This work was carried out to compare the endocrine function of ovarian tissue isolated in vitro, in an identical environment between ewes and ewe lambs. Furthermore, to determine whether the differences in endocrine and reproductive function of ewes and ewe lambs are related to differences in the proteomics of corpora lutea, follicles and oocytes. Oestradiol concentrations in tissue cultured in TCM-199 were similar for ewe and ewe lamb follicles collected post slaughter on day 9 to 12 of the oestrous cycle and cultured for different incubation times but increased with increase in follicular size. Oestradiol secretion was greater (P<0.001) for ewe and ewe lamb follicles cultured in media with FCS. Media progesterone concentrations were higher (P<0.001) for ewe than ewe lamb follicles. Progesterone in media and in follicular fluid was increased with increased follicles size. Ewe CL, collected on day 9 to 12 of the oestrous cycle, produced more progesterone than ewe lamb CL when cultured in TCM-199 with or without FCS, PVA, BSA. Proteomics indicated more large spots, in ewe follicular and CL tissue average gels compared with ewe lamb average gels. The protein spots were estimated to be between 45 to 97 kD, in both tissue and age groups, this range of molecular weight could have affected steroid hormone synthesis. (Chapter.3). Ewe and ewe lamb follicles cultured with FSH and LH produced more oestradiol than without, furthermore, oestradiol concentration increased with follicle size. There was no difference in media oestradiol concentration between age groups after 24 h of culture. However, for follicles cultured for 2, 4 or 6 h, concentrations were greater after 4 and 6 h, in ewes than in ewe lambs. Overall ewe lamb follicles produced more progesterone (P<0.001) than ewe follicles when cultured with FSH and LH when cultured for 24 h, but no difference was observed after 2, 4 and 6 hours between ewes and ewe lambs Overall ewe follicles produced more (P<0.002) progesterone than ewe lambs when cultured with different concentration of hCG although there was no difference between ages with respect to oestradiol concentrations. Ewe CL secreted more progesterone (P<0.002) than ewe lamb CL, when cultured for 0 or 24 h. Furthermore, tissue concentrations were greater in ewe CL than ewe lamb CL after incubation in TCM-199, TCM-199 plus BSA, TCM-199 plus FCS and TCM-199 plus PVA. Ewe lamb CL produced more progesterone than ewe CL in medium containing LH when cultured for 2, 4 and 6 hours, but ewes produced more progesterone than lambs when CL were cultured with different concentrations of hCG. Relative to untreated shells, the protein profiles of the ewe follicular shells treated with FSH and LH changed to a greater extent than that of the ewe lambs treated gels in both ages contained more protein spots than control gels. The largest spots were estimated to be between 30 and 97 kDa (Chapter.4). There was no difference between age groups for follicles from ewes and ewe lambs treated with ovagen in oestradiol and progesterone concentrations observed after 2,4, 6 and 8 h of incubation in TCM-199. However, treatment with ovagen plus hCG resulted in higher oestradiol and progesterone concentrations in the media from ewe follicles compared to ewe lambs. Furthermore, there were more protein spots in the range 30 to 66 kDa marker in gels from ewes treated with either ovagen or ovagen plus hCG than for ewe lambs (Chapter 5). Lamb oocytes were smaller than ewe oocytes and developed to a lesser extent in culture. Furthermore, the addition of FCS to TCM-199 caused greater cytoplasmic and nuclear maturation than other media used in this experiment and ewe lamb oocytes have a similar 1D protein bands compared with ewe oocytes, but contained less protein (Chapter 6).

Ovarian cancers often develop resistance mechanisms against the standard platinum and taxane chemotherapy, which indicates the need for novel therapeutics to improve patient outcome. In vitro assays were performed to assess the effects and mechanism of action of a novel peptide, GAP-107B8, on ovarian cancer cell viability. Xenograft models were used to determine GAP-107B8’s effects on tumour burden in immune-incompetent mice. GAP-107B8 significantly reduced cell viability in ovarian cancer cell lines, although no synergistic effects with carboplatin were observed. This reduction in cell viability was due in part to apoptosis and may involve mechanisms leading to decreased pAKT, but without any change in pPKC levels. In vivo, GAP-107B8 had no effect on ovarian tumour burden, but significantly reduced ascites volume. The findings suggest that GAP-107B8 can reduce some malignant characteristics of cancer cells in vitro and in vivo and should be evaluated further as a potential therapeutic for ovarian cancer.

In Canada, ovarian cancer kills about 67% of diagnosed patients, largely due to difficulties in early diagnosis. Current treatment consists of debulking surgery and intermittent chemotherapy every three weeks. This approach leads to insufficient drug concentrations at disease sites, and long treatment-free intervals cause accelerated tumor proliferation and drug resistance, resulting in a 5-year survival rate of only 25-35%. Drug resistance development is the ultimate cause of the majority of patient deaths. Improvements yielding more effective treatment are fundamental for successful management of this disease. This thesis investigated a continuous chemotherapy strategy devoid of treatment-free intervals for ovarian cancer treatment. A biocompatible, biodegradable polymer-lipid injectable formulation PoLigel, was used for continuous DTX delivery. The formulation was well tolerated; no alterations in body weight, behaviour, histology of peritoneal tissues, or interleukin-6 levels were seen in CD-1 mice treated with the PoLigel. Continuous DTX therapy via the PoLigel was considerably more efficacious than intermittent therapy, resulting in significantly less tumor burden and ascites fluid in models of human and murine ovarian cancer. Continuous therapy resulted in less tumor cell proliferation and angiogenesis, and more tumor cell death than intermittent DTX. The presence and length of treatment-free intervals was shown to contribute to the development of drug resistance. Eliminating these intervals by continuous dosing resulted in superior antitumor efficacy in both chemosensitive and chemoresistant xenograft models of human ovarian cancer, and prevented drug resistance increase after a 21-day treatment period. Survival studies revealed that intermittent dosing led to a mild survival prolongation of 36% and 10% in chemosensitive and chemoresistant models, respectively, whereas continuous DTX prolonged survival by a striking 114% and 95%. Although long-term continuous chemotherapy substantially improved survival, increased drug resistance mechanisms were found at the endpoint. Overall, results presented here encourage the clinical implementation of continuous chemotherapy due to greater achievable therapeutic advantages.

In Canada, ovarian cancer kills about 67% of diagnosed patients, largely due to difficulties in early diagnosis. Current treatment consists of debulking surgery and intermittent chemotherapy every three weeks. This approach leads to insufficient drug concentrations at disease sites, and long treatment-free intervals cause accelerated tumor proliferation and drug resistance, resulting in a 5-year survival rate of only 25-35%. Drug resistance development is the ultimate cause of the majority of patient deaths. Improvements yielding more effective treatment are fundamental for successful management of this disease. This thesis investigated a continuous chemotherapy strategy devoid of treatment-free intervals for ovarian cancer treatment. A biocompatible, biodegradable polymer-lipid injectable formulation PoLigel, was used for continuous DTX delivery. The formulation was well tolerated; no alterations in body weight, behaviour, histology of peritoneal tissues, or interleukin-6 levels were seen in CD-1 mice treated with the PoLigel. Continuous DTX therapy via the PoLigel was considerably more efficacious than intermittent therapy, resulting in significantly less tumor burden and ascites fluid in models of human and murine ovarian cancer. Continuous therapy resulted in less tumor cell proliferation and angiogenesis, and more tumor cell death than intermittent DTX. The presence and length of treatment-free intervals was shown to contribute to the development of drug resistance. Eliminating these intervals by continuous dosing resulted in superior antitumor efficacy in both chemosensitive and chemoresistant xenograft models of human ovarian cancer, and prevented drug resistance increase after a 21-day treatment period. Survival studies revealed that intermittent dosing led to a mild survival prolongation of 36% and 10% in chemosensitive and chemoresistant models, respectively, whereas continuous DTX prolonged survival by a striking 114% and 95%. Although long-term continuous chemotherapy substantially improved survival, increased drug resistance mechanisms were found at the endpoint. Overall, results presented here encourage the clinical implementation of continuous chemotherapy due to greater achievable therapeutic advantages.

Clinical information was collected from 283 randomly chosen ovarian cancer cases from at the Saskatoon Cancer Centre between the years 1983-1995. The data was evaluated for its significance in predicting survival and relapse free survival (RFS) using univariate and multivariate analysis. Several clinical prognostic factors were identified by univariate analysis. Additionally, using Cox's regression model the independent markers of survival and RFS were FIGO stage, and residual disease in 173 and 178 patients respectively. Data on CA 125 serum level, (available in 89 patients) was a marker of prognostic significance in the patients treated with platinum based chemotherapy. CA 125 and CEA antigen expression were also evaluated in seventy one cases. It was found that mucinous neoplasms exclusively expressed CEA antigen. This study indicates that the evaluation of serum level CEA may be a complementary tool for patients with cancers not expressing CA 125. In this retrospective study, DNA from paraffin embedded tissue (PET) in patients with ovarian carcinoma was examined to identify gene abnormalities in p53, p16INK4A, RB-1, p21WAF1/CIP1, Cyclin D1, Erb-B2, and MSH2. Adverse outcome was also examined in addition to survival and RFS, to identify novel molecular prognostic markers. P53 overexpression in 44 of 112 (39%) was associated with reduced survival and RFS (' p' = '0.04' and 'p' = '0.008 '). Aneuploid DNA content, found in 34 of 112 (30%) cases, was associated with shorter survival and RFS ('p' = '0.03' and 'p' = '0.01'). Dot blot hybridization of G1-S control genes (p16INK4A, Cyclin D1, RB-1, and CDK4) did not identify amplification or deletion events to be associated with adverse outcome. A number of gene alterations in 59 of 63 (94%) ovarian cancer cases were detected by dot blot hybridization; the lack of association with clinical outcome indicated that there may be some other genes in addition to those examined that are of prognostic significance. For eighteen cases, microsatellite instability (MSI) was evaluated by using fluorescently labeled primers at nine loci. LOH was a common event in ovarian carcinoma but MSI was infrequent. Molecular and clinical marker multivariate analysis indicated: (a) residual disease for survival, (b) stage and residual disease for RFS, were independent markers of prognosis.

Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

No description available.

There is growing evidence that the different histological types of ovarian cancer have diverse origins. Many high-grade serous tumours (the most common type) are hypothesised to arise from the fallopian tubes, while endometrioid and clear cell tumours are hypothesised to develop from endometriosis, and the origins of mucinous tumours are uncertain. If these hypotheses are true, then the population-level risk factors for the ovarian cancer histotypes should likewise vary - but few epidemiological studies have sufficient cases to explore this. This thesis investigates the association between ovarian cancer and various exposures (including tubal ligation and reproductive factors), in a cohort of 1.3 million women, with 8,000 incident ovarian cancers. Participants completed a questionnaire at recruitment, and were followed up for routinely collected information on cancers and deaths using national registries. Cox proportional hazards models were used to estimate adjusted relative risks of ovarian cancer in women by different exposures. Tubal ligation was associated with reduced risks of cancers of the ovary, and also of the peritoneum and fallopian tube; parity and breastfeeding were also associated with reduced risks of ovarian cancer. These associations with ovarian cancer were highly heterogeneous between the different histological tumour types, consistent with hypotheses of their distinct origins. In particular, risks of endometrioid and clear cell tumours were substantially reduced amongst women with tubal ligation, but substantially increased amongst nulliparous women. For high-grade serous carcinomas, there was a smaller but significant reduction in risk with tubal ligation, and no significant association with parity. I also describe some methodological studies I set up for future tissue-based work: developing techniques in image analysis, and a pilot study of obtaining linked pathology reports and tissue samples. The population-level associations demonstrated by histotype are largely consistent with the new hypotheses of the different origins of ovarian cancer histotypes.

Ovarian cancers are the most lethal gynecological malignancies, responsible for more than 150,000 deaths around the globe annually. Among ovarian cancers, high-grade serous ovarian cancer has a 5-year survival rate of only 40%. This poor survival is due to a widespread lack of understanding of this disease, from suboptimal prevention and screening methods to failures in treatment. Moving towards novel prevention and treatment methods requires better models of ovarian cancer that phenotypically and genetically recapitulate the features of ovarian cancers that are seen clinically. This thesis highlights the characterization of a novel syngeneic model of high-grade serous ovarian cancer that exhibits the growth, expression profile, histology, and a tumor-initiating cell population that closely resembles human disease. We expand on our initial characterization of the STOSE model in a proof-of-principle study using deep learning of second-harmonic generation and two-photon-excited-fluorescence images to classify normal compared to cancerous tissues. The use of deep learning for image classification based on extracellular matrix and cellular structure could have robust application to complementing common histological examination of tissues and in treatment planning. Building on the changes in structure found in normal compared to cancerous ovarian tissue and recent research that showed age-associated fibrosis develops in murine ovaries, we assessed the non-hereditary ovarian cancer risk factors of age and ovulation number for their effects in altering ovarian tissue structure. This thesis concludes with the first evidence of ovarian fibrosis in non-pathological post-menopausal human ovaries. We show that ovarian fibrosis correlates with the development of a pre-metastatic (tumor-permissive) niche, revealing a novel avenue of research into ovarian cancer risk. Interestingly, age-associated fibrosis could be prevented or reversed by metformin use, revealing a possible mechanism for the previously identified ovarian cancer risk reduction seen with metformin use and further supporting the use of metformin for ovarian cancer prevention.