexibles de 4 µm d’épaisseur résolvant les problèmes d’invasivité, de rigidité et de biocompatibilité. Afin d’améliorer le SNR, des sites d'enregistrement en polymère hautement conducteur PEDOT: PSS ont été faits. La qualité des signaux enregistrés in vivo était meilleure que celui obtenu avec de l’or. Puis nous avons validé des sites d'enregistrement en transistors électrochimiques organiques, permettant l'amplification locale des signaux. Les grilles ont été testées in vivo et le SNR a été multiplié par 10. Enfin, nous avons fonctionnalisé les sites avec une enzyme pour mesurer le glucose. Par rapport aux dispositifs classiques, le capteur de glucose a montré une stabilité et une sensibilité inégalée in vitro.En conclusion, l'électronique organique semble être une solution technologique prometteuse pour les limitations des systèmes actuels visant à enregistrer l'activité électrique et moléculaire du cerveau. / Electrophysiological recordings brought considerable information about brain function and dysfunction. Improving recording devices would further our understanding at the basic science level and would be beneficial to patients. Major limitations of current electrodes that are in direct contact with brain tissue include their invasiveness, their poor biocompatibility, their rigidity and a suboptimal signal-to-noise ratio. In addition, it would be desirable to measure simultaneously molecular signals. The coupling between the electrical activity of neurons and metabolism is still poorly understood in vivo. The goal of this work was to provide technological solutions to such challenges in the context of epilepsy. We generate 4 µm thick, totally flexible but resilient grids, thus solving the challenge of invasiveness, rigidity and biocompatibility. In order to improve the signal-to-noise ratio, recording sites were made of the highly conductive polymer PEDOT:PSS. The quality of the in vivo signals recorded was better than that obtained with conventional gold contacts. Going a step further, we made the recording site as an organic electrochemical transistor, which enables local amplification of signals. The grid was tested in vivo and the SNR was increased by a factor of 10. Finally, we functionalized PEDOT:PSS sites with glucose oxidase to measure glucose. Compared to conventional devices, the glucose sensor showed unsurpassed stability and sensitivity in vitro. In conclusion, organic electronics appears to be a promising technological solution to the limitations of current systems designed to record the electrical and molecular activity of the brain.

De plus en plus d’études suggèrent un rôle important du métabolisme énergétique dans la sclérose latérale amyotrophique (SLA). Ainsi, une intolérance au glucose, une résistance à l’insuline et un hypermétabolisme lipidique sont très souvent retrouvés chez les patients et dans les modèles animaux de SLA. Cependant, les bases cellulaires et moléculaires qui sous-tendent ces altérations restent largement méconnues. Une récente étude menée dans un modèle souris de SLA, indique qu’un dysfonctionnement de la pyruvate déshydrogénase (PDH) induirait, dans les muscles rapides, un déséquilibre des voies cataboliques vers une utilisation privilégiée des lipides, et non plus du glucose. Dans ce contexte, nous avons voulu évaluer dans quelles mesures un programme ciblé d’exercice physique pourrait réorienter les voies métaboliques vers une utilisation privilégiée du glucose dans les souris SOD1(G93A), modèle de SLA. Nous avons comparé les effets de deux types d’entraînement, l’un basé sur un exercice de course de faible intensité, assimilable à un exercice d’endurance, l’autre basé sur un exercice de nage à forte intensité, assimilable à un exercice de force/résistance. Contrairement à la course, l’exercice de nage réduit significativement l’intolérance au glucose, augmente le taux sanguin de lactate et le stockage lipidique dans les adipocytes. Dans le muscle rapide tibialis, touché très précocement par la maladie, nous mettons en évidence une altération de l’expression de GLUT4, transporteur musculaire majeur du glucose, et de la glyceraldehyde-3 phopshate deshydrogenase (GAPDH), une enzyme clé de la glycolyse. Ces altérations moléculaires sont retrouvées dans des biopsies musculaires de patients SLA. L’entraînement à la nage des souris SLA stimule l’expression de GLUT4 et de GAPDH, grâce à la réactivation de processus d’autophagie, et réactive les enzymes du métabolisme aérobie, telles que la citrate synthase, enzyme du cycle de Krebs, et la cytochrome oxydase, appartenant à la chaîne respiratoire mitochondriale. L’ensemble de ces données indique qu’un exercice bien calibré peut améliorer le métabolisme énergétique dans un contexte de SLA, et ouvre la voie à une utilisation de l’exercice physique en complément d’une potentielle modulation pharmacologique de la PDH chez les patients. / The use of physical exercise as an intervention for alleviating symptoms in Amyotrophic-Lateral-Sclerosis (ALS) is debated. We have reported that swimming based exercise sustained motor function, induced a significant neuroprotection and extended SOD1(G93A) ALS mouse lifespan in contrast to running based exercise. Because exercise types are expected to differentially alter, in trained skeletal muscles, the energy metabolism, whose defects have been very recently linked to ALS-induced motor-neuron death, we compared the impact of either a daily swimming- or running-based training on several metabolic indicators in ALS mice. We indicate that, unlike running, the swimming-based training of ALS mice led to a significant increase in lipogenesis and glucose tolerance, and reverse the metabolic switch affecting fast-twitch muscles. Besides, our data highlighted detrimental-side effects of the running-based training that further shifted the energetic status towards a more oxidative metabolism. Thus, the present data outline the paramount importance of the choice of the exercise type when designing rehabilitation protocols for ALS patients.

Tsang Po Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 135-152). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.viii / List of Abbreviations --- p.ix / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The Role of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.1 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure of the Glutl Protein --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Tertiary Structure --- p.8 / Chapter 1.3.4 --- Kinetics Properties --- p.11 / Chapter 1.3.5 --- Tissue Distribution --- p.12 / Chapter 1.3.6 --- Multifunctional Property --- p.13 / Chapter 1.3.7 --- Characterization of GLUT1 Gene --- p.13 / Chapter 1.3.8 --- Regulation of GLUT1 Expression --- p.14 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.16 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome (GIutlDS) --- p.19 / Chapter 1.5.1 --- Backgronnd of GIutlDS --- p.19 / Chapter 1.5.2 --- Clinical Features of GIutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.5 --- Manage nent --- p.27 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.27 / Chapter 1.6 --- Hypothesis and Objectives --- p.29 / Chapter Chapter 2: --- Biochemical and Molecular Analysis of GLUT1 in a Suspected GlutlDS Case --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patient --- p.32 / Chapter 2.1.2 --- Blood Samples --- p.32 / Chapter 2.1.3 --- Reagents and Buffers for Reverse Transcription --- p.32 / Chapter 2.1.4 --- Reagents and Buffers for TA Cloning --- p.34 / Chapter 2.1.5 --- Reagents for Genomic DNA Extraction --- p.34 / Chapter 2.1.6 --- Reagents and Buffers for Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.35 / Chapter 2.1.8 --- Reagents for Zero-trans 3-OMG Influx in Erythrocytes --- p.37 / Chapter 2.1.9 --- Reagents for Zero-trans 3-OMG Efflux from Erythrocytes --- p.38 / Chapter 2.1.10 --- Reagents for Erythrocytes Membrane Extraction and Detection --- p.39 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- GLUT1 Gene Analysis --- p.44 / Chapter 2.2.2 --- Zero-trans 3-OMG Influx into Erythrocytes --- p.51 / Chapter 2.2.3 --- Zero-trans 3-OMG Efflux from Erythrocytes --- p.52 / Chapter 2.2.4 --- Glutl Protein Expression --- p.54 / Chapter 2.2.5 --- Statistics --- p.57 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Molecular Analysis of the GLUT1 Gene of a Suspected GlutlDS Patient --- p.58 / Chapter 2.3.2 --- Functional Analysis of the GlutlDS Patient's Glutl Protein --- p.61 / Chapter 2.3.3 --- Glutl Protein Expression in the GlutlDS Patient --- p.64 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3: --- Pathogenicity Studies of GLUT1 Mutations --- p.71 / Chapter 3.1 --- Materials --- p.72 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.72 / Chapter 3.1.2 --- Cell Lire --- p.73 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.73 / Chapter 3.1.4 --- Cell Culture Wares --- p.75 / Chapter 3.1.5 --- Reagents for Transfection --- p.75 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.76 / Chapter 3.1.7 --- Consumables for Confocal Microscopy --- p.77 / Chapter 3.1.8 --- Reagents and Buffers for Flow Cytometry --- p.77 / Chapter 3.1.9 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.77 / Chapter 3.2 --- Methods --- p.79 / Chapter 3.2.1 --- Cell Culture Methodology --- p.79 / Chapter 3.2.2 --- Construction of GLUT1 Mutants --- p.80 / Chapter 3.2.3 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.84 / Chapter 3.2.4 --- Protein Expression Study --- p.85 / Chapter 3.2.5 --- 2-DOG Influx Assay in CHO-K1 Cells --- p.87 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.89 / Chapter 3.2.7 --- Statistics --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Molecular Analysis of 1034-1035Insl2 Mutation --- p.91 / Chapter 3.3.2 --- Expression of the Wild Type and Mutant GFP-Glutl Fusion Proteins --- p.92 / Chapter 3.3.3 --- Functional Analysis of the 1034-1035Insl2 Mutant --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter Chapter 4: --- GLUT1 Promoter Study --- p.100 / Chapter 4.1 --- Materials --- p.101 / Chapter 4.1.1 --- Construction of GLUT1 Promoter Vectors --- p.101 / Chapter 4.1.2 --- Cell Lines --- p.102 / Chapter 4.1.3 --- Cell Culture Media and Other Reagents --- p.103 / Chapter 4.1.4 --- Dual Luciferase Reporter Assay System --- p.103 / Chapter 4.2 --- Methods --- p.105 / Chapter 4.2.1 --- Bioinformatics --- p.105 / Chapter 4.2.2 --- Cell Culture --- p.105 / Chapter 4.2.3 --- Construetion of GLUT1 Promoter Vectors --- p.105 / Chapter 4.2.4 --- 5'-Deletion Analysis of GLUT1 Promoter --- p.108 / Chapter 4.2.5 --- Determination of the Activities of GLUT1 Promoter Fragments --- p.110 / Chapter 4.2.6 --- Statistics --- p.113 / Chapter 4.3 --- Results --- p.114 / Chapter 4.3.1 --- Determination of the Promoter Activity of the 5'-deletion Fragments --- p.114 / Chapter 4.3.2 --- Prediction of Transcription Factors in the 5'-deletion Fragments --- p.119 / Chapter 4.4 --- Discussion --- p.121 / Chapter Chapter 5: --- General Conclusion and Future Perspectives --- p.133 / References --- p.135

Les nanoparticules bimétalliques supportées sont des catalyseurs particulièrement attractifs en raison d’une activité et d’une stabilité accrues par rapport à leurs homologues monométalliques. Dans cette thèse des solides à base d'or ont été étudiés en tant que catalyseurs de l'oxydation sélective du glucose en absence de base. Il a été mis en évidence que la variation du ratio molaire entre l’or et le second métal (Pd, Pt, or Cu) a un impact différent sur les performances catalytiques en fonction de la nature du second métal, du support et de la méthode de préparation. Les séries Au-Pd supportés sur TiO2 et préparés par la méthode de sol-immobilisation et Au-Cu supportés sur TiO2 et préparés par la méthode de précipitation-réduction ont montré un effet synergique important, en particulier lorsque le rapport entre les deux métaux était de 1. Ces catalyseurs convertissent sélectivement le glucose en acide gluconique et leur activité a été trouvée supérieure à celle des contreparties monométalliques. L'analyse XPS a démontré que les espèces Au+δ, Pd+2 et CuOH jouent alors un rôle important dans la réaction étudiée en absence de base. Le bismuth en tant que second métal n'a montré aucun effet bénéfique, au contraire du palladium et du cuivre. Les catalyseurs à base d’Au et de Pt supportés sur ZrO2 se sont avérés quant à eux très stables lorsque la teneur en or était inférieure à 0,3% en masse. La nature du support a un impact très important sur le mécanisme de la réaction conduite en absence base sur des catalyseurs à base d’or. La raison réside dans les interactions que ce support développe avec la phase bimétallique favorisant ainsi la formation des espèces actives / The supported bimetallic nanoparticles are particularly attractive catalysts due to the increased activity and stability over their monometallic counterparts. In this thesis, gold-based solids have been studied as catalysts for the selective base-free oxidation of glucose. It has been demonstrated that the variation of the molar ratio between gold and the second metal (Pd, Pt, or Cu) has a different impact on the catalytic performances depending on the nature of the second metal, the support and the method of preparation. TiO2 supported Au-Pd series prepared by the sol-immobilization method and Au-Cu series prepared by the precipitation-reduction method showed a significant synergistic effect, particularly when the ratio of the two metals was 1. Under the reaction conditions used (T = 60 °C or 80 °C, P = 5 bar air, t = 5 h), these catalysts selectivity to gluconic acid and their activity was found to be greater than that of monometallic counterparts, especially when the catalyst is supported on TiO2. XPS analysis showed that the Au+δ, Pd+2 and CuOH species played an important role in the base-free reaction. Bismuth as the second metal showed no beneficial effect, unlike palladium and copper. The Au-Pt catalysts supported on ZrO2 proved to be still active when the gold content was less than 0.3 wt.%. Ultimately, the nature of the support has a very important impact on the mechanism of the base-free reaction conducted on gold-based catalysts (formation of H2O2 in situ). The reason lies in the interactions of the support with the bimetallic phase thus favoring the formation of the active species

An organisms’ diet represents an exogenous influence that often yields colossal effects on long-term health and disease risk. The overconsumption of dietary sugars for example, has contributed to significant increases in obesity and type-2 diabetes; health issues that are costly both economically and in terms of human life. Individuals who are obese or are type-2 diabetic often have compromised oxygen delivery and an increased vulnerability to oxygen-deprivation related complications, such as ischemic strokes, peripheral arterial disease and myocardial infarction. Thus, it is of interest to identify the molecular changes glucose supplementation or hyperglycemia can induce, which ultimately compromise oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, I determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide biosynthesis and antioxidant activity, modulates anoxia survival. Additionally, a glucose-supplemented diet induces lipid accumulation. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified are homologous to human genes that are differentially regulated in response to metabolic diseases, suggesting that there may be conserved gene expression responses between C. elegans supplemented with glucose and a diabetic and/or obese state observed in humans. These findings support the utility of C. elegans to model specific aspects of the T2D disease process (e.g., glucose-induced sensitivity to oxygen deprivation) and identify potentially novel regulators of common complications seen in hyperglycemic and T2D patients (e.g., macrovascular complications).

Le lactosérum est un résidu liquide laitier qui a lieu pendant la fermentation du fromage. Il est composé par lactose (le solide principal de substance sèche), protéines, vitamines et minéraux. À cause de ces éléments, sa demande biologique et chimique d’oxygène (DBO) et (DCO) est grande (30 < DBO < 50 g/L; and 60 < DCO < 80 g/L). Il est nécessaire donc de traiter le lactosérum avant d’en jeter dans les lacs, les rivières, etc. La valorisation du lactosérum par des bactéries est d’un grand potentiel technique non seulement pour la réduction de DBO et DCO mais aussi pour obtenir des produits comme le 2,3-butanediol (2,3-BD). Des bactéries comme Enterobacter cloacae, Klebsiella pneumoniae et Paenibacillus polymyxa consomment et transforment des saccharides comme lactose en 2,3-BD. Il en est d’autres, comme Escherichia coli, qui doivent être génétiquement modifiées car elles n’ont pas le chemin enzymatique pour produire 2,3-BD. De cette manière l’objectif principal de cette recherche est celui de tester l’habilité d’une souche génétiquement modifié d’E. coli pour transformer le lactose (un disaccharide) en 2,3-BD afin de savoir le potentiel du lactosérum comme une source de lactose. La souche d’E. coli JFR12 (ECGM12) a été utilisée pour fermenter trois concentrations de glucose, galactose et lactose (12.5, 25 and 50 g/L) enrichirent M9. Le rendement de 2,3-BD le plus grand (36% environ, g 2,3-BD/g saccharide) fut obtenu en présence de 25 g/L de glucose et lactose; quoique l’usage de n’importe quelle concentration de galactose produisît des rendements plus pauvres de 2,3-BD. En plus, 2 mélanges de glucose-galactose ont été testés (1:1, w/w) à une concentration finale du 25 et 50 g/L de mélange. Les rendements de 2,3-BD obtenus ont été très similaires à les obtenus avec du galactose comme l’unique source de carbone. Par conséquent, une hypothèse a été formulée: l’usage de galactose entrave la formation de 2,3-BD, alors que les enzymes impliquent dans l’hydrolyse du lactose pourraient neutraliser l’effet du galactose et de cette manière, les rendements de 2,3-BD ont pu être hauts / Whey is a dairy effluent generated during the cheese manufacturing. Its composition presents lactose (the main solid of dry matter), proteins, vitamins and minerals. Due to these compounds, its biological (BOD) and chemical oxygen demand (COD) are high (30 < BOD < 50 g/L; and 60 < COD < 80 g/L). Therefore, it is necessary to treat the whey before releasing it into lakes, rivers, etc. The valorization of whey by bacteria is a potential technique not only for reducing the BOD and COD, but also to obtain products like 2,3-butanediol (2,3-BD). Bacteria like Enterobacter cloacae, Klebsiella pneumoniae and Paenibacillus polymyxa consume and transform saccharides like lactose into 2,3-BD. Other bacteria such as Escherichia coli have to be genetically modified since they do not possess the enzymatic pathway to produce 2,3-BD. In this way, the main objective of this research is to test the ability of a genetically modified strain of E. coli to transform lactose (a disaccharide) into 2,3-BD in order to know the potential of whey as a lactose source. In this way, the E. coli JFR12 (ECGM12) was used to ferment three concentrations of glucose, galactose and lactose (12.5, 25 and 50 g/L) supplemented M9. The highest 2,3-BD yield (around 0.36 g 2,3-BD/g saccharide) was obtained in the presence of 25 g/L of glucose and lactose; whereas the use of whatever galactose concentration provided poorer yields of 2,3-BD compared to those obtained using glucose and lactose. Moreover, 2 mixture of glucose-galactose was tested (1:1, w/w) at a final concentration of 25 and 50 g/L of mixture. The 2,3-BD yields obtained were very similar to those using galactose as a sole carbon source. Therefore, it was hypothesized that galactose impedes the formation of 2,3-BD, whereas enzyme involved in the lactose hydrolysis might counteract the effect of galactose, leading to high 2,3-BD yields

Lai, Ching Janice. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 148-168). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Nervous System and the Blood-Brain-Barrier --- p.3 / Chapter 1.3 --- Glial cells --- p.3 / Chapter 1.4 --- Studying Astrocyte Responses As a New Direction in Neuroscience --- p.4 / Chapter 1.5 --- The Roles of Astrocyte in the CNS --- p.5 / Chapter 1.5.1 --- Energy-Dependent Communication Between Neurons and Astrocytes --- p.7 / Chapter 1.5.2 --- Strategies for Metabolic Exchange Between Astrocytes and Neurons --- p.8 / Chapter 1.5.2.1 --- Provision of Energy Metabolites to Neurons by Astrocytes --- p.9 / Chapter 1.5.2.2 --- Glucose Transporters in the CNS --- p.10 / Chapter 1.5.2.3 --- The Lactate Shuttle Hypothesis --- p.12 / Chapter 1.5.2.4 --- The Regulation of Glucose Uptake at the Blood-Brain-Barrier (BBB) by the Activity of Neurons --- p.14 / Chapter 1.5.3 --- Alternation of Energy Metabolism in Neuropathy --- p.15 / Chapter 1.5.3.1 --- Ketone Body Shuttle Hypothesis --- p.15 / Chapter 1.5.3.2 --- The Utilization of Free Fatty Acids by the Brain --- p.17 / Chapter 1.5.4 --- The Provision of Neurotrophic Factors to Neurons by Astrocytes --- p.17 / Chapter 1.5.4.1 --- Neurotrophins --- p.18 / Chapter 1.5.4.1.1 --- Relationship Between Neurotrophins and Glucose --- p.20 / Chapter 1.5.4.2 --- S100B --- p.21 / Chapter 1.5.5 --- Astrocytic Cholesterol in Astrocytes as a Neurotrophic Factor --- p.22 / Chapter 1.6 --- Neuroprotective Effect of Glucose vi - --- p.23 / Chapter 1.7 --- Diseases Associated with Decreased Glucose Transport at the BBB --- p.24 / Chapter 1.7.1 --- Glucose Transporter Type 1 Deficiency Syndrome (GlutlDS) --- p.24 / Chapter 1.7.2 --- Hypoglycemia with Insulin Therapy for Diabetes Patients --- p.24 / Chapter 1.8 --- Aims and Hypothesis of Study --- p.26 / Chapter Chapter 2. --- 2 Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Cell Culture --- p.27 / Chapter 2.1.1.1 --- Cells --- p.27 / Chapter 2.1.1.1.1 --- C6 cells --- p.27 / Chapter 2.1.1.1.2 --- Primary Astrocytes --- p.27 / Chapter 2.1.1.2 --- Cell Culture Reagent --- p.27 / Chapter 2.1.2 --- Study of Growth Properties --- p.31 / Chapter 2.1.2.1 --- Equipment for Growth Curve Construction --- p.31 / Chapter 2.1.2.2 --- Reagents for Flow Cytometry --- p.32 / Chapter 2.1.2.3 --- Reagents for 3H-thymidine Incorporation Assay --- p.32 / Chapter 2.1.3 --- Study of Neurotrophic Properties --- p.33 / Chapter 2.1.3.1 --- Determination of Neurotrophic Factor Productions --- p.33 / Chapter 2.1.3.1.1 --- Reagents and Buffers for Northern Blot Analysis --- p.33 / Chapter 2.1.3.2 --- Reagents and Buffers for Western Blot Analysis --- p.43 / Chapter 2.1.3.2.1 --- Protein Assay --- p.43 / Chapter 2.1.3.2.2 --- Reagents for SDS Polyacrylamide Electrophoresis of Proteins --- p.44 / Chapter 2.1.3.2.3 --- Reagents for the Transfer of Protein to Membrane and Signal Detection --- p.47 / Chapter 2.1.4 --- Study of Lipid in Glial cells --- p.50 / Chapter 2.1.4.1 --- Determination of Genes Expression in Lipid Metabolism --- p.50 / Chapter 2.1.4.2 --- Reagents for Determination of Cholesterol and Fatty Acid Levels by Gas Chromatography --- p.50 / Chapter 2.2 --- Methods --- p.54 / Chapter 2.2.1 --- Cell culture --- p.54 / Chapter 2.2.1.1 --- Maintenance of C6 cells --- p.54 / Chapter 2.2.1.2 --- Primary Culture of Rat Astrocytes --- p.54 / Chapter 2.2.2 --- Study of Growth Properties of Glial Cells vii - --- p.56 / Chapter 2.2.2.1 --- Construction of cell growth curve --- p.56 / Chapter 2.2.2.2 --- Flow Cytometric Analysis of Cell Cycle Profile --- p.56 / Chapter 2.2.2.3 --- Measurement of DNA Synthesis --- p.57 / Chapter 2.2.3 --- Study of Neurotrophic Properties --- p.58 / Chapter 2.2.3.1 --- Determination of Neurotrophic Facotor Production --- p.58 / Chapter 2.2.3.1.1 --- Northern Blot Analysis --- p.58 / Chapter 2.2.3.1.2 --- Western Blot Analysis --- p.67 / Chapter 2.2.3.2 --- Determination of Gene Expression in Lipid Metabolism --- p.72 / Chapter 2.2.3.2.1 --- Northern Blot Analysis --- p.72 / Chapter 2.2.3.2.2 --- RT-PCR --- p.72 / Chapter 2.2.3.3 --- Study of Lipid Profiles in Glial Cells --- p.73 / Chapter 2.2.3.3.1 --- Sample preparation --- p.73 / Chapter 2.2.3.3.2 --- Total Cholesterol Determination --- p.73 / Chapter 2.2.3.3.3 --- Total Fatty Acid Determination --- p.75 / Chapter 2.2.3.3.4 --- Quantification of Proteins --- p.76 / Chapter 2.2.4 --- Statistical Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.78 / Chapter 3.1 --- The effects of glucose deficiency on cell proliferation --- p.78 / Chapter 3.1.1 --- Direct Cell Count Assay --- p.78 / Chapter 3.1.2 --- Flow Cytometry Assay --- p.83 / Chapter 3.1.3 --- 3H-Thymidine Uptake Assay --- p.85 / Chapter 3.2 --- The Effects of Glucose Deficiency on Neurotrophic Properties of Glial Cells --- p.87 / Chapter 3.2.1 --- The Effects of Glucose Deficiency on mRNA and Protein Expressions of Neurotrophins --- p.88 / Chapter 3.2.1.1 --- Northern Blot Assays --- p.88 / Chapter 3.2.1.2 --- Western Blot Assays --- p.93 / Chapter 3.2.2 --- The Effects of Glucose Deficiency on Lipid Homeostasis --- p.96 / Chapter 3.2.2.1 --- Northern Blot Assays --- p.96 / Chapter 3.2.2.2 --- Gas Chromatography Assays --- p.101 / Chapter 3.2.2.2.1 --- Cholesterol Analyses --- p.102 / Chapter 3.2.2.2.2 --- Fatty Acid Analyses --- p.105 / Chapter Chapter 4 --- Discussion --- p.115 / Chapter 4.1 --- The in vitro Model of Hypoglycorrhachia --- p.115 / Chapter 4.2 --- Decreased Glucose Level Triggers Changes of Gial Cells Proliferation --- p.117 / Chapter 4.3 --- Expression of Neurotrophic Factor under Glucose Deficiency viii - --- p.120 / Chapter 4.3.1 --- Alteration of the Expression of Neurotrophins --- p.120 / Chapter 4.3.1.1 --- NGF --- p.122 / Chapter 4.3.1.2 --- BDNF --- p.123 / Chapter 4.3.1.3 --- NT-3 --- p.126 / Chapter 4.3.1.4 --- NT-4/5 --- p.128 / Chapter 4.3.2 --- Alteration of the mRNA Expression of Calcium Binding ProteinS100B --- p.128 / Chapter 4.4 --- Alteration of Lipid Metabolism in Decreased Glucose Supply --- p.130 / Chapter 4.4.1 --- Up-regulation of ApoE mRNA Expression in Glucose Deficiency --- p.133 / Chapter 4.4.2 --- Cholesterol Homeostasis in Glial Cells --- p.133 / Chapter 4.4.3 --- Fatty Acids Homeostasis in Glial Cells --- p.135 / Chapter 4.4.4 --- Decreased Ketone Bodies synthesis in Glucose Deficiency --- p.143 / Chapter 4.5 --- Limitations of the Current Study --- p.144 / Chapter 4.6 --- Future Directions --- p.145 / Chapter Chapter 5 --- Conclusion --- p.147 / References --- p.148 / Appendix --- p.169

Glucose isomerase (GI) catalyzes the isomerization of glucose to fructose and consequently is one of the bulkiest industrial enzyme for the manufacture of high fructose corn syrup and crystalline fructose. The GI is used in industry mainly in the form of immobilized enzyme. / In this work, the immobilization of GI had been studied by several methods: ion exchange adsorption, covalent binding, alginate cells entrapment and cells cross-linking. Three kinds of carrier support (ion exchange resin, epoxy resin and amino resin) have been used in the immobilization of cells-free enzyme; the whole cells immobilization of GI by cross-linking agents polyethyleneimid and glutaraldehyde were critically examined. The results show that the cells cross-linking is the best method to prepare the immobilized GI products, as it is high in specific activity and thermostability, and low the cost. The method is likely to make significant contribution to the field of immobilization, its application has expanding rapidly in many walks of the society, including environment protection, food and pharmaceutical industries. / Jin, Caike. / "August 2005." / Adviser: Jun Wang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3521. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 125-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.