The G protein-coupled receptor (GPCR) family is the largest group of homologous proteins in the human genome. GPCRs are of prime physiological and medical importance as the actions of a wide range of hormones and drugs are mediated by these receptors. The gonadotropin-releasing hormone (GnRH) receptor is a member of the GPCR family, and plays a central role in the reproductive system. GnRH analogues are used therapeutically in a number of human disorders. All GPCRs contain 7 largely α-helical transmembrane domains. An arginine residue located at the cytosolic boundary of the third transmembrane domain is conserved in all members of the rhodopsin-like subfamily of GPCRs, and is nearly always preceded by an acidic residue (DR motif). This arginine has been proposed to play a critical role in receptor activation. In this thesis, the effects of mutating these residues (Asp¹³⁸ and Arg¹³⁹ respectively, in the mouse GnRH receptor) to neutral amide residues, on coupling of the mouse GnRH receptor, were examined. In addition, the relationship of coupling to internalization in these mutant receptors was explored.

Bibliography: leaves 116-128.

Background: Collection of biologic fluid for laboratory analysis requires relatively large samples, often with additives, and transport in fragile tubes. The analytes or matrices may be unstable so testing needs to be carried out quickly. Collection of these biologic fluids and drying them on filter paper can lower the cost of transporting the sample to the laboratory, avoid instability of the matrix, and degradation of the analytes. Aim: The aim of this project was to develop an inexpensive, convenient, comprehensive and reproducible patient sample collection system which ensures integrity and ease of transport of small-scale samples at room temperature, as well as ensuring convenient long-term storage for subsequent analysis. Methods: Samples (blood, buffy coat, serum, plasma and urine) were collected into various tubes and spotted onto filter paper cards. Concentrations of total cholesterol, triglyceride, phospholipids, glucose, lactate, and protein were measured in the original sample and dried plasma spots (DPS) and the concentration of creatinine was measured in urine and dried urine spots (DUS). Determination of oxidation of lipids by measurement of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) on dried serum spots (DSS) was carried out. Determination of salicylate on serum and dried serum spots and cyanide on whole blood and dried blood spots was carried out. Values obtained from original samples and dried spots were compared. In addition, DNA extracted from a dried buffy coat spot (DBCS) from a familial hypercholesterolemia patient was analysed after spotting. Results: The total cholesterol, triglyceride, phospholipid, glucose, lactate and protein concentration values of 14 samples were compared in whole plasma and DPS stored at different temperatures. These were highly correlated after 1 week and 3 months of collection and storage. Plasma cholesterol, glucose and lactate concentration values for DPS as well as urinary creatinine for DUS at 1 week were not significantly different to that at both 3 and 7 months’ analyses (p>0.05). Plasma triglyceride and phospholipid concentrations were significantly different (p blood vs DBS respectively) for cyanide. Salicylate in DSS and cyanide in DBS were not significantly different to the original samples (paired t-test, p>0.05). Conclusion: Dried filter spots may be used to transport and store biologic fluid samples for analyses of a number of water-soluble and water-insoluble analytes. To protect lipids from being oxidised, the filter paper should be pre-treated with BHT.

Bibliography: leaves 228-252. / The Family Testudinidae have a diverse distribution and, although limited to tropical and subtropical latitudes, are present on all continents with the exception of Australia and Antartica. Their evolutionary history dates back to the late Paleogene at least, and periods of diversification and expansion appear to be closely associated with global climate change, particularly at the border of the Oligocen-Miocene transition.

Includes abstract. Includes bibliographical references (leaves 131-141).

Treatment of SR membranes with mild acid (pH 5.6) (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001) or incubation with millimolar concentrations of ethylene glycol bis (β-aminoethyl ether)-N ,N'-tetraacetic acid (EGTA) at neutral pH and 37°C (McIntosh, D. B. and Berman, M. C. (1978) J. Biol. Chem. 253, 5l40-5146) results in a progressive irreversible inhibition of calcium transport while (Ca²⁺, Mg²⁺)-ATPase activity is unimpaired. Possible conformational changes associated with this uncoupling were monitored by following alterations in kinetic mobility of sulphydryl (-SH) groups either by using 5, 5'-dithiobis- (2-nitrobenzoate) (DTNB) and stopped flow analysis or 1-¹⁴C-N-ethylmaleimide (NEM). Kinetic reactivity with DTNB revealed a total of 20 thiol groups/1.5 x 10⁵ g of SR protein (rontaining 1 mole of ATPase protein) in the presence of sodium dodecyl sulphate, which constitute four kinetic classes. In native control vesicles 4.5 thiol groups were unreactive, 0.4 represented the fast reacting class, 0.8 the moderately fast reacting class and 14.4 the slowly reacting class, displaying pseudo-first order rate constants, k, of 159.0-, 22.0- and 6.23 x 10⁻² sec⁻¹, respectively. Inactivation of calcium transport to the extent of 90%, using mild acid conditions, increased the number of fast and moderately fast reacting groups, each by 1.0 - 1.5 sulphydryl groups / mol ATPase. The number of slowly reacting groups decreased by approximately 3 .0 thiol groups/mol ATPase. The kinetics of the reaction with 1-¹⁴C-NEM was essentially similar to that with DTNB. EGTA inactivation of calcium transport, to the extent of 90% and subsequent 1-¹⁴C-NEM modification, resulted in an increase in the number of fast reacting thiol groups by 0.5-1.0 thiol groups/mol ATPase. The total number of reactive thiol groups decreased by 1.0 -2.0 thiol groups/ mol ATPase, probably due to autoxidation of the newly exposed sulphydryl group. Inactivation of transport carried out in the presence of N-ethylmaleimide to prevent autoxidation resulted in an increase of approximately one thiol group/mol ATPase. The rate constant for the increase in reactivity of this group was 1.45 min⁻¹. This thiol group was localized on the ATPase protein of molecular weight approximately 100 000 daltons. Trypsinization of the ATPase produced four fragments of molecular weights 55 000, 45 000, 30 000 and 20 000. More extensive cleavage resulted in a significant decrease in the 55 000 dalton fragment and increased amounts of the 30 000 and 20 000 dalton subfragments. There was increased labelling on all subfragments of EGTA-treated vesicles compared to control, untreated vesicles. However, the greatest relative increase in labelling appeared to be localized on the 55 000 dalton and 20 000 dalton subfragments. Peptide mapping of the purified ATPase revealed 24 ninhydrin-positive peptides. Five of these were labelled in control and EGTAtreated vesicles, four of which showed increased labelling in the latter preparation. Random labelling of the nonoverlapping fragments may be due to the enzyme being "trapped" in a number of intermediate conformations or due to heterogeneity within the ATPase populations. NEM modification of SR membranes did not affect the tryptic cleavage pattern or the mobilities of the tryptic subfragments. It did however, affect the extent of tryptic cleavage resulting in solubilization of NEM-labelled protein into the medium following centrifugation. This protein fraction was identified as consisting largely of the 55 000 dalton molecular weight species on sodium dodecyl sulphate gel electrophoresis. It is concluded that occupancy of high affinity K₀.₅(Ca²⁺)≈10⁻⁶M) calcium binding sites maintain the (Ca²⁺, Mg²⁺)- ATPase in a stable, coupled conformation. Displacement of this calcium induces a conformational change in the protein which results in the loss of the vectorial component of calcium transport.

Bibliography: p. 83-94. / The number of albatrosses that are killed on longlines in the Southern ocean is conservatively estimated to be 44 000 birds per annum. These numbers are biologically significant since albatrosses are a prime example of an extreme K-selected species. Ongoing long line fishing in the Southern ocean could lead to a decrease in the size of breeding colonies, and is a cause for major concern as it may impact the long-term survival of these birds. Quantifying genetic variation in threatened populations is a valuable application of molecular biology in conservation. In this study genetic variation was quantified using microsatellite analysis in order to investigate the effects of the longline fisheries on seabird populations. In addition, the feasibility of developing diagnostic markers for determining the provenance of birds forming part of the bycatch was also investigated. The inter-population genetic variance of three species of albatross from four distinct breeding colonies is described. Microsatellite markers were found to be highly variable and provided an assessment of the heterzygosity in the distinct populations, and a measure of the gene flow between these populations. Despite the extreme fidelity that adult albatrosses show to their breeding colonies, relatively low levels of genetic differentiation were observed between the colonies. This suggests that an integrated conservation management strategy could be undertaken successfully.

Background: Nucleoside reverse transcriptase inhibitors (NRTIs) interfere with mitochondrial DNA polymerase gamma causing significant toxic effects, including fatal lactic acidosis. Little is known about mitochondrial DNA (mtDNA) in human immunodeficiency virus (HIV) infected children who face a lifetime exposure to these agents. We performed a cross sectional observation of mtDNA levels in whole blood in a pediatric population to ascertain the relationship between mtDNA, NRTI regimens and parameters of HIV-infection severity. Methods: Whole blood mt:nDNA ratios were determined by real-time PCR in three groups: 27 presumed HIV-negative, 89 HIV-infected, NRTI-treated and 62 HIV-infected treatment-naive children. Multivariate analysis was used to identify variables independently associated with mtDNA depletion. Results: Mean mt:nDNA ratios were lower (P < 0.001) at 77% of control in the HIVinfected antiretroviral treatment (ART) Naïve group and 73% of control in the ART group, but not different between the two HIV-infected groups. Mt:nDNA ratios were negatively associated with age (P = 0.029), HIV status (P < 0.0001) and Log10 of the HIV-1 viral load (P = 0.035) and positively associated with CD4 % (p = 0.032). A 6 stavudine vs zidovudine based regimen was associated with lower but not significant levels of mtDNA (P = 0.1). Conclusions: Depletion of whole blood mtDNA in children is associated independently with HIV-infection and markers of HIV infection severity, and does not improve with either stavudine or zidovudine based ART despite virological control, suggesting that these agents also deplete mtDNA.

Includes bibliographical references. / Several studies have shown that patients with low TPMT activity are at risk for severe and potentially fatal haematopoietic toxicity when treated with conventional doses of thiopurine drugs. Genetic polymorphism in the TPMT gene is an important determinant of mercaptopurine toxicity. Patients with mutations in the TPMT gene have a less efficient methylation process, and are therefore, predisposed to severe myelosuppression.

Sixteen of the world's 42 species of land tortoises occur in Africa, 10 of which are endemic to southern Africa. South Africa itself, which occupies 0.8% of the earth's total land mass, has the highest tortoise biodiversity in the world, with 13 species. This is the first study to use molecular techniques to investigate the evolutionary history of this group, which displays an unusually high level of speciation on the continent. Four hundred and fifty base pairs of mtDNA cytochrome b sequence were obtained, using direct PCR-based sequencing, from 32 individual tortoise blood samples, comprising 13 different species from 6 genera. PAUP 3. 1.1, and MEGA were used to infer a phylogeny using Chrysemys scripta elegans (an Emydid) an outgroup. Both phenetic and cladistic methods generated similar results. With the exception of Malacochersus, both morphological and molecular work show largely congruent results. When intra-specific relationships, using the molecular results, were compared to the existing morphological data, Psammobates was the only genus with a consistent topology. Proposals for the re-evaluation of Homopus, Kinixys and Geochelone have been made. Suggestions, based on molecular results, include the distinction between Chersobius and Homopus (Hewitt 1937), incorporating Malacochersus tornieri into Kinixys, and the elevation of Geochelone pardalis pardalis and G.p. babcocki to species level. Sequencing a further nine individuals within Homopus areolatus showed a higher than expected sequence variation, suggesting a distinct population structure and possibly cryptic species.