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Low affinity Fcγ receptors (FcγR) mediate key immune effector mechanisms through the engagement of the Fc portion of immunoglobulin G (IgG). These receptors are involved in multiple biological processes, including clearance of antigen/antibody immune complexes, enhancement of antigen presentation, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, regulation of antibody production, and activation of inflammatory cells. FcγR phenotypic variability modulates these processes through altering receptor IgG subclass binding affinity (FcγRIIa-H131R and FcγRIIIa-F158V), subcellular localization (FcγRIIb-I232T), post-translational modification (FcγRIIIb-HNA1a/b/c), expression of an otherwise pseudogene (FcγRIIc), and receptor surface density (gene copy number variability and promoter haplotypes). Accumulating data suggest that FcγR-mediated effector functions play a significant role in HIV-1 protective immunity, which is substantiated by the association of FcγR phenotypic variants with HIV-1 disease outcome. This study set out to characterize FcγR functional variability in the South African population, and to investigate the potential role thereof in HIV-1 transmission and disease progression in South African Black individuals. Since the only known determinant of FcγRIIIa surface density – FCGR3A gene copy number – is rare, this study investigated novel genetic determinants of FcγRIIIa expression by flow cytometry and nucleotide sequencing. FcγRIIIa expression on peripheral blood mononuclear cells was characterized for 32 South African Caucasian individuals and 22 South African Black individuals (Chapter 3). Significant differences in the proportion of FcγRIIIa-positive monocytes and FcγRIIIa expression levels on natural killer (NK) cells were observed between the population groups. A novel four-variant FCGR3A intragenic haplotype that associated with increased surface expression of FcγRIIIa on NK cells was detectable in Caucasian individuals, but not Black individuals and may account for the observed population differences. Further exploration of genetic diversity at the low affinity FCGR gene locus was extended to include all currently known functional variants of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb using a commercial multiplex ligation-dependent probe amplification assay (Chapter 4). Thirty-two South African Caucasian individuals and 131 South African Black

by Ho, Chung Shun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves [304-328]). / Chapter CHAPTER 1 --- REVIEW ON SODIUM EXCRETION / Chapter L --- Sodium excretion --- p.1-1 / Chapter II. --- Cellular mechanism of sodium reabsorption --- p.1-3 / Chapter III. --- Sensors monitoring ECF volume --- p.1-6 / Chapter IV. --- Factors affecting natriuresis: / Glomerular filtration rate --- p.1-8 / Renal physical forces --- p.1-8 / Sympathetic nervous system --- p.1-10 / Renal dopamine --- p.1-12 / Renin-angiotensin system --- p.1-14 / Aldosterone --- p.1-16 / Renal prostaglandins --- p.1-17 / Renal kallikrein-kinin system --- p.1-18 / Natriuretic peptides --- p.1-19 / Endogenous sodium transport inhibitor --- p.1-21 / Vasopressin --- p.1-22 / Endothelins --- p.1-23 / Endothelin-derived relaxing factor --- p.1-25 / Other hormones --- p.1-25 / Chapter V. --- Conclusion --- p.1-27 / Chapter CHAPTER 2 --- MEASUREMENT OF ENDOGENOUS SODIUM TRANSPORT INHIBITORS / Chapter I. --- Literature review: --- p.2-1 / Pretreatment and purification procedures prior to ESTI measurement --- p.2-1 / Methods of measuring ESTI --- p.2-3 / "Inhibition of purified Na, K-ATPase activity" / Inhibition of sodium pump on intact cells or tissues / Biological effects of sodium pump inhibition / Immunoreactivity with anti-digoxin /anti-ouabain antibodies / Chapter II. --- Method of measurement of ESTI in this study: / Principles of methods --- p.2-11 / Materials and methods --- p.2-14 / Results --- p.2-24 / Discussion --- p.2-53 / Chapter CHAPTER 3 --- MEASUREMENT OF URINARY FREE DOPAMINE / Chapter I. --- Literature review / Properties of dopamine for measurement methods --- p.3-1 / Preservatives used in the urine collection --- p.3-3 / Sample pretreatment procedure before --- p.3-6 / measurement --- p.3-8 / Methods of measurement / Bioassays / Colorimetric method / Fluorometric methods / Radioimmunoassays / Radioenzymatic method / Chromatographic methods --- p.3-16 / Concluding remarks / Chapter II. --- Method of measurement in this study / Principle of the method --- p.3-17 / Materials and methods --- p.3-18 / Results --- p.3-23 / Discussion --- p.3-54 / Chapter CHAPTER 4 --- CROSS SECTIONAL STUDIES IN THE HUMAN / Chapter I. --- Introduction --- p.4-1 / Chapter II. --- Relationship of urinary sodium excretion and plasma ESTI in medical students / Materials and methods --- p.4-2 / Results --- p.4-3 / Discussion --- p.4-6 / Chapter III. --- Excretion of urinary electrolytes and natriuretic factors in young Chinese females / Materials and methods --- p.4-7 / Results --- p.4-8 / Discussion --- p.4-11 / Chapter IV. --- Urinary sodium / DA relationship in Chinese normotensives and hypertensives --- p.4-13 / Materials and methods / Results --- p.4-14 / Discussion --- p.4-17 / Chapter V. --- Urinary DA excretion and plasma ESTI in normotensive and hypertensive NIDDM patients / Materials and methods --- p.4-20 / Results --- p.4-23 / Discussion --- p.4-33 / Chapter CHAPTER 5 --- VOLUME EXPANSION STUDIES IN THE HUMAN / Chapter I --- Introduction --- p.5-1 / Chapter II. --- Volume expansion by headout water immersion / Materials and methods --- p.5-3 / Results --- p.5-5 / Discussion --- p.5-11 / Chapter III. --- Volume expansion by saline infusion / Materials and methods --- p.5-16 / Results --- p.5-19 / Discussion --- p.5-29 / Chapter IV. --- Oral salt loading with free diet / Materials and methods --- p.5-36 / Results --- p.5-38 / Discussion --- p.5-49 / Chapter V. --- Oral salt loading under controlled diet / Materials and methods --- p.5-53 / Results --- p.5-54 / Discussion --- p.5-64 / Chapter CHAPTER 6 --- STUDIES ON THE EFFECTS OF SALT LOADING IN THE RAT / Chapter I. --- Introduction --- p.6-1 / Chapter II. --- Temporal relationship between excretions of DA and ESTI during salt loading in the rat / Materials and methods --- p.6-2 / Results --- p.6-3 / Discussion --- p.6-6 / Chapter III. --- Roles of DA and ESTI in natriuresis in rats treated with carbidopa / Materials and methods --- p.6-8 / Results --- p.6-9 / Discussion --- p.6-14 / Chapter CHAPTER 7 --- CONCLUSION / Measurement of ESTI --- p.7-2 / Measurement of urinary free DA --- p.7-5 / Cross sectional studies in human --- p.7-7 / Volume expansion studies in human --- p.7-11 / Studies on the effects of salt loading in the rat --- p.7-16 / Summary --- p.7-18

Anita K.L. Yiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 93-112). / Acknowledgement --- p.i / List of Abbreviations --- p.ii / Abstract --- p.iv / Table of Contents --- p.vi / Chapter CHAPTER 1. --- Introduction / Chapter 1.1 --- The Renin-Angiotensin System (RAS) --- p.1 / Chapter 1.2 --- Physiology and Pathophysiology of Angiotensin --- p.3 / Chapter 1.3 --- Angiotensin Receptors / Chapter 1.3.1 --- Heterogeneity among Angiotensin Receptors --- p.10 / Chapter 1.3.2 --- Differential Distribution of Subtypes --- p.13 / Chapter 1.3.3 --- Molecular Structure of Subtypes --- p.15 / Chapter 1.3.4 --- Signal Transduction Mechanism --- p.20 / Chapter 1.3.5 --- Physiological Functional Correlates --- p.21 / Chapter 1.4 --- Aim of Study --- p.23 / Chapter CHAPTER 2. --- Preparation of Polyclonal Antibodies Against Angiotensin Receptors / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Preparation of antisera / Chapter 2.2.1.1 --- Preparation of peptide conjugates --- p.25 / Chapter 2.2.1.2 --- Protein determination --- p.27 / Chapter 2.2.1.3 --- Immunization of rabbits with peptide conjugates --- p.27 / Chapter 2.2.1.4 --- Collection of rabbit sera --- p.28 / Chapter 2.2.1.5 --- Affinity purification of antisera --- p.28 / Chapter 2.2.2 --- Enzyme-linked immunosorbent assay (ELISA) / Chapter 2.2.2.1 --- Titer determination --- p.29 / Chapter 2.2.2.2 --- Specificity determination --- p.30 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Preparation of antisera --- p.30 / Chapter 2.3.2 --- Affinity purification of antisera --- p.30 / Chapter 2.3.3 --- ELISA / Chapter 2.3.3.1 --- Titer determination --- p.31 / Chapter 2.3.3.1.1 --- Thy-AT1 antiserum --- p.31 / Chapter 2.3.3.1.2 --- Thy-AT2 antiserum --- p.32 / Chapter 2.3.3.2 --- Specificity determination --- p.32 / Chapter 2.3.3.2.1 --- Thy-AT1 antibodies --- p.32 / Chapter 2.3.3.2.2 --- Thy-AT2 antibodies --- p.49 / Chapter 2.4 --- Discussions --- p.49 / Chapter CHAPTER 3. --- Application of Thy-AT1 Antiserumin Western Blot / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Methods / Chapter 3.2.1 --- Preparation of protein samples --- p.52 / Chapter 3.2.2 --- Protein determination --- p.53 / Chapter 3.2.3 --- SDS-PAGE --- p.53 / Chapter 3.2.3 --- Western blot --- p.54 / Chapter 3.2.5 --- Immunoblotting --- p.54 / Chapter 3.3 --- Results --- p.55 / Chapter 3.4 --- Discussions --- p.58 / Chapter CHAPTER 4. --- Evaluation of Pancreatic Response to Angiotensin II / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods / Chapter 4.2.1 --- Perfusion of pancreas --- p.62 / Chapter 4.2.2 --- Assay of amylase activity --- p.64 / Chapter 4.2.3. --- Calculations --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.4 --- Discussions --- p.65 / Chapter CHAPTER 5. --- Application of Purified Thy-AT2 Antibodies in immunohistochemical studies / Chapter 5.1 --- Introduction --- p.74 / Chapter 5.2 --- Methods / Chapter 5.2.1 --- Preparation of adrenal sections --- p.75 / Chapter 5.2.2 --- Light-microscopic immunohistochemical study --- p.76 / Chapter 5.3 --- Results / Chapter 5.4 --- Discussions / Chapter CHAPTER 6. --- General Discussions --- p.84 / References --- p.93 / Appendix / Chapter A. --- Materials --- p.113 / Chapter B. --- Buffer Compositions --- p.121

Chronic Lymphocytic Leukaemia (CLL) remains the most abundant leukaemia in those aged over 65 years. It is characterised by the expansion of malignant monoclonal B-lymphocytes that were originally described as being functionally incompetent. Identified by immunophenotypic expression of monoclonal light chain restriction, it falls into the classification of chronic B-cell lymphoproliferative disorders (B-LPD). This thesis aims to demonstrate that CD160, an activating NK cell receptor, is aberrantly expressed in B-LPD and can function as a tumour specific antigen, which has clear translation roles within the clinical environment, aiding in the diagnosis of CLL and monitoring of minimal residual disease (MRD). More so, this study aims to provide an insight into the potential biological roles of CD160 within chronic B-cell malignancies. CD160 is an activating NK cell receptor whose major form is a glycosylphosphatidylinositol (GPI)-anchored cell surface molecule with a single immunoglobulin domain. In-vitro studies on a large cohort of B-LPD patients demonstrated that CD160 was primarily restricted to cases of CLL (98%) and Hairy Cell Leukaemia (HCL, 100%) with only a minor population of other B-LPDs expressing the antigen. More so, within the B-cell lineage, CD160 can be considered a tumour specific antigen (TSA) in that when looking for both transcript and protein, they were absent throughout the normal B-cell hierarchy. Many clinical studies base their entry criteria on clinical and biological prognostication, as this provides insights into the biology of CLL and its response to therapy. Disease eradication has been shown to be prognostic. This study demonstrates the feasibility and clinical importance of MRD detection utilising CD160 as novel marker of residual disease. Subsequently, CD160 analysis by flow cytometry (CD160FCA) demonstrated to be as sensitive and specific as other methodologies, and independent of the type of therapy. Further to this the early detection of MRD was correlated with known biological prognostic risk groups. Patients in CR had significantly different EFS based on their MRD status following treatment using the CD160FCA. For those patients with adverse prognostic markers (including CD38, ZAP-70 and M), the time to detection of MRD or relapsing disease ß2using CD160FCA, was significantly shorter than those with a normal or good prognosis. Within normal NK and T lymphocytes, CD160 has a multifunctional role that upon triggering results in a unique profile of cytokine production via the recruitment of Phosphatidylinositol 3-kinase (PI3K). In CLL cells, CD160 stimulation resulted in the recapitulation of these observations including cell survival, an increase in Bcl-2 family antiapoptotic proteins, and cell cycle progression. This thesis has demonstrated that CD160 is aberrantly expressed in malignant B-cells, it has a clear clinical translation role in terms of diagnosis and MRD monitoring, and multiple biological functions which recapitulate those observed in NK-cells.

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Chapter 1 of thesis is a literature review of adenosine research. The central importance of the contributions of both classical pharmacology and, more recently, molecular biology to adenosine research is demonstrated. These disciplines have enabled the classification and characterisation of adenosine receptors and as well an understanding of the physiological significance of endogenous adenosine. The significant benefits of developing therapeutics for regulation of the diverse physiological functions of adenosine, by regulation of adenosine receptors, is outlined. For this therapeutic potential to be realised both high affinity and subtype selective adenosine agonists and antagonists are required. The structure-activity relationships for agonists and xanthine antagonists are discussed. The assimilation of these structure-activity relationships have guided the development of ligand based models of the adenosine receptor pharmacophore. The 'flipped', 'N6-C8' and 'three binding domain' models were described. These models aim to direct the future design of high affinity and selective ligands for adenosine receptors. The development of receptor based models by modelling of the receptor-ligand complex is also presented. The main body of this thesis presents a study of the structure-activity relationships for pyrazolo(3,4-d) pyrimidines binding to adenosine Ai and A2a receptors. Prior to this study few non-xanthine adenosine antagonists had been well defined or optimised in terms of structure-activity relationships. However, the value of such ligands is immense, facilitating further definition of structural requirements for high affinity and selective adenosine receptor binding. These ligands should complement existing agonists and xanthine antagonists in developing an understanding of adenosine receptor binding. The experimental approach to development of the lead compound of this study, a-(6-(l'-carbamoylethylthio)- l-phenylpyrazolo(3,4-d)pyrimidin-4-ylthio)propanamide (5), is outlined in Chapter 2 of this thesis. 5 is substituted at C-4, C-6 and N-i of the pyrazolo(3,4-d)pyrimidine heterocycle. The experimental approach to optiniising 5 was approached in a rational manner, requiring an iterative approach i.e. design of generation I target compounds --synthesis -- biological evaluation -- structure-activity relationships -- design of generation II target compounds, etc. Chapters 3, 4 and 5 of this thesis describe this experimental approach as it relates to optimising the lead compound, 5, for adenosine receptor affinity and subtype selectivity. The importance of receptor interactions with multiple ligand domains, to achieve both potency and selectivity, was recognised so that optimisation of the C-4, C-6 and N-i substituents of the lead compound was targeted and achieved. Previous structure-activity studies with agonists and xanthine antagonists have concentrated on modifying a single ligand domain. Chapter 3 presents twelve generation I target compounds to examine C-4 and C-6 substituent structure-activity relationships. Chapter 4 presents twelve generation II target compounds to further examine C-4 and C-6 substituent structure-activity relationships. Chapter 5 presents sixteen generation ifi target compounds to examine N-I substituent structure-activity relationships. A major outcome from the research presented in these chapters was the development of highly potent and highly selective ligands for the adenosine A1 receptor subtype. a(4-Methylamino- I -phenylpyrazolo(3,4-d)pyrimidin-6-ylthio)hexanamide (29) was the most potent ligand at the Ai receptor identified in this study, and is one of the most potent Ai selective antagonists ever reported. 29 has an A1 K1 value of 0.745±0.045 nM and is 332-fold selective for the A1 receptor over the A2a receptor. a-(1-Phenyl-4-propylthiopyrazolo(3,4-d)pyrimidin-6-ylthio)butanainide (27) was the most selective ligand of this study. It is four orders of magnitude selective for the A1 receptor (up to 16900-fold), and one of the most selective antagonists ever reported. This high selectivity has been achieved with the maintenance of good A1 affinity (A1 K1 = 29.5±6.6 nM). These results prove the value of modifying multiple substituents of adenosine receptor ligands, generating ligands which bind with high potency and selectivity to adenosine Al receptors compared to adenosine A2a receptors.

Changes in the strength of connections between neurons underlie information storage in the mammalian brain. Long-term potentiation (LTP) is a long-lasting form of synaptic plasticity that has been described extensively in the hippocampus, a brain structure that plays a key role in memory formation. The molecular events that realise the persistence of LTP are not well understood, but it is known to require protein synthesis. The aim of this thesis was to investigate synaptic protein expression during the late-phase of hippocampal LTP in freely moving rats. LTP was induced in the perforant path input to the dentate gyrus of Sprague-Dawley rats using high-frequency electrical stimulation paradigms known to produce LTP that persists for many days. To focus on late-occurring events, and complement existing data sets, rats were sacrificed 48 h after LTP induction. A broad survey of synaptic proteins was conducted by two-dimensional gel electrophoresis using postsynaptic density (PSD) fractions. Over 200 protein spots were resolved, from which 163 individual proteins were identified by mass spectrometry. These included cytoskeletal proteins, signalling molecules, molecular chaperones and mitochondrial components, but no transmembrane receptor proteins were detected. Few of the identified proteins were found to undergo significant changes in expression, although proteomic comparison of PSDs prepared from LTP-stimulated and control dentate gyri showed reduced expression of the three component proteins of the mitochondrial voltage-dependent anion channel. While these data complement earlier studies suggesting that the late-phase of LTP is associated with mitochondrial restructuring, they cannot alone explain the persistence of LTP. To directly test whether LTP persistence is associated with increased expression of presynaptic marker proteins, suggesting increased neurotransmitter release; and/or increased postsynaptic receptor expression, implicating an increase in postsynaptic contact area, a targeted approach using Western Blot analysis of dentate gyrus subcellular fractions was undertaken.

Includes bibliographical references (leaves 308-327) / xxviii, 356 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2004?