Doctoral Degrees (Biochemistry)

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Browsing Doctoral Degrees (Biochemistry) by browse.metadata.advisor "Hapgood, J. P."

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    An investigation into the molecular mechanism of action of the progestins, medroxyprogesterone acetate and norethisterone acetate
    (Stellenbosch : Stellenbosch University, 2004-04) Koubovec, Dominique J. B. M.; Hapgood, J. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Although the progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) are widely used in reproductive therapy, the steroid receptors and their target genes involved in the actions of MPA and NET-A are not well understood. Surprisingly, it had not yet been investigated whether doses of MPA and NET-A used for contraception and HRT cause significant side effects through various target genes via the glucocorticoid receptor (GR). In this thesis results of in vitro studies showed that, MPA, like dexamethasone (dex) and prog, significantly repressed tumour necrosis factor (TN F)-stimulated IL-6 protein production, and IL-6 and IL-8 promoter reporter constructs at the transcriptional level in L929sA cells, via interference with nuclear factor KB (NFKB) and activator protein-1 (AP-1) transcription factors. Like dex and prog, MPA did not affect NFKB DNA-binding activity. Furthermore, unlike dex and prog, MPA did not inhibit mitogen-activated protein kinase (MAPK) activity. The antagonistic effects of the GR and progesterone receptor (PR) antagonist, RU486, as well as the MPAinduced nuclear translocation of the GR, strongly suggest that the actions of MPA in these cells are mediated at least in part via the GR. Although the mechanism was not investigated as extensively as for MPA, NET-A was shown to repress IL-8 promoter reporter activity very weakly relative to dex, MPA and prog in Hek293 cells stably transfected with the rat GR. Furthermore, NET-A, like MPA, dex and prog did not interfere with the DNA-binding activity of NFKB. Significant transactivation of a GRE-driven promoter reporter construct by MPA and dex in L929sA via endogenous GR and COS-1 cells via expressed rat GR, and by MPA, dex and prog in Hek293 cells via expressed rat GR was also observed. In contrast, NET-A, unlike MPA, dex and prog showed no transactivation in Hek293 cells. MPA, NET-A and prog were shown to compete with dex for binding to the endogenous human GR in human lung carcinoma A549 cells. Similarly, MPA and NET-A were shown to compete with dex for binding to expressed rat GR in COS-1 cells. MPA displayed a higher relative binding affinity than NET-A for the GR in both systems, and a higher relative binding affinity than prog in A549 cells. Equilibrium dissociation constants (Ki values) for MPA (Ki = 10.8 ± 1.1 nM), NET-A (Ki = 270 ± 1.3 nM) and prog (Ki = 215 ± 1.1 nM) towards the human GR in A549 cells were also established. Furthermore, dose-response curves showed that MPA displays significantly greater GC agonist potency and efficacy than NET-A and prog for both transactivation of a synthetic GRE-reporter construct and transrepression of a synthetic IL-8 reporter construct via expressed rat GR in Hek293 cells, as NET-A showed no transactivation and very weak partial agonist activity for transrepression. Based on these observations, MPA behaves as a GR agonist whereas NET-A is proposed to be a weak antagonist. These results show that MPA and NET-A are not alike and not the same as prog in their mechanism of action via the GR, which may have serious health implications in vivo. Such insights may provide women and their clinicians with more information to facilitate the selection of contraception or reproductive therapy regimes with fewer side effects.
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    Investigation of glucocorticoid and dissociated glucocorticoid activity in hepatoma cell lines with specific reference to regulation of the corticosteroid binding globulin (CBG) proximal promoter'
    (Stellenbosch : Stellenbosch University, 2003-12) Allie-Reid, Fatima; Louw, A.; Hapgood, J. P.; Swart, P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: This study investigated the effect of several hormones on the rat corticosteroid binding globulin proximal promotor and for the first time showed that modulation occurs at the promotor level and can be correlated with changes in corticosteroid binding globulin mRNA and protein levels. The effect of various physical and psychological stressors on rat liver corticosteroid binding globulin mRNA levels was also tested and it was shown that voluntary running had no effect on rat corticosteroid binding globulin levels but that involuntary swimming and immobilization decreased rat corticosteroid binding globulin mRNA levels. Glucocorticoid responsiveness of the corticosteroid binding globulin promoter was investigated further by using truncated contructs of the corticosteroid binding globulin proximal promoter. Glucocorticoid responsiveness was delineated to between -296 and -145bp from the transcription start site an area that contains putative binding sites for D-site binding protein, hepatic nuclear factor-3 and CAAT/enhancer binding protein suggesting that these transcription factors may be involved in glucocorticoid responsiveness of the corticosteroid binding globulin proximal promoter. The dissociative glucocorticoid activity of medroxyprogesterone acetate and Compound A, both putative dissociated glucocorticoids, was compared to standard glucocorticoids by examining transactivation of glucocorticoid response element-containing reporter constructs and transrepression of corticosteroid binding globulin gene expression in hepatic cell lines. Results showed that medroxyprogesterone acetate, but not Compound A, trans activates only in the presence, but not in the absence, of co-transfected glucocorticoid receptor. Medroxyprogesterone acetate down modulated dexamethasone transactivation while the modulatory effect of Compound A depends on the order of addition of Compound A. If added together Compound A has no effect on dexamethasone transactivation, however, if Compound A was added before dexamethasone, Compound A significantly decreased dexamethasone transactivation. Both medroxyprogesterone acetate and Compound A, like glucocorticoids, transrepressed the rat corticosteroid binding globulin proximal promoter. The potency of repression was similar but Compound A repressed with a higher efficacy than medroxyprogesterone acetate. We conclude that Compound A is a completely dissociated glucocorticoid in contrast to medroxyprogesterone acetate that displays only partial dissociation, which is dependent on glucocorticoid receptor levels.
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    Transcriptional regulation of the gonadotropin-releasing hormone receptor (GnRHR) gene by glucocorticoids
    (Stellenbosch : Stellenbosch University, 2007-03) Fernandes, S. M. (Sandra Maria); Hapgood, J. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/- 236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence (TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been previously shown to be involved in regulation of transcription of the mouse GnRHR (mGnRHR) promoter in some cell lines, the role of site 3 has not been previously investigated. This study investigated whether transcription of the mGnRHR gene is regulated by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein- DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1 site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of investigation during this study. Luciferase reporter plasmids containing 600 bp of the mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site 3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into LβT2 cells was carried out to determine the effect of these mutations on the basal expression of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2 cells. This suggests that the AP-1 site is involved in positively mediating the basal transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins involved and the effect of varying doses for varying times of GnRH, as well as the overexpression of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it could be the c-Jun protein that binds to the AP-1 site under basal conditions. Another area that was investigated was whether the mGnRHR promoter can be regulated by dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1 site, as shown by the complete loss of response when these sites were individually mutated, consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells.