Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 101-113. Also available online.

Neurosteroids are an important group of substances that affect communication between neurons. They act as allosteric modulators of membrane receptors for neurotransmitters. One of the most important systems influenced by neurosteroids are NMDA receptors; however, a binding site(s) for their inhibition by steroids have not been found yet. This work is focused on the synthesis of fluorescently labeled photoaffinity probe, which may help explain the structure and location of binding site(s) and simplify the development of new neuroprotectives. A structural analogue of the endogenous neurosteroid, (20S)-20-Azido-5β-pregnan- 3α-yl N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-L-glutamyl 1-ester (8), was prepared. The structure of compound 8 includes photolabile azido group, as well as covalently bounded fluorescent NBD group. In addition, a photoaffinity probe with a modified steroid skeleton - pyridinium 17aα-azido-17α-methyl-17a-homo-5β-androstan-3α-yl 3-sulfate (29) - was synthesized. The ability of compound 8 and 29 to inhibit activated NMDA receptor has been verified for recombinant NR1-1a/NR2B receptors expressed in HEK293 cells using a patch-clamp technique. Additionally, the IC50 values of compounds 8 and 29 have been calculated. (In Czech) Key words: neuroactive steroid, NMDA receptor, photoffinity...

A novel bacterial expression system that is capable of producing high levels of soluble, stable, biologically active human vitamin D3 and estrogen receptors has been developed. The method utilizes ubiquitin fusion technology and a low temperature nalidixic acid induction of the lambda PL promoter. This system can produce large quantities of receptor antigen, but only a small fraction displays wild-type DNA and hormone binding properties. Therefore, the use of this system to overproduce receptors for crystallization studies is not practical. To overcome these problems, a 2 um based ubiquitin fusion system which allows regulated expression of the estrogen receptor in yeast (Saccharomyces cerevisiae) was developed. This system produces the estrogen receptor to a level of 0.2% of the total soluble protein. Moreover, this protein is undegradable, soluble, and biologically active. To test the transcriptional activity of the estrogen receptor produced in yeast, a cis-trans transcription assay was developed. This assay revealed that the transcriptional activity of the human estrogen receptor expressed in yeast was similar to that observed in transfected mammalian cells. This reconstituted estrogen transcription unit in Saccharomyces cerevisiae was utilized to examine the regulation of estrogen receptor functions by antiestrogens, to develop a random and rapid approach for identifying novel estrogen response elements, to characterize estrogen receptor variants cloned from human breast tumors, and to examine the effect of estrogen receptor on the regulation of osteocalcin gene.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Call number: LD2668 .T4 1963 G42 / Master of Science

The metabolism of androgenic anabolic steroids has been studied in the racing greyhound. Various drug preparations have been investigated utilising different derivatisation techniques, coupled with gas chromatographic analysis, to enable the identification of key metabolites in canine post administration samples. This has led to an increased understanding of some of the generic routes of steroid metabolism that take place in the greyhound. This valuable information can help to support metabolism studies in the future. The identification of specific metabolites for each compound investigated, has provided a means for controlling the misuse of these compounds, and contributed valuable enhancements to screening protocols utilised in the canine sports drug testing industry. Utilisation of the techniques described, resulted in the identification of specific major metabolites of the anabolic steroid methyltestosterone, namely 17??-methyl-5??- androstan-3??-17??-diol and 17??-methyl-5??-androstan-3??,16??,17??-triol. 16??- hydroxylation was shown to be a major phase I metabolic pathway in the canine along with phase II conjugation with glucuronic acid. Similar results were obtained during the metabolism study of the progestatgenic steroid norethisterone. Several di- and trihydroxy metabolites were detected in the glucuronic acid fraction of the post administration urines from this study. The norethisterone metabolism study also provided some insight, into the area of trace contaminants of pharmaceutical preparations. Low levels of nandrolone metabolites were also detected in the norethisterone post administration urine samples, leading to the discovery that the administered pharmaceutical tablets contained small quantities of nandrolone and 19- norandrostenedione, albeit below FDA approved contaminant levels. Modern methods of drug screening employ such highly sensitive techniques, that they allow for the detection of metabolites of such trace contaminants, following administration of the drug preparation to the greyhound. It is therefore important to have a broad understanding of the metabolism of various drug preparations, both banned and permitted substances alike; as detection of a trace amount of a banned substance metabolite, arising from the administration of a permitted medication, whose iii metabolite profile is unknown, and therefore potentially not detected, could present an interesting case. In conjunction with research into controlling the use of banned substances for the purposes of suppressing oestrus in the greyhound bitch, an investigation into normal/reference levels of endogenous hormones has been carried out. The endogenous steroid levels in a population of 212 greyhound bitches have been studied with a view to establishing a method for the detection of the exogenous administration of the endogenous anabolic steroid testosterone. The major urinary metabolites investigated were epiandrosterone, 5??-androstane-3??,17??-diol and 5??-androstane-3??,17??-diol. Statistical evaluations have been carried out to support the implementation of a suitable threshold for the key testosterone metabolites, namely 5??-androstane-3??,17??-diol and epiandrosterone. The detection of 5??-androstane-3??,17??-diol was found to be a very good indicator of the exogenous administration of testosterone to the greyhound bitch, when compared with the reference population data for this metabolite. However, further statistical/analytical data evaluation was deemed necessary before an absolute threshold could be implemented for this analyte, for the purposes of controlling the misuse of testosterone in the racing greyhound bitch. To support the understanding of endogenous steroid levels in the female greyhound, yet further, the endogenous reproductive steroid profiles were measured throughout the entire oestrus cycle of a cohort of 33 racing bitches. The results of the study clearly indicate a surge in androgen metabolites during the first 7-10 days of the oestrus cycle, in particular epiandrosterone and 5??-androstane-3??,17??-diol. This unique set of data has provided detailed information regarding the fluctuating concentrations of androgen and progesterone metabolites (following ovulation), at key stages of the canine oestrus cycle. The information obtained from this research can be used to support regulatory decisions regarding the misuse of testosterone in the racing greyhound bitch.

The human endometrium is a complex multicellular tissue, located within the cavity of the uterus. Its luminal surface is defined by a layer of epithelial cells supported on a multicellular stroma containing fibroblasts, glands (lined by a secretory epithelium), blood vessels (lined with endothelial cells) and several populations of immune cells; the latter includes a unique population of natural killer (uNK) cells. The endometrium undergoes dynamic remodelling across the menstrual cycle in response to fluctuating levels of sex steroids secreted by ovarian cells. The phases of the endometrial cycle include an oestrogendominated proliferative phase, a progesterone-dominated secretory phase and menses (endometrial shedding precipitated by falling levels of progesterone). A key feature of the secretory phase is differentiation (decidualisation) of endometrial stromal fibroblasts (ESC) an event characterised by transformation of cell shape, secretion of growth factors/cytokines, angiogenesis/vascular remodelling and an increase in the numbers of resident immune cells. Decidualisation ensures an appropriate nutritional and hormonal environment exists during the establishment of pregnancy. Studies in mice suggest that de novo biosynthesis of oestrogen within the uterus may play an essential role in regulation of decidualisation but no data exist for human. Endometrial endothelial and uNK cells both contain oestrogen receptors but the impact of oestrogens on their function has not been explored. In the current studies three questions have been addressed: 1. Is oestrogen biosynthesis a feature of human endometrial stromal cell decidualisation? 2. What is the impact of oestrogen on uNK cell function? 3. What role (if any) does oestrogen play in the interplay between decidual, immune and vascular cells within the human endometrial stroma? Results obtained provide the first evidence that de novo biosynthesis of oestrogens occurs during decidualisation of human ESC. This was attributed to changes in expression patterns of mRNAs encoding proteins that play a critical role in regulation of oestrogen biosynthesis (STAR, CYP11A1, CYP19A1 [aromatase], HSD17B2 [17βHSD2] and STS [steroid sulphatase]). Changes in the pattern of metabolism were confirmed using thin layer chromatography and analysis of concentrations of oestrone (E1) and oestradiol (E2) in culture media. Secretion of E1 and E2 was reduced by addition of an aromatase inhibitor. Data derived from studies described within this thesis also show for the first time that incubation of uNK cells with E2 not only enhanced cell migration but also stimulated secretion of factors that had a significant impact on endothelial cell angiogenesis. These findings were supported by novel evidence that E2 had a significant impact on expression of genes associated with cell motility and angiogenesis. In addition, factors, including E1/E2, secreted by decidualised stromal cells, stimulated chemotaxis of uNK cells. Future experiments will focus on determining the identity of the angiogenic factors secreted by uNK cells in response to E2 and the mechanisms responsible for uNK cell movement. In summary, new data presented in this thesis provide evidence that local biosynthesis of oestrogens within the endometrial stroma may play a previously unrecognised role in regulating the function of uNK cells and endometrial endothelial cells in women. These results have implications for treatment of disorders such as infertility, heavy menstrual bleeding and endometriosis.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2004. / This study describes: 1. Investigation of product inhibition regarding the metabolism of progesterone in ovine adrenal micosomes. 2. The employment of novel cell culture techniques to study the effect of CYP17 and CYP21 concentration on adrenal progesterone metabolism. 3. The formulation of a mathematical model describing the behaviour of the observed results in point 2.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: 1. Substrate binding assays investigating the effects of methanolic extracts of unfermented and fermented Rooibos on the binding of natural substrates to ovine adrenal microsomal and mitochondrial P450 enzymes, demonstrating the interference of substrate binding in the presence of the Rooibos extracts. 2. The effects of selected flavonoids (quercetin, rutin and aspalathin) on the binding of natural substrates to ovine adrenal microsomal and mitochondrial P450 enzymes, demonstrating interference of substrate binding in the presence of the flavonoid compounds. 3. Substrate conversion assays in non-steroidogenic COS-1 cells to investigate the effects of methanolic extracts of unfermented and fermented Rooibos on the activity of key steroidogenic P450 enzymes (CYP17A1, CYP21A2, CYP11B1, and CYP11B2), demonstrating inhibition of the catalytic activity in the presence of Rooibos extracts. 4. The effects of selected flavonoids on the substrate conversion of the aforementioned key steroidogenic enzymes expressed in COS-1 cells. 5. An investigation of the effect of methanolic extracts of unfermented and fermented Rooibos on steroid hormone production in human adrenal H295R cells under basal and stimulated conditions, demonstrating the modulating effects of unfermented and fermented Rooibos extracts. Basal and stimulated steroid hormone production was decreased in the presence of unfermented and fermented Rooibos. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. Die gebruik van substraatbindings-essais om die effek van metanoliese ekstrakte, van gefermenteerde- en ongefermenteerde Rooibos, op die binding van die natuurlike substrate aan skaap adrenale mikrosomale en -mitochondriale P450 ensieme te bepaal. Daar is getoon dat die ekstrakte 'n beduidende inhiberende effek op ensiemsubstraatinteraksie gehad het. 2. Die die inhiberende effek van geselekteerde flavonoïede (kwersetien, rutien and aspalatien) op die binding van die natuurlike substrate aan skaap adrenale mikrosomale en -mitochondriale P450 ensieme. 3. Die gebruik van substraatomsettings-essais in nie-steroïedogeniese COS-1 selle, om die effek van gefermenteerde- en ongefermenteerde Rooibos ekstrakte op die aktiwiteit van die steroïedogeniese P450 ensieme (CYP17A1, CYP21A2, CYP11B1, and CYP11B2) se katalitiese aktiwiteit te bepaal. Daar kon aangetoon word dat die katalitise aktiwiteite van bg. ensieme beduidend beïnvloed word deur die Rooibos ekstrakte. 4. Die gebruik van substraatomsettings-essais in nie-steroïedogeniese COS-1 selle, om die effek van geselekteerde flavonoïede op die aktiwiteit van bogenoemde steroïedogeniese P450 ensieme te bepaal. 5. 'n Ondersoek na die invloed van metanoliese ekstrakte van gefermenteerde- en ongefermenteerde Rooibos op steroïedhormoon biosintese in die menslike adrenale H295R-selmodel. Die ondersoek, onder basale en gestimuleerde toestande, het getoon dat beide Rooibosekstrakte in bogenoemde toestande steroïedhormoon produksie geinhibeer het.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy