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by Daron Adam Fincham. / Bibliography: leaves [183]-[210] / Thesis (Ph.D.)--Chinese University of Hong Kong, 1985

by Mano Arumanayagam. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 249-265. / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Transport Pathways of Sodium in Human Erythrocytes --- p.2 / Chapter 1.2 --- Active Transport - the Sodium Pump --- p.2 / Chapter 1.2.1 --- Effects of Sodium and Potassium Ions --- p.3 / Chapter 1.2.2 --- Molecular Weight and Subunit Structure --- p.4 / Chapter 1.2.3 --- Inhibitors of the Sodium Pump --- p.6 / Chapter 1.2.4 --- "Measurements of the Sodium Pump/Na+,K+-ATPase" --- p.8 / Chapter 1.3 --- The Passive Fluxes of Sodium & Potassium --- p.13 / Chapter 1.3.1 --- The Na-K-Cl Co-transport System (SPC) --- p.14 / Chapter 1.3.2 --- Sodium-Lithium Countertransport (SLC) --- p.16 / Chapter 1.3.3 --- Ouabain & Frusemide Insensitive Na+ 'leak' --- p.17 / Chapter 1.3.4 --- Methods for the Determination of Passive Na Fluxes --- p.18 / Chapter 1.4 --- Erythrocyte Sodium Transport and Thyroid Hormones --- p.19 / Chapter 1.5 --- Thyroid Hormones and the Na Pump in Other Cells --- p.22 / Chapter 1.6 --- "Mechanism of T3 Induced Increments of the Sodium Pump/Na+,K+-ATPase Activity" --- p.25 / Chapter 1.7 --- Aims of the Project --- p.28 / Chapter CHAPTER 2 --- "ERYTHROCYTE SODIUM FLUXES, OBS AND NA+,K+-ATPASE ACTIVITY IN HYPERTHYROIDISM 29" / Chapter 2.1 --- PREAMBLE --- p.30 / Chapter 2.2 --- MATERIALS & METHODS --- p.31 / Chapter 2.2.1 --- Materials --- p.31 / Chapter 2.2.2 --- Subjects --- p.31 / Chapter 2.2.3 --- Blood Specimen --- p.32 / Chapter 2.2.4 --- Separation of Erythrocytes from Whole Blood --- p.32 / Chapter 2.2.5 --- Erythrocyte Naic and Kic --- p.32 / Chapter 2.2.6 --- Ouabain-sensitive sodium transport --- p.33 / Chapter 2.2.7 --- "Determination of Na+,K+-ATPase activity" --- p.34 / Chapter 2.2.8 --- "Determination SPC, SLC and Na+ 'leak'" --- p.35 / Chapter 2.2.9 --- Determination of OBS and its Kd --- p.37 / Chapter 2.2.10 --- Plasma Thyroid Hormone Analyses --- p.41 / Chapter 2.2.11 --- "Determination of Plasma Concentrations of Sodium, Potassium, Urea and Creatinine" --- p.41 / Chapter 2.2.12 --- "Determination of Hb concentration, Leucocyte and Erythrocyte Counts, and MCV" --- p.41 / Chapter 2.2.13 --- Assessment of the Precision of the Methods --- p.41 / Chapter 2.3 --- STATISTICS --- p.42 / Chapter 2.4 --- RESULTS --- p.44 / Chapter 2.4.1 --- Determination of the Concentration of Ouabain in a Stock of 3H-ouabain --- p.44 / Chapter 2.4.1.1 --- Effect of Incubation Time on Binding of Ouabain to Erythrocytes --- p.46 / Chapter 2.4.1.2 --- Scatchard Plots of Bound Against Ratio of Bound to Free --- p.48 / Chapter 2.4.2 --- "Establishment of the Method for the Simultaneous Determination of SLC, SPC and Na+'leak'" --- p.48 / Chapter 2.4.3 --- Descriptive Statistics of the Subjects in the Study --- p.54 / Chapter 2.4.4 --- "Plasma concentrations of T4, T3, free T4, and free T3 in control subjects and hyperthyroid patients" --- p.54 / Chapter 2.4.5 --- "Plasma Concentrations of Sodium, Potassium, Urea, Creatinine and Ratios of Creatinine to Urea in Control Subjects and Hyperthyroid Patients" --- p.54 / Chapter 2.4.6 --- "Leucocyte Count, Erythrocyte Count, Hb Concentration, MCV and Derived Values for PCV, MCHC and MCH in Control Subjects and Hyperthyroid Patients" --- p.58 / Chapter 2.4.7 --- "Intracellular Sodium Naic, potassium Kic, ouabain-sensitive efflux rate (fo), ouabain-sensitive efflux rate constant (ko), OBS and its dissociation constant (Kd), and Na+,K+-ATPase activity in control subjects and hyperthyroid patients" --- p.58 / Chapter 2.4.8 --- SPC and its Rate Constant --- p.58 / Chapter 2.4.9 --- SLC and its rate constant --- p.63 / Chapter 2.4.10 --- Na+ 'leak' and its rate constant --- p.63 / Chapter 2.4.11.1 --- The Spearman Rank Coefficient of Correlation Matrix for the Characteristics of Sodium Transport Before Treatment --- p.63 / Chapter 2.4.11.2 --- Partial Coefficient of Correlation Between the Rate Constant for SLC and OBS and Naic --- p.69 / Chapter 2.4.11.3 --- Partial Coefficients of Correlation Between the Rate Constants for SLC and SPC with Naic Held Constant --- p.71 / Chapter 2.4.11.4 --- "Partial Coefficients of Correlation (r123) Between Na+ 'leak' and OBS, Na+,K+-ATPase activity and efflux rate constant with Naic Held Constant" --- p.71 / Chapter 2.4.12 --- Spearman Rank Coefficient of Correlation for Plasma Thyroid Function Tests with Sodium Transport Variables --- p.71 / Chapter 2.4.13 --- Effect of Treatment on 11 of the 18 Subjects --- p.74 / Chapter 2.4.13.1 --- "Effect of Treatment on Body Weight, Systolic and Diastolic BP's" --- p.74 / Chapter 2.4.13.2 --- "Plasma Concentrations of Sodium, Potassium, Urea, Creatinine and Ratio of Urea to Creatinine After Treatment" --- p.74 / Chapter 2.4.13.3 --- Hb Concentrations and Other Blood Indices Before and After Treatment --- p.74 / Chapter 2.4.13.4 --- Plasma Concentrations of T4 and Free T3 in patients before and after treatment --- p.78 / Chapter 2.4.13.5 --- The Characteristics of the Sodium Pump --- p.78 / Chapter 2.4.13.6 --- Passive Fluxes of Sodium After Treatment --- p.78 / Chapter 2.4.14 --- "Longitudinal Assessment of Plasma Thyroid Function Tests, Naic, Kic, OBS, Na+,K + -ATPase Activity and Sodium Fluxes in Patients Undergoing Treatment" --- p.87 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter CHAPTER 3 --- "THE EFFECT OF HYPERTHYROIDISM ON IN VIVO AGING OF ERYTHROCYTE OUABAIN BINDING SITES, INTRACELLULAR SODIUM AND POTASSIUM CONCENTRATIONS 105" / Chapter 3.1 --- Review of Literature --- p.106 / Chapter 3.1.1 --- Physical Changes and Methods of Separation --- p.106 / Chapter 3.1.2 --- Biochemical Changes --- p.108 / Chapter 3.1.2.1 --- "Naic,Kic and the Transport of Sodium" --- p.108 / Chapter 3.1.2.2 --- Changes in Other Enzymes/Proteins --- p.111 / Chapter 3.2 --- MATERIALS & METHODS --- p.116 / Chapter 3.2.1 --- Materials --- p.116 / Chapter 3.2.2 --- Subjects --- p.116 / Chapter 3.2.3 --- Plasma Thyroid Hormone Analyses --- p.116 / Chapter 3.2.4 --- Separation of Erythrocytes According to Age --- p.117 / Chapter 3.2.5 --- Determination of MCV & MCHC --- p.118 / Chapter 3.2.6 --- Erythrocyte Creatine Concentration --- p.118 / Chapter 3.2.7 --- Determination of Naic and Kic --- p.119 / Chapter 3.2.8 --- Maximum Number of OBS --- p.119 / Chapter 3.3 --- STATISTICS --- p.119 / Chapter 3.4 --- RESULTS --- p.120 / Chapter 3.4.1 --- "Establishment of the Method for the Separation of Young, Middle and Old Cells" --- p.120 / Chapter 3.4.2 --- Descriptive Statistics of the Subjects in the Study --- p.122 / Chapter 3.4.4 --- Effect of Erythrocyte Age on Markers of Cell Age --- p.124 / Chapter 3.4.4 --- "Effect of Erythrocyte Age on Naic, Kic and OBS" --- p.127 / Chapter 3.5 --- DISCUSSION --- p.133 / Chapter CHAPTER 4 --- THYROID HORMONES AND OUABAIN BINDING SITES OF RETICULOCYTES --- p.140 / Chapter 4.1.1 --- Review of the Literature --- p.141 / Chapter 4.1.2 --- Intracellular Organelles --- p.143 / Chapter 4.1.2.1 --- Ribosomes & RNA --- p.143 / Chapter 4.1.2.2 --- Mitochondria --- p.143 / Chapter 4.1.2.3 --- Other Organelles --- p.144 / Chapter 4.1.3 --- Changes in the Sodium Pump --- p.145 / Chapter 4.1.4 --- Changes in Other Membrane Proteins --- p.146 / Chapter 4.1.5 --- Aim of the Study --- p.147 / Chapter 4.2 --- METHODS --- p.148 / Chapter 4.2.1 --- Animals --- p.148 / Chapter 4.2.2 --- Induction of Reticulocytosis --- p.148 / Chapter 4.2.3 --- Identification of Reticulocytes --- p.148 / Chapter 4.2.4 --- Separation of Reticulocytes from erythrocytes --- p.149 / Chapter 4.2.5 --- Treatment of guinea pigs --- p.150 / Chapter 4.2.6 --- Oxygen Consumption --- p.150 / Chapter 4.2.7 --- Determination of Number of OBS --- p.154 / Chapter 4.2.8 --- STATISTICS --- p.154 / Chapter 4.3 --- RESULTS --- p.155 / Chapter 4.3.1 --- Establishment of the Method for Determinating the Number of OBS --- p.155 / Chapter 4.3.2 --- Scatchard Analysis of Binding of Ouabain to Erythrocytes --- p.155 / Chapter 4.3.3 --- Induction of Reticulocytosis and Treatment of animals --- p.160 / Chapter 4.3.4 --- Weight Loss and O2 Consumption in Control and T3 Treated Guinea Pigs --- p.160 / Chapter 4.3.5 --- Determination of OBS and Kd of Reticulocytes from Guinea Pigs Treated with T3 and Control --- p.164 / Chapter 4.4 --- DISCUSSION --- p.168 / Chapter CHAPTER 5 --- "THYROID HORMONES AND NA+,K+- ATP ASE ACTIVITY OF ERYTHROID CELLS" --- p.171 / Chapter 5.1 --- Review of the Literature --- p.172 / Chapter 5.1.1 --- Erythroid Differentiation and Maturation --- p.172 / Chapter 5.1.2 --- Methods of Separation of Erythroid Cells --- p.178 / Chapter 5.1.3 --- Biochemical Changes During Erythropoiesis --- p.179 / Chapter 5.1.4 --- In vitro Models of Differentiation and the Biochemical Changes During Haemoglobin Synthesis --- p.182 / Chapter 5.1.5 --- Aim of the Study --- p.185 / Chapter 5.2 --- MATERIALS & METHODS --- p.186 / Chapter 5.2.1 --- Materials --- p.186 / Chapter 5.2.2 --- Sterilisation of glassware --- p.186 / Chapter 5.2.3 --- Solutions --- p.186 / Chapter 5.2.4 --- Heat Inactivation of Foetal Bovine Serum --- p.187 / Chapter 5.2.4.1 --- Preparation of Cell Culture Medium (CCM) --- p.187 / Chapter 5.2.4.2 --- Sterility Test of CCM --- p.188 / Chapter 5.2.5 --- Growth of K562 cells --- p.188 / Chapter 5.2.5.1 --- Assessment of Cell Viability --- p.189 / Chapter 5.2.5.2 --- Subculture of Cells --- p.190 / Chapter 5.2.6 --- "Effect of T3 on Na+,K+-ATPase activity of K562 cell line" --- p.190 / Chapter 5.2.7 --- Induction of Haemoglobin Synthesis --- p.191 / Chapter 5.2.8 --- Benzidine Staining for Haemoglobin --- p.192 / Chapter 5.2.9 --- "The Determination of Na+,K+-ATPase activity" --- p.192 / Chapter 5.2.10 --- Determination of Protein --- p.194 / Chapter 5.3 --- RESULTS --- p.195 / Chapter 5.3.1 --- "Effect of Deoxycholate concentration on unmasking Na+,K+-ATPase activity" --- p.195 / Chapter 5.3.2 --- "Effect of varying saponin concentration on Na+, K+-ATPase activity" --- p.195 / Chapter 5.3.3 --- "Effect of Varying Incubation Time on Na+,K+-ATPase activity" --- p.198 / Chapter 5.3.4 --- "Effect of Varying the Amount of Protein on Na+,K+-ATPase activity" --- p.198 / Chapter 5.3.5 --- "Effect of varying ouabain concentration on Na+,K+-ATPase activity" --- p.201 / Chapter 5.3.6 --- Intra- and Inter-Assay Precision --- p.201 / Chapter 5.3.7 --- "Effect of T3 on the Na+,K+-ATPase Activity of K562 Cells" --- p.201 / Chapter 5.3.8 --- "T3 Concentration-Response Relationship for Na+,K+-ATPase Activity of K562 Cells" --- p.204 / Chapter 5.3.9 --- "Time course of Effect of T3 on Na+,K+-ATPase Activity of K562 cell line" --- p.207 / Chapter 5.3.10 --- Induction of Haemoglobin Synthesis in K562 cells --- p.210 / Chapter 5.3.11 --- Effect of Differentiation on T3 Stimulated K562 Cells --- p.210 / Chapter 5.4 --- DISCUSSION --- p.212 / Chapter CHAPTER 6 --- THYROID HORMONES AND THE ATP-DEPENDANT PROTEOLYTIC SYSTEM --- p.217 / Chapter 6.1 --- The ATP-Dependant Proteolytic System --- p.218 / Chapter 6.1.1 --- The Ubiquitin Pathway --- p.219 / Chapter 6.1.2 --- The Non-Ubiquitin Pathway --- p.222 / Chapter 6.1.3 --- ATP-dependant Proteolysis and Erythropoiesis --- p.223 / Chapter 6.1.4 --- Methods Used for Determining Proteolysis --- p.226 / Chapter 6.1.5 --- Aim of the Study --- p.227 / Chapter 6.2 --- MATERIALS & METHODS --- p.228 / Chapter 6.2.1 --- Materials --- p.228 / Chapter 6.2.2 --- Induction of Reticulocytosis and T3 Treatment --- p.228 / Chapter 6.2.3 --- Culture of K562 Cells and Effect of T3 --- p.229 / Chapter 6.2.4 --- Preparation of K562 Cell Lysate --- p.229 / Chapter 6.2.5 --- Preparation of Reticulocyte Extracts --- p.229 / Chapter 6.2.6 --- Iodination of Lysozyme --- p.230 / Chapter 6.2.7 --- Determination of Proteolytic Activity --- p.231 / Chapter 6.3 --- RESULTS --- p.234 / Chapter 6.3.1 --- Proteolytic Activity of Reticulocyte and K562 Cell Lysates --- p.234 / Chapter 6.3.2 --- Induction and Separation of Reticulocytes --- p.234 / Chapter 6.3.3 --- Effect of T3 Treatment on Guinea Pigs --- p.234 / Chapter 6.3.4 --- ATP-dependant proteolytic system of Reticulocytes and K562 Cells Treated with T3 and Controls --- p.239 / Chapter 6.4 --- DISCUSSION --- p.241 / Chapter CHAPTER 7 --- OVERVIEW & FUTURE WORK --- p.244 / Chapter 7.1 --- Overview & Future Work --- p.245 / REFERENCES --- p.249

Typescript (photocopy). / Digitized by Kansas Correctional Industries

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Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 196-221).

Thesis (M. Phil.)--University of Hong Kong, 1975. / Photocopy of typewcript.

Thesis (Ph. D.)--University of Hong Kong, 1984.

Thesis (M. Phil.)--University of Hong Kong, 1978. / Xeorx copy of typescript.

Introduction With the emergence of the H5N1 virus in humans that was of entirely avian origin, a better understanding of potential receptors of the influenza A virus is needed. It is widely accepted that terminally sialylated glycoconjugates on the surface of the red blood cells are receptors to which the influenza A virus binds to and causes the agglutination of red blood cells. By isolating glycans found on red blood cells, perhaps it is possible to find potential receptors that influenza A virus has preferential binding to. There has been a general shift of using turkey red blood cells rather than chicken red blood cells for the haemagglutination of viruses over the past few decades. The influenza virus’s loss in ability to agglutinate chicken red blood cells but keep the ability to agglutinate turkey red blood cells a puzzling mystery. By comparing the differences, perhaps it is possible to elucidate structures influenza viruses prefer to bind to. Methods Mass spectrometricanalysis of purified glycans will allow us to narrow down the structures of the most abundant glycans found on the erythrocyte surface. Lectin staining with flow cytometry is used to identify the receptor specificity of the influenza viruses. Haemagglutination assay in conjunction with glycan binding array data from the CFG will allow us to pinpoint the possible structures that give the viruses the ability to bind using treatments with sialidases. Results The mass spectrometric data was good and the basic glycan structures were elucidated according to their mass to charge ratio. The proportion of the different glycans for each of the erythrocyte type was clearly shown. The lectin staining gave more accurate results have selecting a single cell population and it was clear that turkey red blood cells had more α2,3 and α2,6 linked glycans than the chicken red blood cells. The haemagglutination assay aided the identification of differentiating theα2,3 linked and α2,6 linked liking viruses. The glycan array binding data was obtained but some results were absent. Conclusion It is certain that turkey red blood cells are better than chicken red blood cells for use in the haemagglutination assays as they present some glycans that are present in human bronchial epithelials. The most abundant sialylated glycan are the α2,6 linked sialylated glycans. The most abundant glycans on chicken red blood cells are either absent or in low abundance on the HBEs. Though the turkey red blood cells have some glycans that are present on HBEs, the glycan profile is very different. To agglutinate red blood cells, viruses are likely to bind to the bisecting glycans and shorter antennae glycans. In methodology: for lectin staining experiments, one must take caution in the lectin used as it may affect results. Sialidase S treated RBCs are a good method to distinguish α2,3 linked glycans liking viruses. / published_or_final_version / Pathology / Master / Master of Medical Sciences