Doctoral Degrees (Biochemistry)
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Browsing Doctoral Degrees (Biochemistry) by Subject "Adrenocortical hormones"
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- ItemComparison of two CYP17 isoforms : implications for cortisol production in the South African Merino(Stellenbosch : Stellenbosch University, 2012-03) Hough, Denise; Swart, Pieter; Cloete, Schalk W. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: • the comparison of the enzymatic activities of the two ovine cytochrome P450 17 - hydroxylase/17,20-lyase (CYP17) isoforms expressed in non-steroidogenic COS-1 cells. The Km and Vmax values for the metabolism of pregnenolone and progesterone were determined, while time-dependent metabolism of pregnenolone, 17-hydroxypregenolone, progesterone and 17-hydroxyprogesterone was also reported. The cloning and sequencing of ovine cytochrome b5 is reported and was co-expressed with CYP17. The results showed that the wild type 1 (WT1) isoform of ovine CYP17 produce more cortisol precursors than the wild type 2 (WT2) isoform; • the analysis of the frequency distribution of the CYP17 genotypes within a South African Merino population, which were divergently selected for (H-line) or against (L-line) the ability of a ewe to rear multiple offspring per birthing opportunity. It was observed that the CYP17 frequency distribution was the same within the H- and L-line, with 78.3 % heterozygous WT1/WT2 and 21.7 % homozygous WT1/WT1. No homozygous WT2/WT2 individuals were identified; • the development of a UPLC-MS/MS method for the separation and quantification of all thirteen adrenal steroids that are produced in the adrenal gland; • the relative contribution of the CYP17 genotypes in the total steroidogenic output in adult adrenocortical cells from the adrenal glands of H- and L-line sheep, with particular emphasis on cortisol production. The adrenocortical cells from the H-line sheep showed a marked higher cortisol production than the L-line, while adrenocortical cells from homozygous WT1/WT1 sheep also produced more cortisol than heterozygous WT1/WT2 sheep; • the blood cortisol responses upon the stimulation of the HPA axis by insulin induced hypoglycaemia of the H- and L-line sheep with known CYP17 genotypes. It was observed that the CYP17 genotype and selection line are important factors affecting the cortisol responses of sheep, where L-line heterozygous WT1/WT2 sheep showed the lowest cortisol response and glucose recovery; • the association of the CYP17 genotype with behavioural responses of H- and L-line sheep to flock isolation stress, as well as the association of the CYP17 genotype with ewe reproduction and lamb output. While reproduction seemed to be unaffected by the CYP17 genotype, the behavioural stress responses of sheep to flock isolation correlated with the CYP17 genotype, where the heterozygous WT1/WT2 genotype was associated with a wilder nature.
- ItemThe influence of 3βHSD on adrenal steroidogenesis and the factors which influence its activity(Stellenbosch : Stellenbosch University, 2012-12) Goosen, Pierre; Swart, Pieter; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: This study describes: - the characterization and comparison of the enzymatic activity of both Angora and ovine 3βHSD expressed in non-steroidogenic COS-1 cells. The apparent Km and Vmax values for the metabolism of PREG, 17-OHPREG and DHEA were determined; - the characterization of steroid metabolites produced by COS-1 cells coexpressing either Angora or ovine 3βHSD together with Angora CYP17, in the presence and absence of overexpressed Cyt-b5, following the metabolism of PREG and 17-OHPREG. 3βHSD was identified as an additional factor in causing hypocortisolism in the South African Angora goat; - the influence of Cyt-b5 on the enzymatic activity of both Angora and ovine 3βHSD coexpressed in non-steroidogenic COS-1 cells; - the influence of purified ovine live Cyt-b5 and anti-Cyt-b5 IgG on adrenal microsomal 3βHSD activity. Cyt-b5 was shown to specifically augment 3βHSD activity which represents the first documentation of such augmentation in any species; - the overexpression and purification of Angora 3βHSD using a baculovirus expression system coupled with a detergent based enzyme purification method; - the characterization of both substrate and co-factor kinetics for the individual dehydrogenase and isomerase activities of purified 3βHSD, in the presence and absence of purified ovine liver Cyt-b5. Cyt-b5 was shown to increase the affinity of 3βHSD towards NAD+ during the dehydrogenase reaction whilst having no significant influence on the isomerase reaction. This represents the first documentation of Cyt-b5 influencing co-factor binding in any member of the -ydroxysteroid dehydrogenases; - the FRET analysis of COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP fusion proteins, suggesting an allosteric interaction between 3βHSD and Cyt-b5.
- ItemThe influence of dual CYP17 expression on adrenal steroidogenesis in the South African Angora Goat(Stellenbosch : Stellenbosch University, 2008-12) Storbeck, Karl-Heinz; Swart, P.; Swart, Amanda C.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.This study describes: • the cloning and sequencing of cytochrome P450 17 -hydroxylase/17,20 lyase (CYP17), 3 -hydroxysteroid dehydrogenase (3 HSD) and cytochrome b5 from the South African Angora goat; • the identification of two CYP17 genes encoding two unique CYP17 isoforms in the Angora goat; • the development of a UPLC-APCI-LC method for the separation and quantification of seven adrenal steroids; • the characterisation of the enzymatic activity of the two Angora CYP17 isoforms expressed in non-steroidogenic COS-1 cells. The Km and Vvalues for the metabolism of pregnenolone and progesterone were determined; • the development of a rapid and accurate real-time PCR genotyping test for CYP17 in Angora goats. Three unique genotypes were identified; • the determination of blood cortisol levels upon the stimulation of the HPAaxis by intravenous insulin injection in the three Angora goat genotypes.