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Arginine synthesis is a complex, age-dependant process involving multiple precursors and enzymes, and the interorgan transfer of substrates. The objectives of this thesis were to elucidate arginine synthesis from enteral precursors in newborn infants and in healthy adults using stable isotope methodology. In the first, of four studies, I validated the use of a non-invasive methodology using urinary amino acids for stable isotope studies of arginine synthesis and demonstrated a known, but under-recognised, problem with D-amino acid contamination of tracers. Implementing chiral chromatography, this issue was further investigated using samples from previous studies in our laboratory with several different isotopes. I demonstrated the novel finding that the impact of D-amino acids is dependent on the tracer used, and also on the age of the subject. In the second study, I used a multi-tracer design to assess arginine synthesis from enteral proline or glutamate in healthy preterm infants. Labeled arginine (M+2), proline (M+1) and glutamate (M+3) were given enterally to fifteen stable, growing preterm infants (gestational age at birth 30-35 weeks) at 1-3 weeks’ postnatal age. I found only arginine synthesis from proline, with no synthesis from glutamate. I conclude that enteral proline is the major contributor to arginine synthesis in vivo in human preterm infants. In the third study, I measured arginine synthesis from enteral proline in adults. I have demonstrated that enteral proline contributes significantly, ~25%, to newly synthesised arginine. In the fourth study, I used two glutamine tracers, 1-13C and 2-15N, to determine if glutamine is a carbon or nitrogen donor for arginine. I showed that enteral glutamine contributes ~50% of the carbon skeleton for arginine and that the 2-15N tracer significantly overestimates arginine synthesis, with the labeled N being transferred through transamination from pyrroline-5-carboxylate (to ornithine, rather than directly from glutamate to pyrroline-5-carboxylate. In conclusion, proline is the sole precursor for arginine in human neonates and combines with glutamaine as the dietary precusor in adults. As a precussor for arginine, though, glutamine’s main role is in provision of nitrogen independent of the carbon skeleton.

It has been suggested that membrane characteristics associated with carcinomas could be related to an altered molecular structure of lipids in the plasma membrane. The microsomal fraction, mitochondria and soluble fractions of the cell are major sites of de novo synthesis and elongation of fatty acids. It was the purpose of this study to compare the utilization of AcSCoA in the biosynthetic pathway for saturated fatty acids in tumor and normal tissue, and discern if any deviations in the initial steps of the pathway were responsible forr the observed differences in the plasma membrane of tumors. Labeled 14C-AcSCoA was incorporated into saturated fatty acids in both adenocarcinomas and normal mammary tissue. The distribution and degree of incorporation of labeled 14C-AcSCoA was hoped to demonstrate any deviations in the pathway.Crude supernate, microsomal fraction, mitochondria and soluble fractions were isolated from mammary adenocarcinomas and from normal mammary tissue of Strain A female mice by differential centrifugation. The activity of fatty acid synthetase in the soluble fraction was determined. The crude supernate, the soluble fraction, the mitochondria + soluble fraction and the microsomal fraction + soluble fraction of both the adenocarcinoma and normal mammary tissue were incubated with labeled 14C-AcSCoA, Ma1SCoA and all necessary cofactors. The now labeled fatty acids were extracted from these incubation mixtures. The total percent of incorporated labeled 14C-AcSCoA in each fraction was determined. The percent of incorporation of label into individual saturated fatty acids in each fraction was determined by gas liquid chromatography and liquid scintillation counting. Carrier mixtures of known fatty acids were added to the samples used for GLC analysis to confirm the identity of the labeled fatty acids.Results of this study show that the percent of labeled i4C-AcSCoA incorporated into the various saturated fatty acids was similar in tumor and normal tissue. However, the total uptake of AcSCoA was nearly twice as great in normal tissue. The activity of fatty acid synthetase appearsto be characteristic of each individual mouse. In tumored mice fatty acid synthetase activity appears to be related to tumor weight, that is the larger the tumor the greater the activity. These results do not demonstrate support for a shift in the biosynthesis of saturated fatty acids in carcinomas. It may be that the altered lipid composition of the plasma membrane of tumor cells arises from the carcinoma's ability to utilize exogenous fatty acids.

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The novel substituted pyridylboronic acids 2-bromo-5-pyridylboronic acid 92, 3-hromo-5- pyridylboronic acid 97, 2-chloro-5-pyridylboronic acid 103, 2-fluoro-5- pyridylboronic acid 108, 2-methoxy-5-pyridylboronic acid 123, 2-ethoxy-5- pyridylboronic acid 125, 2-medioxy-3-pyridylboronic acid 131, 3-bromo-6-methoxy-4- pyridylboronic acid 134, 3-chloro-6-methoxy-4-pyridylboronic acid 137 and 3-hromo-6- ethoxy-4-pyridylboronic acid 138 have been synthesised and shown to undergo palladium-catalysed Suzuki cross-coupling reactions with a vast variety of heteroaryl bromides to yield novel heteroarylpyridine derivatives.5-Formylfnran-2-boronic acid 170 has been synthesised and isolated and has been shown to undergo palladium-catalysed Suzuki cross-coupling reactions with a variety of heteroaryl bromides to yield novel heteroaryl substituted furyl derivatives. These derivatives have been shown to undergo efficient functionalisation by Wittig chemistry.

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BACKGROUND: The protein tyrosine phosphatase non receptor type 22 (PTPN22) gene inhibits T-cell activation. A functional single nucleotide polymorphism (SNP) Arg620Trp (rs2476601), resulting from a C→T substitution at nucleotide position 1858, is a significant risk factor for rheumatoid arthritis (RA) in European populations. The variant allele results in a gain of function that alters the threshold for T-cell signalling and abnormal T regulatory cell function. AIM: To investigate the role of the PTPN22 R620W polymorphism in disease susceptibility and severity in Black South Africans (BSA) with RA. METHODS: A cohort of 187 BSA patients with RA and 93 ethnically matched Black and 60 White controls with no history of RA or other autoimmune diseases were studied. Genotyping was performed by the polymerase chain reaction and pyrosequencing. RESULTS: The rs2476601 SNP was nonpolymorphic in both Black patients and Black control subjects with total absence of the variant ‘T’ allele. In White control subjects the frequency of the ‘T’ allele was 0.092, with T/T, C/T and C/C genotype frequencies of 0.00, 0.183, and 0.817, respectively. CONCLUSION: This study shows that the rs2476601 SNP of the PTPN22 gene is nonpolymorphic in BSA and therefore not associated with RA but the minor ‘T’ allele frequency in White South Africans is similar to that in other European populations. However, since variations in the rest of the gene were not investigated, these results do not exclude other PTPN22 polymorphisms from playing a role in RA susceptibility in BSA.

Bismuth(III) has been used in the medicinal industry for many years, but its mechanism of action is not fully understood and there is very little information on thermodynamic and kinetic parameters for complex formation. Amino acids are the building blocks of life and so, by initially simply determining the complexing ability of various amino acids with bismuth, an indication of how bismuth could interact in the body can slowly be developed and could assist in the eventual development and design of more effective bismuth containing drugs.