Lamprey adults may be parasitic or non-parasitic, but the genetic basis of life history type is unknown. Although external differences between types are not apparent until metamorphosis, previous studies have suggested histological differences during ovarian differentiation. This study examined potential differences between parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey I. fossor before, during, and after ovarian differentiation, using histological examination of the gonad, quantitative reverse-transcriptase PCR, and RNA sequencing. There were no observable differences in the timing or nature of ovarian differentiation. However, there was evidence of differential expression of individual genes associated with growth, apoptosis, and fecundity and of sets of genes associated with energy and lipid metabolism. The sequence resources developed in this project will be useful for future examination of the genetic basis of lamprey life history type and of the genes controlling sex differentiation in these ancient vertebrates.

BACKGROUND. Polycystic ovarian syndrome is one of the commonest endocrinopathies in women of reproductive age. The prevalence of the disease is estimated to be around 5 % in general population (Azziz, 2004). Literature on the prevalence of PCOS in Black women is limited (Knochenhauer, 1998). This syndrome is a diagnostic conundrum due to the phenotypic variability of these women. The PCOS woman also has a greater disposition for impaired glucose homeostasis as well as hyperlipidaemia. OBJECTIVE. The hormonal and metabolic profiles of South African women with PCOS have not been described. Ethnic differences in the prevalence of PCOS have also not been well explored. Our study aims to describe and compare the phenotypic profile of African and Indian women with PCOS and to determine the frequency of insulin resistance and hyperlipidaemia in these women. METHODS. A retrospective audit of all patients attending gynaecology endocrine and infertility clinics over the period June 2005 to June 2009 was carried out. The biochemical and clinical profiles were analysed and a comparative analysis between the two largest groups, Indian and Black women were done. All women that attended these clinics were subjected to a fasting lipogram and fasting serum glucose. An abnormal fasting serum glucose would have necessitated a full glucose tolerance test. RESULTS. A total of 110 patients were analysed in this study. There were 87 Indian patients, 16 Black patients, 5 Coloured patients and 2 White patients. Eighty nine percent of PCOS women studied had an increased body mass index (>25). There was an increased LH:FSH in 66 (75.9%) of Indian women and 13 (81.3%) of Black women. Increased androgens were present in 26 (30.2%) in Indian women and 6 (37.5%) of Black women. An increase in fasting insulin was found in 48 (55.2%) of the Indian women and 5 (31.3%) of the Black women. Twenty five (29.1%) Indian women had an increase in fasting serum glucose compared to 1 (6.3%) in Black women. In the Indian population, 13 (14.9%) were found to have Diabetes Mellitus, and 9 (10.3%) had an impaired glucose tolerance test. In the Black population only 1 patient had impaired glucose tolerance. There were no Black patients with Diabetes Mellitus. No Black women were found to have hyperlipidaemia, however 12 (14.3%) Indian women were affected. None of these differences between the races were statistically significant. The major limitation of the study was the sample size of Black women. This is an ongoing study, and aims to recruit more Black women. This will be able to adequately address the correct perspective regarding the metabolic and cardiovascular abnormalities in these women. CONCLUSION. The prevalence of insulin resistance and hyperlipidaemia in local women with PCOS was 50.9%.and 11.3% respectively. Menstrual irregularities and infertility are the most frequent presenting complaints of women with PCOS. Features of hyperandrogenism are not common presenting complaints in South African women. There are no differences in the hormonal and clinical profile of South African Indian and Black women with PCOS, however, there is a trend toward Indian women having a greater prevalence of glucose abnormalities than Black women. We recommend further studies in the management of the metabolic abnormalities in local women with PCOS, in an attempt to develop a protocol to manage the metabolic complexities of PCOS. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2010.

Lamprey adults may be parasitic or non-parasitic, but the genetic basis of life history type is unknown. Although external differences between types are not apparent until metamorphosis, previous studies have suggested histological differences during ovarian differentiation. This study examined potential differences between parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey I. fossor before, during, and after ovarian differentiation, using histological examination of the gonad, quantitative reverse-transcriptase PCR, and RNA sequencing. There were no observable differences in the timing or nature of ovarian differentiation. However, there was evidence of differential expression of individual genes associated with growth, apoptosis, and fecundity and of sets of genes associated with energy and lipid metabolism. The sequence resources developed in this project will be useful for future examination of the genetic basis of lamprey life history type and of the genes controlling sex differentiation in these ancient vertebrates.

Ovarian high-grade serous cancer (HGSC) is the most lethal gynecologic malignancy and well-characterized models may improve patient outcomes. Patient-derived xenografts (PDXs) recapitulate disease heterogeneity; however, to be useful in predicting response to novel chemotherapeutics, they must reflect the response of the donor tissue to standard chemotherapy. The objectives of this study were: first, to evaluate the response of PDXs’ to platinum therapy and compare this response to that of the donor; and second, to determine whether treatment with chemotherapy enriches for tumourigenic cells. Eighteen samples formed tumours in the mammary fat pads of NOD-Scid-IL2Rγnull mice and were treated with Carboplatin. There was a 100% concordance between sample status and PDXs response to chemotherapy. HGS histology was confirmed for all cases. A conclusion regarding post-chemotherapy tumourigenicity could not be made due to inadequate statistical power. PDXs represent useful tools for evaluation of novel therapies and identification of patients who are platinum-resistant/sensitive.

Follicular growth and maturation are tightly regulated processes, which involve the participation of endocrine, autocrineparacrine factors and intracellular molecules. Due to the numerous research efforts, a large number of regulators and their mechanisms of regulation of follicular growth and differentiation have been established. Although the abnormal expression and activities of some of these regulators are believed to be associated with ovarian dysfunction diseases, such as polycystic ovarian syndrome (PCOS), the etiology and pathogenesis of this syndrome are not completely understood. In this thesis, we have identified two novel regulators of follicular growth and differentiation and examined the cellular and molecular mechanisms that contribute to the folliculogenesis. We present here that chemerin reduces FSH-induced steroidogenic enzyme expression and steroid hormone production in follicles and granulosa cells. Prohibitin expression is upregulated by chemerin and knockdown of prohibitin attenuates the suppressive role of chemerin on steroidogenesis, an action regulated by Akt. Using an androgenized rodent model, we also present the dysregulation of chemerin and prohibitin and their association with dysregulated follicular steroidogenesis. Our data and preliminary clinical studies demonstrate the potential involvement of chemerin and prohibitin in the etiology of PCOS. These studies significantly improve the knowledge of ovarian functions and the pathophysiology of PCOS, and provide important clues for the development of novel diagnosis biomarkers and new treatment strategies for this complex syndrome.

High-grade serous ovarian cancer (HG-SOC) is the most lethal gynecologic malignancy. Although most respond to initial therapy, the vast majority of patients eventually recur and die of their disease. Understanding intra-tumor cellular heterogeneity and inter-patient variability is necessary to effectively cure HG-SOC. The work described in this thesis should help to speed the progress of ovarian cancer research in several ways. First, I generated a robust xenograft model that recapitulates the cellular heterogeneity of HG-SOC. In addition, I performed gene expression profiling on a subset of xenografts and showed that they recapitulate the inter-patient diversity of this disease. Second, I applied this model to pre-clinical testing of a folate-targeted imaging agent and showed that it can identify metastatic studding by PET/CT and fluorescence imaging. Using my xenograft model, I investigated the properties of tumor-initiating cells (TIC) and demonstrated that TIC in HG-SOC are rare. Furthermore, although CD133 marks most TIC, heterogeneity in the phenotype is observed within individual tumors and between different patients. Finally, I used a transformative technology, CyTOF, to develop a novel pipeline for prioritization of candidate TIC markers, as well as for characterization of cellular heterogeneity in primary HG-SOC samples.

Proteins secreted or shed by tumors can be found in serum. Detecting these proteins by mass spectrometry (MS) is difficult, due to the wide dynamic range of protein concentrations in serum. To circumvent this issue, we mined the conditioned media of epithelial ovarian cancer (EOC) cell lines which is a less complex fluid to work with. We hypothesize that some of the proteins shed or secreted by EOC cell lines are similar to those secreted or shed by EOC tumors and that some of these proteins can be used as biomarkers. We mined the conditioned medium of four ovarian cancer cell lines (HTB75, TOV-112D, TOV-21G and RMUG-S) by two-dimensional liquid chromatography-mass spectrometry. Our study identified 1208, 1252, 885, and 463 proteins from the HTB-75, TOV-112D, TOV-21G, and RMUG-S cell lines respectively. In all, we identified 2039 proteins from which we focused on 420 extracellular and plasma membrane proteins. High abundance proteins such as albumin and immunoglobulins, which are problematic for serum proteomics, did not interfere with our study. Several known markers of EOC including CA-125, HE4, Mesothelin, and KLK6, were identified in this study. The list of 420 extracellular and membrane proteins was cross-referenced with the proteome of ascites fluid to generate a final list of 51 potential candidates. According to Ingenuity Pathway Analysis, two of the top 10 diseases associated with our list of 51 proteins were cancer and reproductive diseases. Of the 51 candidates, 10 proteins were selected for verification in sera from ovarian cancer patients and healthy individuals. Clusterin showed a significant difference between cancer patients and normal, with sera from cancer patients showing higher levels. Another protein, NPC2, did not show a difference in sera between cancer and normals. Protein expression studies using immunohistochemistry showed that NPC2 is highly expressed in ovarian cancer tissue and absent in normal ovarian surface epithelium. In summary, clusterin and NPC2 appear to play a role in ovarian cancer pathobiology and their role in EOC need to be studied further.

High-grade serous ovarian cancer (HG-SOC) is the most lethal gynecologic malignancy. Although most respond to initial therapy, the vast majority of patients eventually recur and die of their disease. Understanding intra-tumor cellular heterogeneity and inter-patient variability is necessary to effectively cure HG-SOC. The work described in this thesis should help to speed the progress of ovarian cancer research in several ways. First, I generated a robust xenograft model that recapitulates the cellular heterogeneity of HG-SOC. In addition, I performed gene expression profiling on a subset of xenografts and showed that they recapitulate the inter-patient diversity of this disease. Second, I applied this model to pre-clinical testing of a folate-targeted imaging agent and showed that it can identify metastatic studding by PET/CT and fluorescence imaging. Using my xenograft model, I investigated the properties of tumor-initiating cells (TIC) and demonstrated that TIC in HG-SOC are rare. Furthermore, although CD133 marks most TIC, heterogeneity in the phenotype is observed within individual tumors and between different patients. Finally, I used a transformative technology, CyTOF, to develop a novel pipeline for prioritization of candidate TIC markers, as well as for characterization of cellular heterogeneity in primary HG-SOC samples.

The cancer antigen 125 (CA125) is a cell-surface mucin which is over-expressed by the majority of ovarian cancers. However, its biology and the role it plays in ovarian cancer is largely unknown, although other cell-surface mucins have been shown to play a role in apoptosis, cell growth and tumour immune evasion. To analyse the function of CA125 in ovarian cancer, we initially knocked down the expression of CA125 using RNA interference. Knocking down CA125 expression using in vitro transcribed short interfering RNAs (siRNAs) induced a potent cell death response, which has been well characterised in the literature as an induction of an interferon response and resulting in cell apoptosis. Subsequently, using the short hairpin RNA expression vector, pSUPER, which has been shown to knock down genes with high efficiency with reduced off-target affects, we generated stable sub-lines of the ovarian cancer cell line, OVCAR-3, which had been transfected with pSUPER constructs targeting CA125. Intriguingly, these sub-lines had a range of abnormal mitotic events and nuclear defects. However, there was no clear association with the level of CA125 knock down. This could be either due to clonal selection from the parent OVCAR-3 cell line or in addition to CA125 knock down, additional genetic changes are required to occur to favour a state of survival. Similar to the in vitro data, xenografts of the sub-clones into SCID mice generated inconclusive results as to whether CA125 knock down contributes to tumour growth, invasion and metastasis in vivo. More recently, we have been able to achieve high levels of short-term CA125 knock down using synthetic siRNAs designed to reduce off-target affects. These preliminary in vitro and in vivo experiments conducted with pSUPER sub-lines should be repeated using synthetic siRNAs to confirm the role of CA125 in this context. Given the role which the cytoplasmic tail of cell-surface mucins plays in its function, we generated a polyclonal antibody recognising the CA125 cytoplasmic tail, designated M16.1. Immunofluorescence imaging of CA125 in ovarian cancer cell lines, OVCAR-3 and PEO-1, using the OC125 extracellular domain antibody indicated cell-surface localisaton of CA125. However, in addition to the cell-surface localisation, the M16.1 antibody localised to the cell cytoplasm, indicating cleavage and release of the CA125 cytoplasmic tail into the cytosol. Additionally, M16.1 co-localised with α-tubulin at perinuclear sites and to areas resembling microtubule organising centres. However, M16.1 did not co-localise with γ-tubulin at the centrosome, indicating association with non-centrosomal microtubules. Furthermore, depolymerisation of microtubules on ice for 1 hour resulted in loss of diffuse cytoplasmic M16.1 staining but co-localisation between M16.1 and α-tubulin at non-centrosomal sites remained. Intriguingly, when microtubules were allowed to reform at 37oC in PEO-1 cells which had CA125 knocked down by synthetic siRNAs, the ability to reform radial asters was impaired, possibly indicating the CA125 cytoplasmic tail involvement in anchoring microtubules to non-centrosomal sites. Furthermore, we also cloned a portion of CA125 encompassing the cytoplasmic tail, transmembrane domain and 9 tandem repeats. When this construct was transfected into COS-1 cells, the CA125 cytoplasmic tail localised to microtubule bundles during metaphase. Mitotic involvement of the endogenous CA125 cytoplasmic tail was confrmed in OVCAR-3 and PEO-1 cells using M16.1. Given this association and also the results from the pSUPER sub-lines with CA125 knockdown, CA125 may be involved in controlling the fidelity of mitosis, which is grossly altered during tumourigenesis. More recently, it was identified that galectin-1 (Gal-1), an S-type lectin, is a ligand for CA125. Gal-1 is a potent inducer of T cell apoptosis and has been implicated as playing a major role in immune evasion for cancer cells. Consequently, we analysed the expression of CA125 and Gal-1 in ovarian cancer and confirmed the two molecules were expressed concurrently at the mRNA level by RT-PCR. Moreover, immunofluorescence studies also confirmed that CA125 and Gal-1 interacted with each other at the cell-surface of 27/87 cells, an ovarian cancer cell-line. Therefore, we hypothesised that CA125 presents Gal-1 to the immune system, which then induces T cell apoptosis and allows the tumour to escape the immune system. However, CA125 did not protect tumour cells from recognition or killing by T cells, which was shown by no differences in IFN-γ secretion or tumour lysis by cytotoxic T cells using influenza peptide pulsed pSUPER sub-lines with CA125 knockdown. The work described in this thesis suggests that CA125 plays a major role in the aetiology and progression of ovarian cancer through its actions on mitosis, microtubule organisation and immune evasion.

Abstract Ovarian cancer is the 6th most common cancer in women worldwide and mortality from this cancer is high, because early diagnosis is difficult (Sankaranarayanan et al. 2006). Thus, identification of modifiable factors contributing to its aetiology is important in reducing the burden of a very fatal form of women’s cancer. The overall aim of this thesis was to investigate the association between diet and ovarian cancer risk within the context of a framework ‘from nutrients to lifestyle’. The study examined dietary and lifestyle factors that had not previously been investigated comprehensively in four main areas: nutrients (retinol, beta-carotene, vitamins E, C, and B), foods (fruit, vegetables, dairy products, eggs, meat and liver), diet patterns and lifestyle (smoking, alcohol, and body-mass-index). The study used the data from two population-based case-control studies of women aged 18-79 years conducted in Australia 10 years apart; the Survey of Women’s Health (SWH, 1990-1993) involves 683 cases and 777 controls, and the Australian Ovarian Cancer Study (AOCS, 2002-2005) includes 1329 cases and 1397 controls. Cases were recruited from gynaecological oncology treatment centres and controls were selected at random from the electoral roll. Detailed information on non-dietary risk factors was obtained using a questionnaire and dietary information was obtained via a semi-quantitative food frequency questionnaire. Multivariable-adjusted odds ratios (ORs) (adjusting for age, parity, oral contraceptive use, education, and energy intake) for ovarian cancer risk were estimated separately for each study with logistic regression modelling and weighted pooled risk estimates for the two studies were calculated using fixed-effects models. Principal components analysis of around 40 food groups was performed to identify dietary patterns in each study separately. There was an increased risk of ovarian cancer associated with retinol intake in both studies (combined OR for the highest vs. lowest quartile =1.42, 95%CI 1.19-1.69), while intake of beta-carotene was inversely related to cancer risk (combined OR=0.80, 95%CI 0.67-0.96). The associations between retinol and beta-carotene and risk appeared to be independent. Liver was also associated with an increased risk; however this association seemed to be explained by the high levels of retinol in liver. Dairy products and eggs are other good sources of retinol, but no clear relations were seen and additional adjustment for saturated fat further attenuated the associations. High vitamin E intake (combined OR=0.73, 95%CI 0.61-0.87) was associated with a decreased risk, but no overall association was seen for vitamin C (combined OR=1.06, 95%CI 0.89-1.27). It is noteworthy that vitamin C seemed to be more beneficial for current smokers than for never/past smokers. For the B vitamins, a significant inverse association was apparent only for niacin intake (combined OR=0.69, 95%CI 0.58-0.82). While there was no association between cancer risk and sources of niacin such as total meat and red meat, other niacin-rich foods such as poultry (combined OR=0.77, 95%CI 0.66-0.89) and fish (combined OR=0.83, 95%CI 0.71-0.97) were significantly inversely associated with risk. In contrast to poultry and fish, high consumption of processed meat was associated with a 24% increase in risk (combined OR=1.24, 95%CI 1.06-1.45). Total fruit (combined OR=0.75, 95%CI 0.60-0.94) and total vegetables (combined OR=0.69, 95%CI 0.52-0.92), specifically cruciferous vegetables (combined OR=0.79, 95%CI 0.63-0.98) and green leafy vegetables (combined OR=0.79, 95%CI 0.67-0.94), were associated with a modestly decreased risk, whereas the inverse association between red/yellow vegetables and risk did not quite reach statistical significance (combined OR=0.84, 95%CI 0.64-1.08). High fruit intake was, like vitamin C intake, somewhat more beneficial for current/past smokers than for never smokers. Exclusion of women who took dietary supplements did not substantially change the observed associations between nutrients and risk. Three major eating patterns were identified: ‘snacks and alcohol’, ‘fruit and vegetable’, and ‘meat and fat’. Significant inverse associations between the snacks and alcohol pattern and risk were attenuated after further adjustment for white/red wine intake in both studies; it thus appeared that the observed association was at least partly due to wine intake. A significant association between the fruit and vegetable pattern and risk was seen only in the more recent study. A diet characterized by high meat and fat was associated with an increased risk of ovarian cancer, although the observed association was stronger in the SWH There was no evidence that the associations between diet pattern and cancer risk varied by women’s lifestyle. Although there was some variation in the analyses stratified by the histologic subtype of ovarian cancer, no consistent patterns of variation were observed for either study. These findings provide additional evidence that a healthy diet defined by high intake of fruit, vegetables, particularly cruciferous and green leafy vegetables, white meat and fish and low in meat and fat, especially processed meats might be beneficial against ovarian cancer.