Dissertations / Theses on the topic 'Facilitative Glucose Transport Proteins'

Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.

Hydrodynamic analysis and electron microscopy of GLUT1/lipid/detergent micelles and freeze fracture electron microscopy of GLUT1 proteoliposomes support the hypothesis that the glucose transporter is a multimeric (probably tetrameric) complex of GLUT1 proteins. Some detergents (e.g. octylglucoside) maintain the multimeric complex while other detergents (e.g. CHAPS and dodecylmaltoside) promote the dissociation of GLUT1 oligomers into smaller aggregation states (dimers or monomers). GLUT1 does not appear to exchange rapidly between protein/lipid/detergent micelles but is able to self-associate in the plane of the lipid bilayer. Quantitatively deglycosylated GLUT1 displays aberrant electrophoretic mobility, but each protein band contains full-length GLUT1 and the less mobile species, when treated with additional detergent and reductant, converts to the more mobile species. Preliminary structural analysis suggests that denaturing detergent- and thiol chemistry-related changes of α-helical content may mirror mobility shifts. Limited proteolysis of membrane-resident GLUT1 (± ligands) releases membrane-spanning α-helical domains suggesting that (i) some bilayer-resident helices are highly solvent exposed; (ii) membrane-spanning domains 1, 2, & 4 and 7, 8, & 10 are destabilized upon ligand binding; and (iii) helix packing compares well with high-resolution structures of prokaryotic transporters from the same superfamily. Results are consistent with a central, hydrophilic, translocation pathway comprised of amphipathic, membrane-spanning domains that alter associations upon ligand/substrate binding. We have resolved technical difficulties (heterogeneity, lipid/detergent removal, glycosylation, small molecule contamination) associated with GLUT1 analysis by mass spectrometry; and we map global conformational changes between sugar uptake and sugar efflux.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
Vascular anomalies constitute a distinct group of lesions, but they may present similar clinical and histopatological characteristics, which can lead to diagnostic mistakes. This study aimed by histopathology and immunohistochemical expression of human glucose transporter protein (GLUT-1), correctly identify and classify oral vascular anomalies, besides analyzing the immunoexpression of markers proliferation and apoptosis (Ki-67 and Bcl-2). All cases diagnosed as "oral hemangiomas" belonging to the archives of the Service of Pathological Anatomy from the subject of Oral Pathology of the Department of Dentistry (DOD), of the Federal University of Rio Grande do Norte (UFRN) were reviewed, totalizing 77 cases. Immunohistochemical analysis for GLUT-1 showed that only 26 (33.8%) of the specimens were true infantile hemangiomas (IHs). The 51 (66.2%%) GLUT-1 negative specimens were then reclassified as pyogenic granulomas (PGs) and vascular malformations (VMs) from their histopathologic characteristics,totalizing 26 (33.8%) cases of IHs, 20 (26.0%) of PGs and 31 (40.2) cases of oral VMs. The cases analyzed by the marker Ki-67 showed different median IH (13,85), PG (33,70) and VM (4.55) with statistically significant differences between them (p <0.001). In relation to the protein Bcl-2, the groups also showed different median of the established scores IH (1.00), PG (1.50), VMs (0.0) demonstrating statistically significant differences between them (p<0,001). No statistically significant correlation between the indexes of positivity for Ki-67 and the scores of immunoexpression of Bcl-2 were observed in any group. Thus, we can conclude that it is necessary a careful and parameterized review of cases of vascular anomalies making use of auxiliary tools such as GLUT-1, since the histopathological findings alone, sometimes, are not sufficient to differentiate some anomalies. Furthermore, analysis of the expressions of markers involved in the levels of proliferation of lesions is important for a better understanding of its biological behavior aspect
As anomalias vasculares constituem um grupo de les?es distintas, mas que podem apresentar caracter?sticas cl?nicas e histopatol?gicas semelhantes, que podem levar a equ?vocos diagn?sticos.Este estudo objetivou por meio da histopatologia e da express?o imuno-histoqu?mica daprote?na humana transportadora de glicose (GLUT-1), identificar e classificar corretamente as anomalias vasculares orais, al?m de analisar a imunoexpress?o de marcadores de prolifera??o e apoptose (Ki-67 e Bcl-2).Todos os casos diagnosticados como hemangiomas orais pertencentes aos arquivos do Servi?o de Anatomia Patol?gica da disciplina de Patologia Oral do Departamento de Odontologia (DOD) da Universidade Federal do Rio Grande do Norte (UFRN) foram revisados, totalizando 77 casos. A an?lise imuno-histoqu?mica para GLUT-1 revelou que apenas 26 (33,8%) dos esp?cimes tratavam-se de hemangiomas da inf?ncia (HIs) verdadeiros. Os 51 (66,2%%)esp?cimes GLUT-1 negativos foram ent?o reclassificados em granulomas piog?nicos (GPs) e malforma??es vasculares (MVs) a partir de suas caracter?sticas histopatol?gicas, totalizando 26 (33,8%) casos de HIs, 20 (26,0%) de GPs e 31 (40,2) casos de MVs orais. Os casos submetidos ? an?lise do marcador Ki-67 apresentaram medianas diferentes HI (13,85), GP (33,70) e MV (4,55) com diferen?as estatisticamente significantes entre elas (p<0,001). Em rela??o ? prote?na Bcl-2, os grupos tamb?m apresentaram diferentes medianas dos escores estabelecidos HI (1,00), GP (1,50), MVs (0,0), demonstrando diferen?as estatisticamente significantes entre elas (p<0,001). N?o foi observada correla??o estatisticamente significante entre os ?ndices de positividade para o Ki-67 e os escores de imunoexpress?o de Bcl-2 em nenhum grupo.Dessa maneira, podemos concluir que se faz necess?rio uma revis?o criteriosa e parametrizada dos casos de anomalias vasculares utilizando ferramentas auxiliares, como a GLUT-1, uma vez que os achados histopatol?gicos sozinhos, ?s vezes, n?o s?o suficientes para diferenciar algumas anomalias. Al?m disso, a an?lise das express?es de marcadores envolvidos nos n?veis de prolifera??o das les?es ? um aspecto importante para o melhor entendimento do seu comportamento biol?gico

Studies have demonstrated that under conditions of chronic metabolic stress, GLUT1-mediated sugar transport is upregulated at the blood-brain barrier by a number of mechanisms. Although acute metabolic stress has also been shown to increase GLUT1-mediated transport, the mechanisms underlying this regulation remain unclear. This work attempts to explain how GLUT1-mediated sugar uptake is increased during acute metabolic stress, as well as explore the factors involved in this modulation of sugar transport in blood-brain barrier endothelial cells. Glucose depletion, KCN and FCCP were applied to brain microvascular endothelial cell line bEnd.3 in order to induce acute metabolic stress by ATP depletion. Kinetic sugar uptake measurements in combination with qPCR, whole cell lysate western blots, and cell-surface biotinylation were employed to probe for changes in GLUT1-mediated sugar uptake, GLUT1 expression levels, and GLUT1 localization during metabolic stress. Finally, the role of AMP-activated kinase (AMPK) in the bEnd.3 cell response to acute stress was examined using the specific AMPK activator AICAR and inhibitor Compound C. The data presented in this thesis supports the following two conclusions: 1. GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress is increased 3-7 fold due to translocation of intracellular GLUT1 to the plasma membrane, with no change in expression of total GLUT1 protein, and 2. AMPK plays a direct role in modulating increases in GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress by regulating trafficking of GLUT1 to the plasma membrane.

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.

The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure. A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions. This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding.

The importance of insulin delivery and action is best characterized in Type 2 Diabetes, a disease that is becoming a pandemic both nationally and globally. Obesity is a principal risk factor for Type 2 Diabetes, and adipocyte function abnormalities due to adipose hypertrophy and hyperplasia, have been linked to obesity. Numerous reports suggest that the intracellular and systemic consequences of adipocyte function abnormalities include adipocyte insulin resistance, enhanced production of free fatty acids, and production of inflammatory mediators. A hallmark of adipocyte insulin sensitivity is the stimulation of glucose transporter isoform 4 (GLUT4) trafficking events to promote glucose uptake. In the Type 2 diabetic and insulin resistant states the mechanism behind insulin-stimulated GLUT4 trafficking is compromised. Therefore, understanding the role of factors involved in glucose-uptake in adipose tissue is of great importance. Studies from our laboratory suggest an important role for the unconventional myosin, Myo1c, in promoting insulin-mediated glucose uptake in cultured adipocytes. Our observations suggest that depletion of Myo1c in cultured adipocytes results in a significant reduction in the ability of adipocytes to take up glucose following insulin treatment, suggesting Myo1c is required for insulin-mediated glucose uptake. A plausible mechanism by which Myo1c promotes glucose uptake in adipocytes has been suggested by further work from our laboratory in which expression of fluorescently-tagged Myo1c in cultured adipocytes induces significant membrane ruffling at the cell periphery, insulin-independent GLUT4 translocation to the cell periphery, and accumulation of GLUT4 in membrane ruffling regions. Taken together Myo1c seems to facilitate glucose uptake through remodeling of cortical actin. In the first part of this thesis I, in collaboration with others, uncovered a possible mechanism through which Myo1c regulates adipocyte membrane ruffling. Here we identified a novel protein complex in cultured adipocytes, comprising Myo1c and the mTOR binding partner, Rictor. Interestingly our studies in cultured adipocytes suggest that the Rictor-Myo1c complex is biochemically distinct from the Rictor-mTOR complex of mTORC2. Functionally, only depletion of Rictor but not Myo1c results in decreased Akt phosphorylation at serine 473, but depletion of either Rictor or Myo1c results in compromised cortical actin dynamic events. Furthermore we observed that whereas the overexpression of Myo1c in cultured adipocytes causes remarkable membrane ruffling, Rictor depletion in cells overexpressing Myo1c significantly reduces these ruffling events. Taken together our findings suggest that Myo1c, in conjunction with Rictor, modulates cortical actin remodeling events in cultured adipocytes. These findings have implications for GLUT4 trafficking as GLUT4 has been previously observed to accumulate in Myo1c-induced membrane ruffles prior to fusion with the plasma membrane. During our studies of adipocyte function we noticed that current siRNA electroporation methods present numerous limitations. To silence genes more effectively we employed a lentivirus-mediated shRNA delivery system, and to standardize this technology in cultured adipocytes we targeted Myo1c and MAP4K4. Using this technology we were able to achieve clear advantages over siRNA oligonucleotide electroporation techniques in stability and permanence of gene silencing. Furthermore we showed that the use of lentiviral vectors in cultured adipocytes did not affect insulin signaling or insulin-mediated glucose uptake events. Despite our inability to use lentiviral vectors to achieve gene silencing in mice we were able to achieve adipose tissue-specific gene silencing effects in mice following manipulation of the lentiviral conditional silencing vector, and then crossing resulting founders with aP2-Cre mice. Interestingly however, only founders from the MAP4K4 conditional shRNA vector, but not founders from the Myo1c conditional shRNA vector, showed gene knockdown, possibly due to position-effect variegation. Taken together, findings from these studies are important because they present an alternative means of achieving gene silencing in cultured adipocytes, with numerous advantages not offered by siRNA oligonucleotide electroporation methods. Furthermore, the in vivo, adipose tissue-specific RNAi studies offer a quick, inexpensive, and less technically challenging means of achieving adipose tissue-specific gene ablations relative to traditional gene knockout approaches.

The purpose of this investigation was to determine the effects of post-exercise carbohydrate-protein feedings on muscle glycogen restoration following exhaustive cycle ergometer exercise. Seven male collegiate cyclist (age=25.6±3.3y, ht.=180.9±8.5cm, wt.=75.4±10.7kg, VO2max=4.20±0.4 1•miri 1) performed three trials, each separated by -lwk, 1) 100% (x-D glucose (CHO), 2) 70% carbohydrate-20% protein-10% fat (CHOPRO), and 3) 86% carbohdyrate-14% amino acid (CHO-AA). All feedings were eucaloric, based upon 1.0 g•kgb.W.'1•hr"1 of carbohydrate, and administered every half hour during a four hour muscle glycogen restoration period in an 18% wt./vol. solution. Muscle biopsies were obtained immediately and four hours post exercise. Following the exhaustive exercise and every half hour for four hours a blood sample was drawn. Muscle glycogen concentrations increased 53%, 47%, and 57% for the CHO, CHO-PRO, and CHO-AA feedings, respectively, however no differences among the feedings were apparent in muscle glycogen restoration. The plasma glucose and insulin concentrations demonstrated no differences throughout the restoration period among the three feedings. These results suggest that muscle glycogen restoration does not appear to be enhanced with the addition of either protein or amino acids to an eucaloric carbohydrate feeding following an exhaustive cycle exercise. However, it appears that if adequate amounts of carbohydrates are consumed (greater than 0.70 g•kgb,W,."'•hf' carbohydrate) following exhaustive exercise, maximal muscle glycogen restoration occurs.
School of Physical Education

Integral membrane proteins (IMPs) assume critical roles in cell biology and are key targets for drug discovery. Given their involvement in a wide range of diseases, the structural and functional characterization of IMPs are of significant importance. However, this remains notoriously challenging due to the difficulties of stably purifying membrane-bound, hydrophobic proteins. Compounding this, many diseases are caused by IMP mutations that further decrease their stability. One such example is cystic fibrosis (CF), which is caused by misfolding or dysfunction of the epithelial cell chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Roughly 70% of CF patients world-wide harbor the ΔF508-CFTR mutation, which interrupts CFTR’s folding, maturation, trafficking and function. No existing treatment sufficiently addresses the consequences of ΔF508, and the substantial instability that results from this mutation limits our ability to study ΔF508-CFTR in search of better treatments. To that end, my colleagues at Sanofi generated homology models of full-length wild-type and ΔF508-CFTR +/- second-site suppressor mutations (SSSMs) V510D and R1070W, and performed molecular dynamics (MD) simulations for each model. Using information obtained from this analysis, I tested several hypotheses on the mechanism by which ΔF508 destabilizes full-length CFTR and how SSSMs suppress this effect. Leveraging studies of the purified NBD1 subdomain and of full-length CFTR in a cellular context, I confirmed the prediction of a key salt-bridge interaction between V510D and K564 important to second-site suppression. Furthermore, I identified a novel class of SSSMs that support a key prediction from these analyses: that helical unraveling of TM10, within CFTR’s second transmembrane domain, is an important contributor to ΔF508-induced instability. In addition, I developed a detergent-free CFTR purification method using styrene-maleic acid (SMA) copolymer to extract the channel directly from its cell membrane along with the surrounding lipid content. The resulting particles were stable, monodisperse discs containing a single molecule of highly-purified CFTR. With this material, I optimized grid preparation techniques and carried out cryo-EM structural analysis of WT-hCFTR which resulted in 2D particle class averages which were consistent with an ABC transporter shape characteristic of CFTR, and a preliminary 3D reconstruction. This result establishes a foundation for future characterization of ΔF508-CFTR in its native state. I have also applied this SMA-based purification method to the facilitated glucose transporter GLUT1 (SLC2A1). SLC2A1 mutations contribute to a rare and developmentally debilitating disease called GLUT1-deficiency syndrome. Using SMA, I successfully extracted GLUT1 in its native state. With the application of this method, I was able to purify endogenous GLUT1 from erythrocytes, in complex with several associated proteins as well as the surrounding lipids, in its monomeric, dimeric and tetrameric forms without the use of cross-linking or chimeric mutations. These results point to the potential for studying isolated IMPs without the use of destabilizing detergents and thereby offer a pathway to analysis of wild-type and mutant membrane protein structure, function and pharmacodynamics.

Hebert and Carruthers (1992) showed that the human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive noncovalent subunit interactions. The GLUT1 tetramer dissociates into dimers upon exposure to reductant but subunits are not associated via disulfide bridges. Each subunit of SDS-denatured tetrameric GLUT1 exposes only two thiols while reduced denatured GLUT1 exposes all six sulfhydryl groups. They hypothesized that glucose transporter oligomeric structure and cooperative catalytic function resulted from noncovalent subunit interactions promoted or stabilized by intramolecular disulfide bridges. These interactions give rise to an antiparallel arrangement of substrate binding sites within the transporter complex. In the present studies, we tested aspects of this model. Specifically, we wanted 1) to understand why the native, noncovalent, homotetrameric GLUT1 complex is sensitive to reductant, 2) to determine whether the tetramer is more catalytically efficient than the dimer in situ, and 3) to test the hypothesis that it is the antiparallel arrangement of substrate binding sites between subunits that provides the transporter with its catalytic advantage. We used biochemical and molecular biological approaches to isolate specific determinants of transporter oligomeric structure and/or transport function in purified isolated transporter preparations, in intact red cells and in CHO cells. We have also examined the hypothesis that net sugar transport in the human erythrocyte is rate limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules. Our findings support the hypothesis that each subunit of the parental glucose transporter contains a single intramolecular disulfide bridge located between cysteine residues 347 and 421. This disulfide seems to be necessary for GLUT1 tetramerization. Our findings suggest that GLUT1 N-terminal residues 1 through 199 provide contact surfaces for subunit dimerization but are insufficient for subunit tetramerization. Our studies also show that in situ disulfide disruption by cell impermeant reductants results in the loss of cooperative subunit interactions and a 3 to 15-fold reduction in the transport efficiency of the transporter. We further find that in situ GLUT1 is susceptible to exofacial proteolysis. Exofacial trypsin cleavage eliminates cooperativity between subunits but does not affect transporter oligomeric structure or transport activity. Thus catalytic efficiency does not derive directly from cooperative interactions between substrate binding sites on adjacent subunits. We have confirmed that 30MG transport in human erythrocytes is a diffusion limited process. We find that steady-state sugar uptake in red cells and K562 cells measures two processes - sugar translocation and intracellular sugar binding. We propose a model for native GLUT1 structure and function.

O diabetes melito gestacional (DMG) está relacionado ao crescimento fetal exagerado. Entender a influência de fatores relacionados ao crescimento fetal auxilia na identificação dos fetos com maior risco de desvios da normalidade. Objetivo: comparar fatores clínicos, laboratoriais e a expressão placentária de transportadores de glicose segundo o crescimento fetal em pacientes com DMG. Método: Para análise dos fatores clínicos e laboratoriais foi realizado um estudo retrospectivo com 425 gestantes com DMG do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC FM-USP) no período de janeiro de 2003 a novembro de 2009. Para a análise da expressão placentária dos transportadores de glicose dos tipos 1 (GLUT1), 3 (GLUT3) e 4 (GLUT4) foram selecionados todos os casos de recém-nascidos grandes para idade gestacional (RNGIG) pareados com um caso controle de recém-nascido adequado para idade gestacional (RNAIG). Foram incluídas apenas gestações únicas e com DMG diagnosticado pelo teste de tolerância à glicose oral de 100 gramas, sem malformações fetais e com idade gestacional definida e confiável. Todas as gestantes realizaram dieta para diabetes, controle glicêmico diário e uso de insulina quando necessário. Os critérios de seguimento e tratamento seguiram rigorosamente as normas do Protocolo de Condutas do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do HC-FMUSP. As gestantes foram divididas para análise dos dados em dois grupos: Fatores clínicos e laboratoriais com: 376 RNAIG e 49 RNGIG num total de 425 DMG. Expressão Placentária dos Transportadores de Glicose: 50 RNAIG e 44 RNGIG. Foram realizados testes de associação e médias das variáveis e relacionadas com os grupos de RNAIG e RNGIG. Resultados: Na análise univariada, dos fatores clínicos e laboratoriais, não houve diferenças entre os grupos quanto a: idade materna, antecedente familiar de diabetes, antecedente pessoal de hipertensão arterial, número de gestações, valores de glicemia de jejum e 1 hora no TTGO-100g, idade gestacional no parto, sexo do RN, tipo de parto e índice de Apgar no 1º e 5º minutos. Houve diferenças estatisticamente significativas entre os grupos quanto a: índice de massa corpórea pré-gestacional (p < 0,02); uso de insulina (p < 0,041); macrossomia anterior (p < 0,001); idade gestacional do diagnóstico do DMG (p < 0,001); glicemias de duas e três horas no TTGO-100g respectivamente com (p < 0,003) e (p < 0,026). Na análise de regressão logística foram considerados preditores independentes da ocorrência de RNGIG: o índice de massa corpórea pré - gestacional, a macrossomia anterior, aidade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas. Em relação a expressão dos transportadores de glicose não diferiram entre os grupos em relação a expressão de GLUT1 na decídua, GLUT3 na decídua e vilosidades e GLUT4 na decídua e vilosidades. Houve diferença entre os grupos quanto à: a expressão do GLUT1 nas vilosidades. Conclusões: O índice de massa corpórea pré - gestacional, a macrossomia anterior, a idade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas foram preditores da ocorrência de RNGIG. A expressão de GLUT1 nas vilosidades coriônicas teve relação com a ocorrência de RNGIG
Gestational diabetes mellitus (GDM) is related to excessive fetal growth. Knowing the influence of factors related to fetal growth assists in the identification of fetuses at high risk of deviations from normality. Objective: To compare clinical and laboratory tests and the placental expression of glucose transporters according to fetal growth in patients with GDM. Method: A retrospective study of clinical and laboratory factors related with large for gestational age newborns, included 425 pregnant women with GDM was carried out at Sector Endocrine Clinic of Obstetrics Hospital of the School of Medicine, University of São Paulo (HC-FMUSP), between January 2003 to November 2009. For the analysis of placental expression of glucose transporters types 1 (GLUT1), 3 (GLUT3) and 4 (GLUT4) were selected all cases of newborns large for gestational age (LGA) paired with a case control newly born appropriate for gestational age (AGA). We included only patients with singleton pregnancies and GDM diagnosed by OGTT-100g, with newborns without malformations and birth weight classified as adequate or large for gestational age. All pregnant women received diet for diabetes, daily glycemic control and insulin when necessary. The criteria for monitoring and treatment followed strictly the standards of Conduct Protocol Endocrine Obstetric Clinic of the Clinic Hospital, School of Medicine, University of São Paulo. The pregnancies were divided for analysis into two groups: 376 cases of newborns AGA and 49 cases of newborns LGA. Data were analyzed and considered the probability value p <0.05. Results: In the univariate analysis of clinical and laboratory factors, there were no differences between the groups regarding maternal age, family history of diabetes, personal history of hypertension, number of pregnancies, blood fasting glucose and 1 hour in- OGTT 100g, gestational age at delivery, gender of the newborn, type of delivery, Apgar score at 1st and 5th minutes. There were statistically significant differences between the groups regarding: body mass index before pregnancy (p <0.02), insulin (p <0.041), previous macrosomia (p <0.001), gestational age at diagnosis of GDM (p <0.001), blood glucose levels two and three hours at 100 g OGTT, respectively, with (p <0.003) (p <0.026). In logistic regression analysis were considered independent predictors of the occurrence of LGA: body mass index before pregnancy, previous macrosomia gestational age at diagnosis of GDM and two hours after glucose overload 100 grams. Regarding the expression of glucose transporters, the groups did not differ regarding the expression of GLUT1 in the decidua, GLUT3 in the decidua and villi and GLUT4 in the decidua and villi. There were differences between the groups regarding the expression of GLUT1 in the villi. Conclusions: The body mass index before pregnancy, previous macrosomia, gestational age of diagnosis of GDM and two hours after glucose overload 100 grams were predictors of the occurrence of LGA. The expression of GLUT1 in chorionic villi was related to the occurrence of LGA newborn

Wong Hei Yi.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 157-175).
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.ix
List of Abbreviations --- p.x
Table of Contents --- p.xiii
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- Importance of Glucose in Biological System --- p.1
Chapter 1.2 --- Glucose Transporter Families --- p.2
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 --- p.7
Chapter 1.3.2 --- Secondary Structure --- p.8
Chapter 1.3.3 --- Membrane Topology --- p.8
Chapter 1.3.4 --- Tertiary Structure --- p.9
Chapter 1.3.5 --- Kinetics Properties --- p.11
Chapter 1.3.6 --- Affinity Reagents --- p.12
Chapter 1.3.7 --- Tissue Distribution --- p.13
Chapter 1.3.8 --- Multifunctional Property --- p.14
Chapter 1.3.9 --- Characterization of GLUT1 Gene --- p.14
Chapter 1.3.10 --- Regulation of GLUT1 Expression --- p.15
Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.17
Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome --- p.20
Chapter 1.5.1 --- Background of GlutlDS --- p.20
Chapter 1.5.2 --- Clinical Features of GlutlDS --- p.23
Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24
Chapter 1.5.4 --- Diagnosis --- p.26
Chapter 1.5.4.1 --- Erythrocyte Glucose Transporter Activity --- p.26
Chapter 1.5.4.2 --- Molecular Genetic Testing of GLUT1 Gene --- p.27
Chapter 1.5.4.3 --- Glucose Concentration --- p.27
Chapter 1.5.5 --- Management --- p.28
Chapter 1.5.5.1 --- Ketogenic Diet --- p.28
Chapter 1.5.5.2 --- Medication --- p.29
Chapter 1.5.5.2.1 --- Glutl Activator --- p.29
Chapter 1.5.5.2.2 --- Glutl Inhibitor --- p.29
Chapter 1.6 --- Hypothesis and Objectives --- p.31
Chapter Chapter 2: --- Identification of the First Two Asian GlutlDS Cases --- p.33
Chapter 2.1 --- Materials --- p.34
Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patients --- p.34
Chapter 2.1.2 --- Blood Samples --- p.35
Chapter 2.1.3 --- Reagents for Zero-trans Influx of 3-OMG Uptake in Erythrocytes --- p.35
Chapter 2.1.4 --- Reagents for Zero-trans Efflux of 3-OMG Uptake in Erythrocytes --- p.37
Chapter 2.1.5 --- Reagents for Glutl Gene Analysis --- p.37
Chapter 2.1.6 --- Reagents and Buffers for Reverse Transcription --- p.38
Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.39
Chapter 2.1.8 --- Reagents for Erythrocytes Membrane Preparation and Detection --- p.41
Chapter 2.2 --- Methods --- p.46
Chapter 2.2.1 --- Zero-trans Influx of 3-OMG Uptake in Erythrocytes --- p.46
Chapter 2.2.2 --- Zero-trans Efflux of 3-OMG out of Erythrocytes --- p.47
Chapter 2.2.3 --- Glutl Protein Expression --- p.48
Chapter 2.2.4 --- GLUT1 Gene Analyses --- p.51
Chapter 2.2.5 --- Statistics --- p.58
Chapter 2.3 --- Results --- p.59
Chapter 2.4 --- Discussions and Conclusions --- p.69
Chapter Chapter 3: --- Pathogenicity of GLUT1 Mutations --- p.78
Chapter 3.1 --- Materials --- p.79
Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.79
Chapter 3.1.2 --- Cell Lines --- p.80
Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.81
Chapter 3.1.4 --- Cell Culture Wares --- p.83
Chapter 3.1.5 --- Reagents for Transfection --- p.83
Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.83
Chapter 3.1.7 --- Reagents and Buffers for Flow Cytometry --- p.84
Chapter 3.1.8 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.84
Chapter 3.1.9 --- Reagents and Consumables for Confocal Microscopy --- p.85
Chapter 3.2 --- Methods --- p.86
Chapter 3.2.1 --- Cell Culture Methodology --- p.86
Chapter 3.2.2 --- Construction of Glutl-Encoding Vectors --- p.87
Chapter 3.2.3 --- Construction of Glutl Mutants --- p.91
Chapter 3.2.4 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.92
Chapter 3.2.5 --- Glucose Influx Assays in CHO-K1 Cells --- p.96
Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.97
Chapter 3.2.7 --- Statistics --- p.98
Chapter 3.3 --- Results --- p.99
Chapter 3.4 --- Discussions and Conclusions --- p.112
Chapter Chapter 4: --- Effects of Anticonvulsive Compounds on Cellular Glucose Transport --- p.117
Chapter 4.1 --- Materials --- p.118
Chapter 4.1.1 --- Cell Lines --- p.118
Chapter 4.1.2 --- Cell Culture Media --- p.118
Chapter 4.1.3 --- Blood Sample --- p.119
Chapter 4.1.4 --- Anticonvulsive Compounds --- p.119
Chapter 4.1.5 --- Reagents for Zero-trans Influx of 3-OMG Uptake in Fibroblasts --- p.120
Chapter 4.1.6 --- Reagents for Zero-trans Influx of 2-DOG Uptake in Primary Astrocytes --- p.120
Chapter 4.1.7 --- Reagents for Total RNA Isolation --- p.121
Chapter 4.1.8 --- Reagents and Consumables for Real-Time PCR --- p.122
Chapter 4.2 --- Methods --- p.123
Chapter 4.2.1 --- Cell Culture --- p.123
Chapter 4.2.2 --- Drug Concentrations --- p.123
Chapter 4.2.3 --- Zero-trans Influx of 3-OMG Uptake in Erythrocytes --- p.123
Chapter 4.2.4 --- Zero-trans Influx of 3-OMG Uptake in Fibroblasts --- p.124
Chapter 4.2.5 --- Zero-trans Influx of 2-DOG Uptake in Primary Astrocytes --- p.125
Chapter 4.2.6 --- Gene Expression Study --- p.127
Chapter 4.2.7 --- Statistics --- p.130
Chapter 4.3 --- Results --- p.131
Chapter 4.4 --- Discussions and Conclusions --- p.148
Chapter Chapter 5: --- General Conclusions and Future Perspectives --- p.154
References --- p.157

by Tsui Hong Teng.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 174-181).
Abstracts in English and Chinese.
A CKNO WLED GMENTS --- p.7
ABSTRACT --- p.8-10
Chapter Chapter 1: --- Introduction: --- p.11-44
Chapter 1.1) --- Glucose transporters
Chapter 1.2) --- Glucose transporters and cancers
Chapter 1.3) --- Antisense strategies
Chapter 1.4) --- Cellular uptake of oligonucleotides
Chapter 1.5) --- Hyperthermia and combined treatments
Chapter Chapter 2: --- Materials and methods --- p.45-60
Chapter 2.1) --- Materials:
Chapter 2.1a) --- Cell lines and culture media
Chapter 2.1b) --- Oligonucleotides synthesis
Chapter 2.1c) --- Chemicals
Chapter 2.2) --- Methods:
Chapter 2.2a) --- Oligonucleotide design
Chapter 2.2b) --- Oligonucleotide treatment
Chapter 2.2c) --- Flow cytometry
Chapter 2.2d) --- Confocal microscopy
Chapter 2.2e) --- MTT assay for cytotoxicity or cell proliferation
Chapter Chapter 3: --- Cellular uptake of oligonucleotide spontaneously and Lipofectin-aided: --- p.61-85
Chapter 3.1) --- Introduction
Chapter 3.2) --- Flow cytometric studies
Chapter 3.3) --- Confocal microscopic studies
Chapter 3.4) --- Cytotoxic effect of Lipofectin alone on MCF-7 cells
Chapter 3.5) --- Discussion
Chapter Chapter 4: --- Hyperthermia can enhance oligonucleotide uptake: --- p.86-118
Chapter 4.1) --- Introduction
Chapter 4.2) --- Flow cytometric studies
Chapter 4.3) --- Confocal microscopic studies
Chapter 4.4) --- Cytotoxic effect of hyperthermia on MCF-7 cells
Chapter 4.5) --- FITC-ODN uptake in survival cells by propidium iodide (PI) exclusion method for hyperthermia
Chapter 4.6) --- Discussion
Chapter Chapter 5: --- The antiproliferative effects of antisense molecules against Glut-1 and 5 on MCF- 7 cells transfected by Lipofectin: --- p.119-146
Chapter 5.1) --- Introduction
Chapter 5.2) --- The growth curve of MCF-7 cells
Chapter 5.3) --- The calibration of MTT assay
Chapter 5.4) --- The effect of antisense Glut-1 concentration without Lipofectin on MCF-7 cells
Chapter 5.5) --- The effect of antisense Glut-1 concentration with Lipofectin on MCF-7 cells
Chapter 5.6) --- The effect of antisense Glut-5 concentration without Lipofectin on MCF-7 cells
Chapter 5.7) --- The effect of antisense Glut-5 concentration with Lipofectin on MCF-7cells
Chapter 5.8) --- The effect of transfection time of antisense Glut-1 on MCF-7 cells
Chapter 5.9) --- The effect of transfection time of antisense Glut-5 on MCF-7 cells
Chapter 5.10) --- The effect of transfection time of antisense Glut-5 for higher concentration on MCF-7 cells
Chapter 5.11) --- The effect of antisense Glut-1 to Lipofectin (w/w) ratio on MCF-7 cells
Chapter 5.12) --- The effect of antisense Glut-1 to Lipofection (w/w) ratio for higher transfection time on MCF-7 cells
Chapter 5.13) --- The effect of antisense Glut-5 to Lipofectin (w/w) ratio on MCF-7 cells
Chapter 5.14) --- Discussion
Chapter Chapter 6: --- Combined treatments: --- p.147-162
Chapter 6.1) --- Introduction
Chapter 6.2) --- The effect of combined treatment of antisense Glut-1 combined with antisense Glut-5 on MCF-7 cells
Chapter 6.3) --- The chronic effect of hyperthermia for 5 hours on MCF-7 cells
Chapter 6.4) --- The effect of combined treatment between antisense Glut-1 and hyperthermia on MCF-7 cells
Chapter 6.5) --- The net effect of antisense Glut-1 in combined treatment between hyperthermia and antisense Glut-1 on MCF-7 cells
Chapter 6.6) --- The effect of combined treatment between antisense Glut-5 and hyperthermia on MCF-7 cells
Chapter 6.7) --- The net effect of antisense Glut-5 in combined treatment between hyperthermia and antisense Glut-5 on MCF-7 cells
Chapter 6.8) --- Discussion
Chapter Chapter 7: --- Discussion: --- p.163-173
Chapter Chapter 8: --- References: --- p.174-181

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

Chan Chung Sing.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 93-101).
Abstracts in English and Chinese.
Acknowledgement --- p.i
Abstract --- p.ix
List of Abbreviations --- p.xvii
Chapter CHATPER ONE --- INTRODUCTION
Chapter 1.1 --- Glucose Homeostasis --- p.1
Chapter 1.1.1 --- Function --- p.1
Chapter 1.1.2 --- Origins and regulation of glucose --- p.2
Chapter 1.1.3 --- Glucoregulatory factors --- p.4
Chapter 1.1.4 --- Insulin --- p.6
Chapter 1.1.4.1 --- Function of Insulin --- p.7
Chapter 1.1.4.2 --- Discovery and Production of Insulin --- p.7
Chapter 1.1.4.3 --- Insulin Signaling Pathway --- p.8
Chapter 1.1.4.3.1 --- Insulin Receptor --- p.8
Chapter 1.1.4.3.2 --- MAPK Pathway --- p.9
Chapter 1.1.4.3.3 --- Phosphatidylinositol 3-kinase (PI3-K) Pathway --- p.10
Chapter 1.1.5 --- Glucose Transporters --- p.11
Chapter 1.1.6 --- Role of skeletal muscle in glucose homeostasis --- p.13
Chapter 1.1.7 --- Insulin Resistance --- p.14
Chapter 1.1.8 --- Glucose abnormality and its complications --- p.16
Chapter 1.2 --- Actin --- p.19
Chapter 1.2.1 --- Function of Actin --- p.20
Chapter 1.2.2 --- Actin Accessory Protein --- p.22
Chapter 1.2.3 --- Actin Polymerization --- p.23
Chapter 1.3 --- "Interaction between Insulin, GLUT4 and Actin in Glucose Homeostasis" --- p.24
Chapter 1.3.1 --- Insulin-Induced Actin Remodeling --- p.25
Chapter 1.3.2 --- Actin Remodeling and Insulin-Induced GLUT4 Translocation --- p.26
Chapter 1.3.3 --- Involvement of Insulin Signaling Molecules in Actin Remodeling --- p.27
Chapter 1.3.4 --- Actin Remodeling and Insulin Resistance --- p.30
Chapter 1.4 --- Hypothesis and Objective --- p.30
Chapter 1.4.1 --- Rationale --- p.30
Chapter 1.4.2 --- Hypothesis --- p.31
Chapter 1.4.3 --- Objective --- p.31
Chapter CHAPTER TWO --- MATERIALS AND METHODS
Chapter 2.1 --- Materials --- p.33
Chapter 2.2 --- Cell Culture --- p.36
Chapter 2.2.1 --- Cell Culture --- p.36
Chapter 2.2.2 --- Reagents Preparation and Incubation --- p.39
Chapter 2.3 --- 2-Deoxyglucose Uptake --- p.39
Chapter 2.4 --- Immunofluorescence Microscopy --- p.41
Chapter 2.4.1 --- Permeabilized cell staining --- p.41
Chapter 2.4.2 --- Membrane-intact cell staining --- p.43
Chapter 2.4.3 --- The analysis of actin remodeling reduction --- p.44
Chapter 2.5 --- Live Image Microscopy --- p.44
Chapter 2.6 --- Transmission Electron Microscope Study --- p.44
Chapter 2.7 --- Statistical Analysis --- p.46
Chapter CHAPTER THREE --- RESULTS
Chapter 3.1 --- Cell Growth --- p.48
Chapter 3.2 --- Acute Effect of Insulin on L6 myotubes --- p.48
Chapter 3.2.1 --- Immunofluorescence Microscopy --- p.49
Chapter 3.2.1.1 --- The time profile of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.49
Chapter 3.2.1.2 --- The concentration effect of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.50
Chapter 3.2.1.3 --- Relationship between actin cytoskeleton and GLUT4mycin permeabilized L6 myotubes --- p.51
Chapter 3.2.1.4 --- Translocation of GLUT4myc in membrane-intact L6 myotubes --- p.51
Chapter 3.2.1.5 --- "Effect of methyl-β-cyclodextrins, MeOH or EtOHin permeabilized and membrane-intact L6 myotubes" --- p.52
Chapter 3.2.2 --- 2-Deoxyglucose Uptake --- p.52
Chapter 3.2.2.1 --- "Effects of insulin, methyl-β-cyclodextrins, MeOH and EtOH in L6 myotubes" --- p.52
Chapter 3.2.3 --- TEM Study --- p.53
Chapter 3.2.3.1 --- Effects of insulin on actin cytoskeleton and GLUT4myc in L6 myotubes --- p.53
Chapter 3.3 --- Effect of high glucose and high insulin incubation in L6 myotubes --- p.54
Chapter 3.3.1 --- Immunofluorescence Microscopy --- p.54
Chapter 3.3.1.1 --- High insulin and high glucose preincubation in permeabilized L6 myotubes --- p.55
Chapter 3.3.1.2 --- Effect of high insulin and high glucose incubationin membrane-intact L6 myotubes --- p.55
Chapter 3.3.2 --- 2-Deoxyglucose Uptake --- p.56
Chapter 3.3.2.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.56
Chapter 3.3.3 --- TEM Study --- p.57
Chapter 3.3.3.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.57
Chapter 3.4 --- Effect of FFA incubation in L6 myotubes --- p.58
Chapter 3.4.1 --- Immunofluorescence Microscopy --- p.58
Chapter 3.4.1.1 --- FFA preincubation in permeabilized L6 myotubes --- p.58
Chapter 3.4.1.2 --- FFA incubation in membrane-intact L6 myotubes --- p.59
Chapter 3.4.2 --- 2-Deoxyglucose Uptake --- p.59
Chapter 3.4.2.1 --- FFA incubation in L6 myotubes (24 hours) --- p.60
Chapter 3.4.3 --- TEM Study --- p.62
Chapter 3.4.3.1 --- FFA incubation in L6 myotubes --- p.62
Chapter 3.5 --- Effect of CHO incubation in L6 myotubes --- p.62
Chapter 3.5.1 --- Immunofluorescence Microscopy --- p.62
Chapter 3.5.1.1 --- CHO preincubation in permeabilized L6 myotubes --- p.63
Chapter 3.5.1.2 --- CHO incubation in membrane-intact L6 myotubes --- p.63
Chapter 3.5.2 --- 2-Deoxyglucose Uptake --- p.64
Chapter 3.5.2.1 --- CHO incubation in L6 myotubes (24 hours) --- p.64
Chapter 3.5.3 --- TEM Study --- p.65
Chapter 3.5.3.1 --- CHO incubation in L6 myotubes --- p.65
Chapter 3.6 --- Overall changes in glucose uptake after preincubation experiment --- p.65
Chapter CHAPTER FOUR --- DISCUSSION
Chapter 4.1 --- Effect of insulin on L6 myotubes --- p.69
Chapter 4.2 --- "Effect of methyl-β-cyclodextrins, MeOH and EtOH on L6 myotube" --- p.75
Chapter 4.3 --- Effect of pretreatment of cells in conditions of insulin resistance --- p.76
Chapter 4.3.1 --- Effect of high glucose and high insulin preincubation on L6 myotubes --- p.76
Chapter 4.3.2 --- Effect of FFA preincubation on L6 myotubes --- p.78
Chapter 4.3.3 --- Effect of CHO preincubation on L6 myotubes --- p.82
Chapter 4.3.4 --- Effect of cell preincubation in conditions of insulin resistance on L6 myotubes (TEM) --- p.83
Chapter 4.4 --- Summary of the effects of cell preincubation in conditions of insulin resistance --- p.84
Chapter 4.5 --- Possible mechanisms involved in insulin resistance induction --- p.86
Chapter 4.5.1 --- Possible changes in GLUT expression and activities --- p.87
Chapter 4.5.2 --- Possible changes in insulin signaling propagation --- p.88
Chapter 4.5.3 --- Altered functioning of various actin accessory proteins --- p.89
Chapter 4.6 --- Limitation of the study --- p.90
Chapter 4.7 --- Conclusion --- p.90
Chapter 4.8 --- Future study --- p.91
REFERENCES --- p.93
TABLES

by Au Kwong Keung.
Thesis (Ph.D.)--chinese University of Hong Kong, 1998.
Includes bibliographical references (p. 212-227).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

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

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

by Chan Ka Kui.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 104-113).
Abstracts in English and Chinese.
ABSTRACT --- p.1
Chapter 1 --- INTRODUCTION --- p.5
Chapter 1.1 --- Incidence rate of breast cancer in Hong Kong --- p.5
Chapter 1.2 --- Estrogen and breast cancer --- p.6
Chapter 1.3 --- The relation between glucose transporters and breast cancer --- p.7
Chapter 1.4 --- Antisense oligonucleotide --- p.10
Chapter 1.5 --- Action mechanisms of antisense oligonucleotide --- p.11
Chapter 1.6 --- Modification of the oligonucleotide --- p.13
Chapter 1.7 --- Length --- p.16
Chapter 1.8 --- Sequence selection of the antisense oligonucleotide --- p.16
Chapter 1.9 --- Delivery means in antisense oligonucleotide --- p.18
Chapter 1.10 --- The therapeutic role of antisense oligonucleotide --- p.19
Chapter 1.11 --- Objective of the project --- p.21
Chapter 2 --- MATERIAL AND METHODS --- p.23
Chapter 2.1 --- Materials --- p.23
Chapter 2.2 --- Methods --- p.26
Chapter 3 --- RESULTS --- p.37
Chapter 3.1 --- The characteristics of MCF-7 and MDA-MB-231 cells --- p.37
Chapter 3.2 --- Trend of uptake of antisense oligonucleotides in MCF-7 and MDA- MB-231 cells --- p.41
Chapter 3.3 --- The integrity of the oligonucleotide in serum-free medium during transfection --- p.48
Chapter 3.4 --- Detection of effects of Glut5 antisense oligonucleotides of breast tumor cells-MTT assay --- p.50
Chapter 3.5 --- Detection of the antiproliferative effect by trypan blue exclusion assay and thymidine incorporation --- p.56
Chapter 3.6 --- Cell cycle analysis and DNA extraction --- p.61
Chapter 3.7 --- Suppression of Glut5 mRNA detected by RT-PCR --- p.66
Chapter 3.8 --- Suppression of translation of Glut5 proteins as indicated by Western blotting --- p.73
Chapter 3.9 --- Measurement of the fructose and glucose uptake in MCF-7 and MDA -MB-231 cells after antisense treatment --- p.76
Chapter 3.10 --- Change of the phosphofructokinase-1 (PFK-1) activities in MDA- MB-231 cells --- p.82
Chapter 3.11 --- Measurement of the change in the intracellular pH of the breast tumor cells --- p.84
Chapter 4 --- DISCUSSION --- p.89
Chapter 4.1 --- The insights of Glut5 antisense oligonucleotide into cancer therapy --- p.89
Chapter 4.2 --- The uptake pattern of Glut5 antisense oligonucleotides in breast tumor cells --- p.90
Chapter 4.3 --- Stability of antisense oligonucleotide during transfection --- p.92
Chapter 4.4 --- Effects of Glut5 antisense oligonucleotide on MCF-7 and MDA-MB- 231cells --- p.93
Chapter 4.5 --- Proofs of undergoing antisense action mechanism --- p.95
Chapter 4.6 --- Physiological changes in breast tumor cells after antisense treatment --- p.97
Chapter 5 --- CONCLUSION --- p.103
Chapter 6 --- References --- p.104

Lam Mei Wah.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 172-181).
Abstracts in English and Chinese.
Abstract --- p.i
論文撮要 --- p.iv
Acknowledgement --- p.vii
Table of contents --- p.viii
List of tables --- p.xi
List of figures --- p.xii
Abbreviations --- p.xvii
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- The facilitative glucose transporter family --- p.2
Chapter 1.2 --- Overexpression of glucose transporters in tumor cells --- p.5
Chapter 1.3 --- Antisense strategy --- p.8
Chapter 1.3.1 --- Modifications of oligonucleotides --- p.9
Chapter 1.3.2 --- Delivery system for oligonucleotides --- p.13
Chapter 1.3.3 --- Factors influencing antisense activity --- p.16
Chapter 1.3.4 --- Mechanism of action of antisense oligonucleotides --- p.17
Chapter 1.3.5 --- Clinical trials of antisense treatment --- p.21
Chapter 1.4 --- Objective of present study --- p.23
Chapter Chapter 2: --- Materials and Methods --- p.24
Chapter 2.1 --- Materials --- p.25
Chapter 2.1.1 --- Reagents and buffers --- p.25
Chapter 2.1.2 --- Reagents for Western blot analysis --- p.26
Chapter 2.1.3 --- Culture medium --- p.28
Chapter 2.1.4 --- Chemicals --- p.29
Chapter 2.1.5 --- Culture of cells --- p.31
Chapter 2.1.5.1 --- Differentiated Human Hepatoblastoma cell line (HepG2) --- p.31
Chapter 2.1.5.2 --- "Multi-drug resistant hepatoma cell line, R-HepG2 cells" --- p.32
Chapter 2.1.6 --- Animal Studies --- p.33
Chapter 2.2 --- Methods --- p.34
Chapter 2.2.1 --- In vitro studies --- p.34
Chapter 2.2.1.1 --- Design of oligonucleotide sequence --- p.34
Chapter 2.2.1.2 --- Transfection --- p.35
Chapter 2.2.1.3 --- MTT assay --- p.36
Chapter 2.2.1.4 --- Flow cytometry --- p.37
Chapter 2.2.1.5 --- H-thymidine incorporation assay --- p.45
Chapter 2.2.1.6 --- 2-Deoxy-D-[l-3H] glucose uptake assay --- p.46
Chapter 2.2.1.7 --- Adenosine-5'-triphosphate (ATP) assay --- p.47
Chapter 2.2.1.8 --- Western blot analysis --- p.50
Chapter 2.2.2 --- In vivo studies --- p.55
Chapter 2.2.2.1 --- Animal studies --- p.55
Chapter (i) --- Lactate dehydrogenase (LDH) assay --- p.58
Chapter (ii) --- Creatine kinase (CK) assay --- p.60
Chapter (iii) --- Aspartate transaminase (AST) assay --- p.62
Chapter (iv) --- Alanine transaminase (ALT) assay --- p.64
Chapter Chapter 3: --- Results --- p.67
Chapter 3.1 --- In vitro studies --- p.68
Chapter 3.1.1 --- Characteristics of the multi-drug resistant cell line (R-HepG2) developed in our laboratory --- p.68
Chapter 3.1.2 --- Effect of lipofectin on cell viability --- p.77
Chapter 3.1.3 --- Cellular uptake of antisense oligonucleotide --- p.82
Chapter 3.1.4 --- Effect of Glut 2 antisense oligonucleotides on human hepatoma HepG2 and its multidrug resistant (R-HepG2) cells by MTT assay --- p.87
Chapter 3.1.5 --- Suppression of Glut 2 protein expression by antisense oligonucleotides as revealed by Western blot analysis --- p.96
Chapter 3.1.6 --- Uptake of glucose in HepG2 and R-HepG2 after Glut 2 antisense treatment --- p.100
Chapter 3.1.7 --- ATP content in HepG2 and R-HepG2 was lowered after treating the cells with antisense oligonucleotides --- p.108
Chapter 3.1.8 --- Antisense oligonucleotides against Glut 2 exhibited antiproliferative effect on HepG2 and R-HepG2 cells --- p.117
Chapter 3.1.9 --- Change in cell cycle pattern after antisense treatment --- p.125
Chapter 3.1.10 --- Glut 2 antisense oligonucleotides did not induce apoptosis --- p.131
Chapter 3.2 --- In vivo studies --- p.135
Chapter 3.2.1 --- Effect of antisense oligonucleotides on the tumor weight in nude mice bearing HepG2 cells or R-HepG2 cells --- p.135
Chapter 3.2.2 --- Assessment of any side effect of antisense drug done on normal tissues of nude mice --- p.139
Chapter 3.2.2.1 --- Treatment on tumor bearing nude mice with Glut 2 antisense or sense oligonucleotides did not cause myocardial injury --- p.139
Chapter 3.2.2.2 --- Liver injury was not detected in Glut 2 antisense or sense oligonucleotides treated tumor bearing nude mice --- p.147
Chapter Chapter 4: --- Discussion --- p.151
Chapter 4.1 --- In vitro study of the effect of antisense oligonucleotides against Glut 2 on HepG2 and its multi-drug resistant R-HepG2 cell lines --- p.152
Chapter 4.1.1 --- Design of antisense oligonucleotides against Glut 2 --- p.154
Chapter 4.1.2 --- Conditions for antisense inhibition by oligonucleotides --- p.155
Chapter 4.1.3 --- Biological effects of antisense oligonucleotides --- p.158
Chapter 4.2 --- In vivo study of the effect of antisense oligonucleotides against Glut 2 on HepG2 or R-HepG2 cells bearing nude mice --- p.166
Chapter 4.2.1 --- Effect of Glut 2 antisense oligonucleotides on tumor weight --- p.167
Chapter 4.2.2 --- In vivo side effects of oligonucleotides --- p.168
Chapter 4.3 --- Conclusion --- p.169
Bibliography --- p.172