Given the volatile, generally high price of crude oil, as well as environmental concerns associated with its use as a fuel, development of alternative energy sources is currently of considerable interest. Lignocellulose-derived energy has the potential to supplant traditional fossil fuels in the future because of its economic and environmental advantages. Lignocellulosic biomass is abundant and renewable. Lignocellulose is primarily composed of cellulose, hemicellulose and lignin, which can be converted by acid hydrolysis to simple sugars used in fermentation to produce biofuels. In this study, hemicellulose was hydrolyzed with different concentrations of hydrochloric acid at different temperatures. The resulting components were analyzed by high performance liquid chromatography (HPLC). The hydrolysis of cellulose was similarly characterized, with two additional parameters, the degree of polymerization (DP) and the crystallinity index (CrI), which were analyzed by Ubbelohde viscometer and X-ray diffraction respectively. The experimental results indicate that the hydrolysis rate of hemicellulose and the generation rate of furfural and 5-hydroxymethylfurfural (HMF) increased with increasing hydrochloric acid concentrations and reaction temperatures. In the selected five monosaccharides, xylose, glucose, mannose, arabinose and galactose, xylose has the highest hydrolysis rate and the accumulation of furfural during xylose hydrolysis is also the highest. Moreover, the hydrolysis rate of cellulose and the generation rate of glucose also increased with increasing hydrochloric acid concentrations and reaction temperatures. DP and CrI, both decreased when the cellulose was treated in concentrated hydrochloric acid. The rate of change of DP increased with the concentrations of acid and the reaction temperatures. The change rate of CrI increases by increasing concentration of acid and the temperature when it is above 0℃, while the CrI index decrease sharply when the reaction temperature was kept below 0℃. Experimental results also show that the hydrolysis rate of cellulose is much lower than that of hemicellulose.

Given the volatile, generally high price of crude oil, as well as environmental concerns associated with its use as a fuel, development of alternative energy sources is currently of considerable interest. Lignocellulose-derived energy has the potential to supplant traditional fossil fuels in the future because of its economic and environmental advantages. Lignocellulosic biomass is abundant and renewable. Lignocellulose is primarily composed of cellulose, hemicellulose and lignin, which can be converted by acid hydrolysis to simple sugars used in fermentation to produce biofuels. In this study, hemicellulose was hydrolyzed with different concentrations of hydrochloric acid at different temperatures. The resulting components were analyzed by high performance liquid chromatography (HPLC). The hydrolysis of cellulose was similarly characterized, with two additional parameters, the degree of polymerization (DP) and the crystallinity index (CrI), which were analyzed by Ubbelohde viscometer and X-ray diffraction respectively. The experimental results indicate that the hydrolysis rate of hemicellulose and the generation rate of furfural and 5-hydroxymethylfurfural (HMF) increased with increasing hydrochloric acid concentrations and reaction temperatures. In the selected five monosaccharides, xylose, glucose, mannose, arabinose and galactose, xylose has the highest hydrolysis rate and the accumulation of furfural during xylose hydrolysis is also the highest. Moreover, the hydrolysis rate of cellulose and the generation rate of glucose also increased with increasing hydrochloric acid concentrations and reaction temperatures. DP and CrI, both decreased when the cellulose was treated in concentrated hydrochloric acid. The rate of change of DP increased with the concentrations of acid and the reaction temperatures. The change rate of CrI increases by increasing concentration of acid and the temperature when it is above 0℃, while the CrI index decrease sharply when the reaction temperature was kept below 0℃. Experimental results also show that the hydrolysis rate of cellulose is much lower than that of hemicellulose.

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive. / Master of Science

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Iminosugars are polyhydroxylated alkaloids, and can be viewed as sugar analogues in which the endocyclic oxygen atom has been replaced with nitrogen. These compounds are highly medically relevant and their biological activity is largely due to their inhibition of glycosidases. Several examples of the iminosugar class are currently marketed as drugs, and many more are in earlier stages of development for a variety of diseases and disorders. The most fruitful approaches to the chemical synthesis of iminosugars have utilised carbohydrate starting materials as optically pure chiral building blocks, or chirons. Most of the monosaccharides are not readily available, but the relatively few naturally abundant cheap sugars have been exploited as chirons for over a century. The availability of the rare sugars is growing with the development of a new biotechnological approach to their synthesis, known as Izumoring. This thesis is primarily concerned with the chemical synthesis of iminosugars from carbohydrate starting materials. The synthesis of unnaturally functionalised sugar polyols and their suitability as substrates for the Izumoring process is also discussed. Chapter 1 provides a brief general overview of the history, natural occurrence and therapeutic application of iminosugars. General strategies for their synthesis from carbohydrate chirons are discussed. Chapter 2 concerns divergent syntheses of several iminosugar targets from both enantiomers of glucuronolactone and their biological evaluation. A new scaleable synthesis of the natural product 1-deoxynojirimycin is presented that has since been adopted for commercial purposes, as well as an efficient strategy for the synthesis of both enantiomers of 2,5-dideoxy-2,5-imino- mannitol and their novel amino acid analogues. Access to hexosaminidase inhibiting acetamido- substituted piperidines is presented, including 2-acetamido-1,5-imino-1,2,5-trideoxy-D- galactitol, which has been found to be one of the few known potent and specific inhibitors of α- N-acetyl-galactosaminidase. This inhibitory profile may allow the compound’s use for further investigation of a strategy for cancer treatment. Chapter 3 concerns the synthesis of carbon branched pyrrolidines and their biological evaluation. A novel and highly potent α-glycosidase inhibitor has been discovered, synthesised by a strategy that utilises the benzhydryl ether as key protecting group. A mild method for the introduction of this protecting group has been shown to be general to a range of sterically congested and/or acid/base sensitive carbohydrate lactones. Chapter 4 concerns the synthesis of deoxygenated and fluorinated sugar alcohols and their successful biotechnological transformation into ketoses by the Izumoring process. Publications arising from this work are included in the Appendix.

L’identification de marqueurs susceptibles de mettre en évidence et quantifier l’altération du bois par différents agents de dégradation peut présenter un grand intérêt dans la mise au point de méthodes de contrôle du matériau. Dans la première partie de ce travail, la caractérisation et le dosage des composés extractibles de deux essences non durables, le hêtre et le chêne soyeux, exposées à différents champignons de pourritures blanches ou brunes préalablement traitées ou non avec du propiconazole a été effectué. L’évolution de la quantité et de la nature des extractibles dépend directement de la présence du biocide et permet de différencier clairement les éprouvettes en fonction du taux de dégradation de ces dernières. C’est le cas notamment de la catéchine présente dans les extraits de hêtre, qui disparaît rapidement lorsque le bois est exposé à un agent de pourriture et qui peut à ce titre constituer un marqueur de l’attaque du bois. Dans la seconde partie de ce travail, l’évolution de la composition chimique du bois traité par pyrolyse douce à 240°C sous azote a été étudiée. Bien que les résultats obtenus confirment en grande partie ceux décrits précédemment dans la littérature, le dosage des différentes fractions du bois (lignine et holocellulose) et l’analyse des monosaccharides constitutifs de la fraction holocellulose du bois ont permis de mettre en évidence un important phénomène de carbonisation rapporté jusqu’à présent pour des températures plus élevées. Ce phénomène confirmé par RMN13C peut être à l’origine de certaines des nouvelles propriétés du matériau. / The identification of markers able to highlight and quantify wood deterioration by different degradation agents can be of great interest in the development of material control methods. In the first part of this work, the characterization and the quantification of the two non durable wood species extractives, beech and silky oak, treated or not with propiconazole and exposed to various white rot or brown rot fungi were carried out. The evolution of the quantity and the nature of extractives depend directly on the presence of biocide and allowed to clearly differentiate degraded and non-degraded samples. This is particularly true in the case of catechin identified in the extracts of beech, which is rapidly degraded by rotting fungi constituting therefore a valuable marker of wood degradation. In the second part of this work, the evolution of the chemical composition of wood treated by mild pyrolysis at 240°C under nitrogen was investigated. Although the results mainly confirm those described previously in the literature, isolation of lignin and holocellulose fractions and analysis of monosaccharides constitutive of holocellulose fraction highlighted an important carbonization of wood reported up to now for higher temperatures. This phenomenon is confirmed by 13C NMR and could be at the origin of some of the new properties of the material.

by Lai Mei Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 130-136). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / 論文撮耍 --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Abbreviations --- p.xii / Table of content --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Facilitative glucose transporters --- p.1 / Chapter 1.1.1 --- Predicted Secondary structure of Glutl --- p.1 / Chapter 1.1.2 --- The tissue-specific distribution of glucose transporters --- p.2 / Chapter 1.2 --- Increase of glucose uptake in cancer cells --- p.5 / Chapter 1.3 --- Antisense oligonucleotide therapeutics --- p.7 / Chapter 1.3.1 --- Chemical modifications of oligonucleotides --- p.7 / Chapter 1.3.2 --- Cellular Uptake of Oligonucleotide --- p.11 / Chapter 1.3.3 --- Mechanism of action --- p.13 / Antisense-mediated RNA Cleavage --- p.14 / """Occupancy-only"" mediated mechanism" --- p.15 / Chapter 1.3.4 --- Antisense treatment in vivo --- p.17 / Chapter 1.4.5 --- Human Studies of Antisense Treatment --- p.18 / Chapter Chapter 2 --- Materials & Methods --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Cell Culture --- p.21 / Chapter 2.2.1 --- Human colon adenocarcinoma cell Line (Caco-2) --- p.21 / Chapter 2.3 --- General Methodology for treatment of cells with antisense oligonucleotides --- p.22 / Chapter 2.3.1 --- Treatment of cells with oligonucleotides --- p.22 / Chapter 2.4 --- Cytotoxicity Assay --- p.23 / Chapter 2.4.1 --- MTT assay --- p.23 / Chapter 2.4.2 --- 3H-thymidine incorporation --- p.23 / Chapter 2.5 --- RNA extraction --- p.24 / Chapter 2.6 --- Competitive Reverse-transcription polymerase chain reaction (RT-PCR) of glucose transporters --- p.25 / Chapter 2.7 --- Measurement of 2-deoxy-D-glucose and Fructose transport --- p.27 / Chapter 2.8 --- Western blotting --- p.28 / Chapter 2.9 --- Flow cytometry --- p.30 / Chapter 2.9.1 --- Measurement of cellular accumulation of fluorophore-labeled oligonucleotide --- p.30 / Chapter 2.10 --- Design of antisense oligonucleotide --- p.31 / Chapter 2.11 --- ATP assay --- p.34 / Chapter 2.12 --- Animals studies --- p.35 / Chapter Chapter 3 --- Optimization of phosphorothioate antisense oligonucleotide delivery by Lipofectin --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Measurement of oligonucleotide uptake --- p.38 / Chapter 3.2.1 --- Lipofectin as a delivery system for the oligonucleotide uptake --- p.39 / Chapter 3.2.2 --- Effect of Lipofectin ratio on the oligonucleotide uptake --- p.41 / Chapter 3.2.3 --- Effect of oligonucleotide concentration on the oligonucleotide uptake --- p.41 / Chapter 3.2.4 --- Effect of incubation time on the oligonucleotide uptake --- p.44 / Chapter 3.2.5 --- Effect of oligonucleotide length on cellular uptake --- p.44 / Chapter 3.3 --- Effect of Lipofectin on cell viability --- p.47 / Chapter Chapter 4 --- In vitro effect of Antisense Oligonucleotides against glucose transporters on Caco-2 Cell --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Design of Antisense Oligonucleotides against Glucose Transporters gene --- p.50 / Chapter 4.3. --- Antisense effect of different regions of antisense oligonucleotide --- p.52 / Chapter 4.4 --- Antisense and Sense effect of oligonucleotide against start codon (G5 7015) on Caco-2 cells --- p.59 / Chapter 4.4.1 --- Effect of oligonucleotide to Lipofectin ratio on cell viability --- p.59 / Chapter 4.4.2 --- Dose-Response Study: effect of concentration of antisense - oligonucleotide on cell viability --- p.61 / Chapter 4.4.3 --- Effect of length´ؤof oligonucleotide on cell viability --- p.61 / Chapter 4.4.4 --- Time-Response Study: effect of antisense oligonucleotide on cell viability --- p.66 / Chapter 4.5 --- "The effect of antisense oligonucleotide against Glut1, Glut3 and Glut5 on cell viability of Caco-2 cells" --- p.70 / Chapter 4.6 --- Analysis of ATP content in Caco-2 cells by using antisense oligonucleotide flanking start codon (G5 7015) --- p.72 / Chapter 4.7 --- Effect of G5 7015 on HepG2 cells --- p.72 / Chapter Chapter 5 --- Effect of antisense oligonucleotides against Glut5 on mRNA and Protein levels of Glut5 gene --- p.76 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- RT-PCR of Glut isoform in Caco-2 cells --- p.77 / Chapter 5.3 --- Effect of antisense oligonucleotides against Glut 5 on mRNA level in Caco-2 cells --- p.77 / Chapter 5.3.1 --- Effect of oligonucleotides targeted different region of Glut5 gene on Glut5 message level --- p.77 / Chapter 5.3.2 --- Reduction in expression of mRNA level of Glut5 by using antisense oligonucleotides targeting start codon (G5 7015) --- p.81 / Chapter 5.3.3 --- Study of the dose and time dependence on inhibition of mRNA expression in G5 7015 treated Caco-2 cells --- p.83 / Chapter 5.3.4 --- Cross-Inhibition of antisense targeting glucose transporter isoforms --- p.83 / Chapter 5.4 --- Reduction in Glut5 protein level using G5 7015 antisense oligonucleotide --- p.86 / Chapter 5.5 --- Inhibition of Glut5 activity using G57015 oligonucleotide --- p.88 / Chapter 5.6 --- Inhibition of Glut5 mRNA level in vivo --- p.93 / Chapter Chapter 6 --- The possible role for Glucose Transporters in the Modification of Multidrug Resistance in Tumor cells --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials & Methods --- p.97 / Chapter 6.2.1 --- Cell culture --- p.97 / Chapter 6.2.2 --- Chemicals --- p.98 / Chapter 6.2.3 --- Measurement of doxorubicin uptake --- p.99 / Chapter 6.3 --- The expression of P-glycoprotein and Doxorubicin resistance of R-HepG2 cells --- p.99 / Chapter 6.4 --- Comparison of H3-2-deoxyglucose uptake between HepG2 and R-HepG2 cells --- p.99 / Chapter 6.5 --- Quantification of Glut1 and Glut3 expression by RT-PCR --- p.102 / Chapter 6.6 --- Comparison of doxorubicin between HepG2 and R-HepG2 cells cultured accumulation in glucose free medium --- p.104 / Chapter 6.7 --- The time course of doxorubicin accumulation in R-HepG2 cells culturing in glucose free medium --- p.106 / Chapter 6.8 --- "Cell viability of R-HepG2 cells after treatment of glucose transporter inhibitors, phloretin (PT), cytochalasin B (CB) and mitochondrial inhibitor，2,4-Dinitrophenol (DNP)" --- p.106 / Chapter 6.9 --- "Effect of glucose transporter inhibitors (PT, CB) and mitochondrial inhibitor (DNP) on doxorubicin accumulationin R-HepG2" --- p.110 / Chapter 6.10 --- Effect of antisense oligonucleotide against Glutl on doxorubicin accumulation in R-HepG2 cell --- p.113 / Chapter 6.11 --- "Analysis of ATP content and 3H-2-deoxy-D-glucose uptakein R-HepG2 after treatments of PT, CB and DNP" --- p.115 / Chapter Chapter 7 --- Discussion --- p.117 / Chapter 7.1 --- Antisense oligonucleotide against glucose transportersin Caco-2 cell --- p.117 / Chapter 7.2 --- Cellular uptake of oligonucleotide --- p.119 / Chapter 7.3 --- In vitro study of using antisense oligonucleotide against Glut5 --- p.121 / Chapter 7.4 --- In vivo study of using antisense oligonucleotide against Glut5 --- p.126 / Chapter 7.5 --- Possible role of inhibition of glucose transport in reversing P- gp --- p.127 / Chapter Chapter 8 --- References --- p.130

Chung Ka Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 151-162). / Abstracts in English and Chinese. / Contents --- p.i / Acknowledgements --- p.v / Abstract --- p.vi / 論文摘要 --- p.ix / List of Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Breast Cancer --- p.2 / Chapter 1.1.1 --- Incidence Rate of Breast Cancer --- p.2 / Chapter 1.1.2 --- Risk Factors Lead to Breast Cancer --- p.5 / Chapter 1.1.3 --- Conventional Treatments --- p.5 / Chapter 1.2 --- Relationship between Breast Cancer and Glucose Transporters --- p.7 / Chapter 1.2.1 --- Importance of Glucose and Fructose --- p.7 / Chapter 1.2.2 --- Facilitative Glucose Transporters (Gluts) and The Relationship with Breast Cancer --- p.7 / Chapter 1.3 --- Antisense Oligonucleotides --- p.13 / Chapter 1.3.1 --- Characteristics of Antisense Oligonucleotides --- p.13 / Chapter 1.3.2 --- Action Mechanism of Antisense Oligonucleotides --- p.15 / Chapter 1.3.3 --- Sequence Selection --- p.19 / Chapter 1.3.4 --- Chemical Modifications of Antisense Oligonucleotides --- p.20 / Chapter 1.3.5 --- Uptake and Delivery Means of Antisense Oligonucleotides --- p.24 / Chapter 1.4 --- Objectives of Present Study --- p.26 / Chapter Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.32 / Chapter 2.1.2 --- Buffers and Reagents --- p.33 / Chapter 2.1.3 --- Reagents for Transfection --- p.34 / Chapter 2.1.4 --- Reagents for D-[U14C]-Fructose and 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.35 / Chapter 2.1.5 --- Reagents for ATP Assay --- p.35 / Chapter 2.1.6 --- Reagents for RT-PCR --- p.36 / Chapter 2.1.6.1 --- Reagents for RNA Extraction --- p.36 / Chapter 2.1.6.2 --- Reagents for Reverse Transcription --- p.36 / Chapter 2.1.6.3 --- Reagents for Gel Electrophoresis --- p.37 / Chapter 2.1.7 --- Reagents for Real Time-PCR --- p.38 / Chapter 2.1.8 --- Reagents and Chemicals for Western Blotting --- p.39 / Chapter 2.1.8.1 --- Reagents for Protein Extraction --- p.39 / Chapter 2.1.8.2 --- Reagents for SDS-PAGE --- p.39 / Chapter 2.1.9 --- Reagents for Flow Cytometry --- p.42 / Chapter 2.1.10 --- In Vivo Study --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Oligonucleotide Design --- p.44 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.47 / Chapter 2.2.3 --- Transfection --- p.47 / Chapter 2.2.4 --- MTT Assay --- p.47 / Chapter 2.2.5 --- D-[U14C]-fructose and 2-deoxy-D-[l-3H] Glucose Uptake Assay --- p.48 / Chapter 2.2.6 --- Detection of Intracellular ATP Concentration --- p.49 / Chapter 2.2.7 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.2.7.1 --- RNA Extraction by TRIzol Reagent --- p.51 / Chapter 2.2.7.2 --- Determination of RNA Concentration --- p.51 / Chapter 2.2.7.3 --- Reverse Transcription --- p.52 / Chapter 2.2.7.4 --- Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.2.8 --- Real-Time PCR --- p.55 / Chapter 2.2.8.1 --- Analysis of the Real-Time PCR Data --- p.57 / Chapter 2.2.9 --- Western Blot Analysis --- p.58 / Chapter 2.2.9.1 --- Protein Extraction --- p.58 / Chapter 2.2.9.2 --- Protein Concentration Determination --- p.58 / Chapter 2.2.9.3 --- Western Blotting --- p.60 / Chapter 2.2.10 --- Flow Cytometry --- p.62 / Chapter 2.2.10.1 --- Detection of Cell Cycle Pattern with PI --- p.62 / Chapter 2.2.10.2 --- Detection of Apoptosis with Annexin V/PI --- p.62 / Chapter 2.2.11 --- In Vivo Study --- p.63 / Chapter 2.2.11.1 --- Establishment of Tumor-Bearing Animal Model --- p.63 / Chapter 2.2.11.2 --- Treatment Schedule --- p.63 / Chapter 2.2.11.3 --- Toxicity of Antisense Oligonucleotides --- p.64 / Chapter Chapter 3 --- Results --- p.66 / Chapter 3.1 --- In Vitro Study --- p.67 / Chapter 3.1.1 --- Effect of Tamoxifen on MCF-7 cells and MDA-MB-231 cells --- p.67 / Chapter 3.1.2 --- Cytotoxicity of Antisense Oligonucleotides against Glut 5 on MCF-7 cells and MDA-MB-231 cells by MTT Assay --- p.69 / Chapter 3.1.3 --- Effect of Antisense Oligonucleotides against Glut 5 on Fructose and Glucose Uptake of MCF-7 cells and MDA-MB-231 cells by D-[U14C]-Fructose & 2-Deoxy-D-[l-3H] Glucose Uptake Assay --- p.77 / Chapter 3.1.4 --- Effect of Antisense Oligonucleotides against Glut 5 on Intracellular ATP Content of MCF-7 cells and MDA-MB-231 cells by ATP Assay --- p.81 / Chapter 3.1.5 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 RNA Expression of MCF-7 cells and MDA-MB-231 cells by RT-PCR and Real-Time PCR --- p.83 / Chapter 3.1.5.1 --- RT-PCR --- p.83 / Chapter 3.1.5.2 --- Real-Time PCR --- p.87 / Chapter 3.1.6 --- Effect of Antisense Oligonucleotides against Glut 5 on Glut 5 Protein Expression of MCF-7 cells and MDA-MB-231 cells by Western Blot Analysis --- p.89 / Chapter 3.1.7 --- "Effect of Antisense Oligonucleotides against Glut 5 on Change in Cell Cycle Pattern of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using PI Stainning" --- p.93 / Chapter 3.1.8 --- "Effect of Antisense Oligonucleotides against Glut 5 on Induction of Apoptosis of MCF-7 cells and MDA-MB-231 cells by Flow Cytometry, Using Annexin V-FITC Stainning" --- p.98 / Chapter 3.2 --- In Vivo Study --- p.101 / Chapter 3.2.1 --- Animal Model: Nude Mice --- p.101 / Chapter 3.2.2 --- Effect of Antisense Oligonucleotides against Glut 5 on the MCF-7 cells-Bearing Nude Mice --- p.101 / Chapter 3.2.2.1 --- Change of Weight of the Tumor-Bearing Nude Mice --- p.101 / Chapter 3.2.2.2 --- Tumor Growth Rate --- p.105 / Chapter 3.2.2.3 --- Glut 5 RNA Expression by Real-Time PCR --- p.109 / Chapter 3.2.2.4 --- Glut 5 RNA Expression by Western Blotting --- p.111 / Chapter 3.2.3 --- "Assessment of Side Effects of Antisense Oligonucleotides against Glut 5, by Measuring the Plasma Enzyme Level" --- p.113 / Chapter Chapter 4 --- Discussion --- p.118 / Chapter 4.1 --- Antisense Oligonucleotides against Glut 5 on Human Breast Cancer --- p.119 / Chapter 4.1.1 --- Antisense Oligonucleotides Strategy --- p.119 / Chapter 4.1.2 --- Role of Glut 5 in Breast Cancer --- p.123 / Chapter 4.1.3 --- Effects of Tamoxifen on MCF-7 and MDA-MB-231 --- p.126 / Chapter 4.2 --- In Vitro Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.127 / Chapter 4.3 --- In Vivo Study of Antisense Oligonucleotides against Glucose Transporter 5 on Breast Cancer Cells --- p.135 / Chapter 4.3.1 --- Effects of Antisense Oligonucleotides against Glut 5 on Body Weight and Tumor Size --- p.137 / Chapter 4.3.2 --- Expression Level of Glut 5 of the Tumor --- p.138 / Chapter 4.3.3 --- Assessment of Side Effects of Antisense Oligonucleotides against Glut 5，by Measuring the Plasma Enzymes Level --- p.140 / Chapter 4.4 --- Possible Mechanism of Antisense Oligonucleotides against Glut 5 on Breast Cancer --- p.141 / Chapter Chapter 5 --- Future Prospectus and Conclusions --- p.143 / Chapter 5.1 --- Future Prospectus of Antisense Oligonucleotides --- p.144 / Chapter 5.1.1 --- Antisense Oligonucleotides and Treatment of Breast Cancer --- p.144 / Chapter 5.1.2 --- Role of Glut 5 in Breast Cancer --- p.147 / Chapter 5.2 --- Conclusions and Remarks --- p.148 / References --- p.151

The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation? The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro. This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation.

Insulin stimulates glucose uptake in adipose and muscle cells via translocation of the intracellular vesicles containing GLUT4. It was largely unknown whether and/or how the signaling molecules such as PI 3-kinase and Akt regulate the mechanical movements of the GLUT4-containing vesicles. Hence, this study was performed to test the hypothesis that actin and microtubules function in translocating GLUT4 vesicles. Treatments of insulin as well as endothelin-1 (ET-1), an insulin-mimicking peptide which does not act through PI 3-kinase, induced polymerization of actin without affecting the microtubular network. By mass spectrometry, the tyrosine kinase PYK2 was identified to be tyrosine phosphorylated specifically by ET-1 but not by insulin. Expression of the carboxyl-terminal fragment (CRNK) PYK2, but not wild type nor kinase-deficient PYK2 mutants, inhibited ET-1-stimulated actin polymerization while expression of all three PYK2 constructs had no effect on insulin-stimulated actin polymerization. More importantly, expression of CRNK, but not wild type nor kinase-deficient PYK2 constructs, blocked ET-1- but not insulin-stimulated GLUT4 translocation to the plasma membrane. These suggest that ET-1 and insulin stimulate actin polymerization via distinct signaling pathways, and that the actin polymerization is required for GLUT4 vesicle translocation. In order to test the possible involvement of microtubule in GLUT4 vesicle translocation, time lapse imaging of 3T3-L1 adipocytes expressing GLUT4-YFP and tubulin-CFP was performed. GLUT4-YFP vesicles move long-range bi-directionally on microtubules, which suggests the presence of molecular motors on the vesicles. Moreover, insulin increased the number of vesicle movements on microtubules without changing the velocities. Interestingly, the stimulatory action of insulin appears to be independent of PI 3-kinase activation. Conventional kinesin was identified as a highly expressed kinesin isotype in adipocytes. Notably, expression of dominant negative mutants but not wild type kinesin inhibited insulin-stimulated long-range GLUT4 vesicle movements and GLUT4 translocation to the plasma membrane in live and fixed cells, respectively. These data indicate that insulin signaling induces the movement of GLUT4 vesicles on microtubule which is mediated by conventional kinesin. Overall, the data presented here provide evidence supporting the hypothesis that actin and microtubule cytoskeletons are required for insulin to mobilize GLUT4 vesicles in adipocytes.