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Nonmammalian vertebrates possess some unusual features in their hormonal systems/ when compared to mammals. As a consequence, they can make an important contribution in investigations concerning the fundamental mechanisms operating in endocrinology. Such studies concerning androgens include inter alia their effects on developmental aspects in the brain of birds and related singing behaviour; the role of neural enzymes in reproductive processes in fish; and the relation between androgens and the stages of spermatogenesis in amphibia, The present thesis examines the biochemistry of androgens in the Australian lizard Tiliqua rugosa. The major compounds studied were testosterone and epitestosterone, which are known to be present in high concentrations in the plasma of the male animal. Previous investigations are expanded, particularly in the areas of steroid identification and testicular biosynthesis. In addition, preliminary studies on the metabolism in the brain (and other tissues) and plasma protein binding are reported. The presence of epitestosterone as a major free androgen in the plasma of the male lizard was confirmed. Other steroids were found in the sulphate fraction. Testosterone sulphate was the most rigorously identified compound, while some evidence was also found for the presence of conjugated 5-androstene-3β,17-diols, etiocholanolone and dehydroepiandrosterone (DHA). Epitestosterone does not appear to be extensively conjugated in this animal. Steroids were not found to be conjugated as glucuronides. The identification studies employed a novel method of electrochemical detection of steroids. This technique was investigated and extended in the current thesis. Biosynthetic studies were carried out on androgen interconversions in the testis, in vitro. The major enzyme activities detected were 17α-arid 17β-oxidoreductases (17α-OR and l7β-OR) and 3β-hydroxysteroid dehydrogenase (3β-HSD)/isonerase. No evidence was found for the presence of a steroid-17-epimerase that would directly interconvert testosterone and epitestosterone. The 17-oxidoreductases were found to be dependent on the cofactor NBDFH. Testosterone appears to be formed mainly via the 4-ene pathway, whereas epitestosterone is formed from both the 4- and 5-ene routes. The compound 5-androstene-3β, 17α-diol was found to be an intermediate in the synthesis of epitestosterone from DHA. Temperature was found to significantly affect 17α-OR activity (maximum at 32°C). In contrast,17β-OR activity was independent of this factor in the testis. Androgen metabolism in the testis was found to be regulated by cofactors, temperature and season. The major enzyme activities found in the male brain were 17α- and 17β-OR. 3βHSD/isomerase was not found; however a low activity of 5α-reductase was identified. Aromatase activity was not positively identified, but preliminary results suggest that it may be present at low levels. The 17-oxidoreductases were widespread throughout the brain. The 17α-OR was significantly lower in the forebrain than other brain sections. The 170-OR activity did not vary significantly throughout the organ, although there was a trend for its activity to be higher in the midbrain region (containing the hypothalamus in these sections). The concentration of endogenous steroids in brain tissue was estimated by radioimmunoassay. Epitestosterone was found throughout the organ structure, whereas testosterone was found mainly in the midbrain (containing hypothalamic regions in these sections). Correlations between enzyme activities and steroid concentrations in brain regions suggested that the main function of 17α-OR is to produce epitestosterone, whereas the 17β-OR may catalyse a more reversible reaction in vivo. Temperature was found to significantly affect both 17α- and 17β-OR activities in the brain. In contrast to the testis, the maximum activity of the brain enzymes occurred at 37°C. The level of 17α-OR activity in the male lizard (100 nmol/g tissue/h) is the highest reported for this enzyme in vertebrates. Both activities were found to be quantitatively similar in the whole brain homogenates of male and female animals, and did not vary seasonally when examined in the male. The 17-oxidoreductases were also found in most other tissues in T.rugosa, including epididymis, adrenal, kidney and liver (but not blood). This suggests that the high activities of both 17α-OR and 17β-OR are dominant features of the steroid system in this animal. The formation of 11-oxygenated compounds was found in the adrenal, in addition to the formation of polar metabolites in the kidney and liver (possibly polyhydroxylated and conjugated steroids). A preliminary investigation into the plasma binding of androgens was carried out. The insults suggest that there are several binding sites for testosterone; one with high affinity and low capacity; the other with low affinity and high capacity. Binding experiments were carried out at 32°C. At this temperature, specific binding was greater than at 25 or 37°C. From the results of competition studies it was suggested that epitestosterone (with a K(i)= 3 X 10 (-6)M for testosterone binding) regulates the binding of testosterone (K(i)=10(-7)M) and hence the concentrations of the latter steroid as a free compound in plasma. In general, the study has shown that the biochemistry of androgens in the reptile T.rugosa is largely similar to that found in other vertebrates. The major difference is a greatly increased activity of 17α-OR, which causes a higher concentration of 17α-compounds to be present in the tissues of this lizard. The physiological roles for epitestosterone are not yet clear. However it appears from this study that this steroid regulates testosterone concentrations in several tissues by either steroidogenic or binding mechanisms. Several major influences on this regulation include temperature, availability of cofactors and seasonal effects.

Includes list of articles published or accepted for publication during the period of PhD candidature. / "July, 2004" / Includes bibliographical references (leaves 255-293) / xxviii, 293 leaves : ill., plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 2004?

Bibliography: leaves 182-210.

The vagina is the port of entry for sexually transmitted diseases in women. Its epithelium constitutes the luminal border, thus comprising an important defence barrier. The objective of this work was to investigate the mechanisms of importance in the immune defence of the vaginal epithelium of healthy, fertile women, and possible menstrual cycle changes. Effects of hormonal contraceptive usage on oestrogen receptor (ER) and progesterone receptor (PR) expression were studied. The contribution of epithelial cell to the immune defence was estimated by assaying their expression of antimicrobial defensins and the epithelial thickness. Vaginal biopsies and serum samples were collected during the follicular and luteal phases in regularly menstruating women (controls) and in users of combined oral contraceptives (COCs), levonorgestrel implants (LNGs), or depot-medroxyprogesterone acetate injections (DMPAs). Fifteen healthy women (aged 20–34 years) were enrolled in each group. Morphometry was performed on vaginal tissue stained with haematoxylin/eosin and by immunohistochemistry using monoclonal antibodies against immune cell markers, PR, and ER. Expression of mRNA for human α-defensins HD-5 and HD-6, and human β-defensins (HBD) 1 to 4 were determined by real-time qRT-PCR and in situ hybridization. In controls, the epithelium was 261 ± 16 μm thick and harboured 241 ± 35 leukocytes (CD45+) per mm2. T lymphocytes (CD3+) dominated. Both αβ T cells and γδ T cells were present with an approximate 4-fold dominance of αβ T cells. Cytotoxic T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7 ± 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes and granulocytes were generally absent. No differences were found between the follicular and luteal phase. All four β-defensins analysed for were detected in vaginal epithelium and most samples expressed at least two. HBD-2 and HBD-3 were most frequent. HBD-3 and HBD-4 expressing cells were localized in the parabasal and intermediate cell layers. α-defensins were not detected. The epithelium was significantly thicker (333 ± 9 μm) in COC, LNG, and DMPA users than in controls, and commonly showed hyperplasia. In DMPA and LNG users the frequency of intraepithelial leukocytes (CD45+) was increased, explained by increased frequencies of both αβ and γδ T cells. In DMPA users there was also a selective increase in CD8+ T cells. PR expression was significantly reduced in DMPA users compared with controls, COC and LNG users. COC and particularly DMPA users often had undetectable levels of serum E2. In conclusion, both adaptive immunity, i.e. intraepithelial T cells, and innate defence mechanisms, i.e. intraepithelial macrophages and β-defensins, are believed to contribute to the immune defence in the human female lower genital tract. These parameters did not change during the menstrual cycle but hormonal contraceptive usage, especially DMPA, affected the quality of the epithelium. The use of DMPA and LNG was correlated with the accumulation of T cells within the epithelium. The effects of these changes on the risk of contracting infections are yet to be determined.

Graduation date: 1992

It is the responsibility of humans, as environmental stewards, to monitor our impact on the environment so that efforts can be made to remediate the effects of our actions and change behaviors. To better understand our environmental footprint, sensitive and simple analytical methods are needed to quantify the contaminants that we discharge into our natural surroundings. Emerging environmental contaminants are of particular concern because there is limited or no information available on their occurrence, fate, and toxicity. As a result, the implications of using these chemicals are not well understood. Therefore, accurate environmental data are needed to help scientists and government policy-makers make informed decisions on research directions and chemical regulation. However, challenges exist for the analysis of emerging contaminants, including a lack of suitable analytical standards and internal standards, their broad range of chemical properties, and that they are frequently present at trace levels and in complex environmental matrices. The work presented within this dissertation focuses on the development, validation, and comparison of analytical methodologies based on large-volume injection high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS) for the analysis of emerging environmental contaminants in aqueous environmental matrices. Large-volume injection (e.g. 900 μL to 4,500 μL) is an analytical technique that eliminates sample preparation associated with pre-concentration by injecting larger-than-traditional volumes of sample directly onto a HPLC column. In Chapter 2, a direct aqueous large-volume injection method was developed and validated for the quantification of natural and synthetic androgenic steroids in wastewater influent, wastewater effluent, and river water. This method was then applied to hourly composite samples of wastewater influent that were taken over the course of a single day. This work expands on the research of the endocrine-disrupting chemicals that occur in wastewater and provides an estimate of the community use/abuse of synthetic androgenic steroids. Environmental analytical methods should be as environmentally friendly as possible and efforts should be made to reduce the waste generated during analysis while maintaining analytical performance. In Chapter 3, a method based on large-volume injection was compared to two methods based on solid-phase extraction. The purpose of this comparison was to demonstrate that the same method performance could be achieved by large-volume injection as that by solid-phase extraction while reducing waste, labor, and costs. Estrogens and perfluorinated chemicals were used as model analytes and wastewater influent was used as a model matrix. The results of this study provide convincing reasons for analysts to adopt large-volume injection as an alternative to solid-phase extraction. In Chapter 4, a novel analytical method was developed and validated to quantify newly-identified and legacy fluorinated chemicals in groundwater. The final method combined micro liquid-liquid extraction, non-aqueous large-volume injection, and orthogonal chromatographic separations. Ground water samples collected from six different U.S. military bases was used to demonstrate the method. This is the first report on the occurrence of these newly-identified fluorinated chemicals in any environmental media and serves as a rational for conducting future research on their environmental fate and toxicity. The breadth of the research presented in this dissertation advances the field of environmental analytical chemistry in several areas. First, classes of environmental contaminants for which there is limited (synthetic androgenic steroids) or no (newly-identified fluorochemicals) environmental data were studied. Second, novel methods based on direct-aqueous and non-aqueous large-volume injection were developed and validated to identify and quantify those contaminants. Third, it was demonstrated that solid-phase extraction is not a "necessary evil" needed to develop methods for emerging environmental contaminants in aqueous matrices. Finally, this work is a platform on which other environmental chemists can use to develop large-volume injection methods in the future. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Aug. 2, 2012 - Feb. 2, 2013

Steroidogenic factor (SF1) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Recent studies have demonstrated that phospholipids are ligands for SF1. In the present study our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using mass spectrometry we show that in H295R adrenocortical cells SF1 is bound to sphingosine (SPH) under basal conditions and that cAMP stimulation decreases the amount of SPH bound to the receptor. We also show that silencing both acid and neutral ceramidases using siRNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. In vitro analysis of ligand binding using scintillation proximity assays show that several sphingolipids and phospholipids, including phosphatidic acid (PA), can compete with [3H]SPH for binding to SF1, suggesting that SF1 may have more than one ligand and binding specificity may change with the changes in intracellular fluxes of phospholipids. Further, phosphatidic acid (PA) induces SF1-dependent transcription of CYP17 reporter constructs. Inhibition of diacyglycerol kinase (DAGK) activity using R59949 and silencing DAGK- expression attenuates SF1-dependent CYP17 transcriptional. We propose that PA is an activating ligand for SF1 and that cAMP-stimulated activation of SF1 takes place by displacement of SPH.

The aim of this research was to study the correlation between tissue tumor marker expression and HR-HPV infection, smoking, hormonal contraceptive use and sex steroids in women with cervical intraepithelial neoplasia or normal epithelium. The study investigated the expression of 11 tumor markers in cervical biopsies obtained from 228 women with different diagnoses ranging from normal cervical epithelium to various stages of CIN. 188 women were recruited at our colposcopy clinic (out-patient surgery, Department of Obstetrics and Gynecology, Falun Hospital) for laser cervical conization or a directed punch biopsy, either because of a vaginal smear (Pap smear) that showed cytological findings suggesting CIN, or because of repeated findings showing atypical squamous cells of undetermined significance (ASCUS). For 40 volunteers, punch biopsies were taken from the normal cervical epithelium. The time period for this study was 2005-2007. Study I :  228 women, of whom 116 were tested, 64 were positive to HR-HPV. The results showed that Ki67 tumor cell proliferation index was the only marker that independently correlated to both the presence of HR-HPV and the severity of cervical lesions. Study II:  228 women, of whom 83 were smokers (36, 9%). Smokers showed lower expression of p53, FHIT (tumor suppressor markers) and interleukin-10 .Higher expression of Cox-2 and Ki-67 (tumor proliferation markers). Study III:  195 women who were premenopausal. There was increased p53 expression (tumor suppressor) in the progestin-IUD users compared to non-users. Decreased IL-10 expression (immunological marker) was observed in both COC users and any progestin-only users. Study IV: Serum from 80 premenopausal women was available. The main finding was that the increased levels of serum progesterone and estradiol were associated with increased Cox-2 expression (proliferation marker). Serum progesterone and estradiol levels influence cellular and extracellular proteins which have been associated with neoplastic development in normal epithelium and CIN. Conclusion: The results of these studies support previous epidemiological findings on the role of smoking, contraceptive use and sex steroids as co-factors in development of CIN and that tumor marker expression varies in different grades of CIN.