Since high-risk Human Papillomavirus (HPV) plays a critical role in cervical carcinogenesis, it is essential to improve our understandings on the role of HPV-related molecular markers in cervical neoplasia. The aim of this study was to investigate the expression and prognostic significance of selected HPV-related markers, including HPV16/18 E2 binding sites (E2BS) methylation, Pak 1, c-FLIP, Notch 1 and Brd4 expressions, as well as the potential functions of Pak 1 in cervical neoplasia. First, the differential expressions of these markers among clinical samples of normal cervical epithelium, low-grade and high-grade cervical intraepithelial neoplasia (CINs) and cervical cancer were studied. Methylation status of E2BS 1, 2 and 4 was determined by pyrosequencing. Expressions of the target proteins were assessed by immunohistochemistry. Both HPV16/18 E2BS 1&2 and E2BS4 methylation progressively increased from normal cervix through CINs to cancer. More importantly, HPV16 E2BS1&2 (for transcriptional repression of E6/E7 oncoproteins) became more heavily methylated than E2BS4 (for transcriptional activation of E6/E7) in cervical cancer, favouring the differential binding of E2 protein to E2BS4. Pak 1, c-FLIP, Notch 1 and Brd4 were all overexpressed in cervical cancer. Their expressions increased progressively from normal cervix to low-grade +/- high-grade CINs. Pak 1 and c-FLIP expression was positively correlated with HPV18 E6 and HPV16 E7 expression respectively. Notch 1 expression was inversely correlated with HPV16 E7 and HPV18 E6 expressions. Brd4 expression was positively correlated with HPV16 E2 and inversely correlated with HPV16 E7 expressions. The prognostic significance of the molecular markers was investigated by correlation with clinical parameters. Heavier methylation at E2BS1&2 relative to E2BS4 was associated with better overall and disease-free survival in cervical cancer patients. HPV16 E2BS1&2 hypermethylation, weak cytoplasmic Brd4 expression, strong c-FLIP or Notch 1 expression were associated with higher risk of recurrent abnormal smears after treatment of CINs. The role of Pak 1 in cervical cancer was further explored by comparing its functions between cervical cancer cell lines with and without transient knock-down of Pak 1 by siRNAs. It was demonstrated that the significant functions of Pak 1 in cervical cancer were on promoting cell proliferation and inhibiting apoptosis. Transient HPV16 E6 inhibition showed no effect on total / phosphorylated Pak 1 expressions. Lastly, the function of Pak 1 on the regulation of cervical cancer cell radiosensitivity was also investigated. Pak 1 inhibition increased cell sensitivity in response to irradiation by enhancing apoptosis and inhibiting cell proliferation. Conclusively, differential methylation status at HPV16/18 E2BS, as well as Pak 1, c-FLIP, Notch 1 and Brd4 proteins contribute to cervical carcinogenesis, and are potential prognostic markers in cervical cancer and CIN patients. Pak 1 inhibitor may be a potential adjunctive agent to improve radiotherapy. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

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Cervical cancer is the most common malignancy after breast cancer in women worldwide. It accounts for 83% of all new cases and 85% cancer death in developing countries. In South Africa, cervical cancer is the common cancer in women with an annual crude incidence rate of 30.2 per 100,000 women and the highest rate were found to be in women between the ages of 66-69 years. Approximately 6800 women in S.A face new case of cervical cancer while accounting for 3700 cancer death annually. Because of unequal access to the health facilities, socio-economic differences, HPV and HIV infection, the rate of cervical cancer in black women is higher (42.1%) compared to the low rate in white women. Because of the name “cancer bush’ given to Sutherlandia Frutescence(S.F) plant by the traditional healers as well as Xhosas, Zulu, Sotho and cape Dutch for its anti-cancer activity, the plant was in this study to confirm its cytotoxic effect on the cervical cancer cell lines. Aim of the study: to evaluate the methanolic extracts of Sutherlandia Frutescens on cervical cancer cell lines. Materials and Methods: The MTT assay was performed to evaluate SiHa cell lines treated with methanolic extract of S.F (50μg/ml, 100μg/ml, 150μg/ml and 200μg/ml). The three compounds (Canavanine, GABA and Pinitol) were also evaluated for its anti-tumour activity. The cell growth was then showed in real time using Xcilligence. Flow cytometry was employed to determine the mode of action. Caspase 3/7 assay was performed to confirm if cell death was via caspase-dependent or independent and ATP was done to assess the ATP level in S.F treated cells. Results: MTT shows a significant decline in cells treated with 50μg/ml, 100μg/ml and 200μg/ml of the extract and 50μg/ml was concluded to be the concentration at which 50% of the cells die. The ATP results are inconsistent with MTT result; the ATP level increased in S.F treated cells. Cell index which represents the quantitative measure of cell growth in real time decline upon treatment with 50μg/ml. Flow cytometry showed cells are dying by apoptosis and the cell cycle arrest is mostly in the S phase. The cell death was caspase-dependent as it shows an increased luminescence which is proportional to the number of caspase. The concentrations of the compounds used, Canavanine (1000μM, 1500μM and 2500μM), GABA (100μM, 300μM and 500μM) and Pinitol (30μM, 90μM and 120μM) induce cell death and cell death shows to decrease after the maximum concentration. Conclusion: Sutherlandia Frutescence has proven with number of research that it induces cell death via apoptosis. After evaluating its cytotoxicity, the plant shows to be a promising anti-tumor agent that needs to be clinically proven. / Life Sciences / M. Sc. (Life Science)

published_or_final_version / Nursing Studies / Master / Master of Nursing

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Gauteng, Johannesburg, 2016. / Overexpression of RBBP6 in cancers of the colon, lung and oesophagus makes it a potential target in anticancer therapy. This is especially important because it associates with the tumour suppressor gene p53, inactivation of which has been linked to over 50% of all cancer types. Cancer is an enormous burden of a disease globally. Today, more people die from cancer than HIV/AIDS, tuberculosis and malaria combined. And in females, breast and cervical malignancies remain the most common types. Currently, cervical cancer is the most diagnosed gynaecological cancer type, whose mortality rate is the highest in developing countries due to the asymptomatic nature of the disease coupled with inadequate cancer control tools and facilities. Breast cancer incidence rate has increased beyond that of lung cancer, making it the most common malignancy among women. / GR2016

MSc., Faculty of Science, University of the Witwatersrand, 2011 / Aim: This study aimed at evaluating the accuracy of the treatment setup margin in external beam radiotherapy in cervical cancer patients treated supine with or without the CIVCO “kneefix and feetfix”TM immobilizing devices. Methods and materials: 2 groups of 30 cervical cancer patients each, who were treated supine with two parallel opposed fields or a four-field “box” technique were selected randomly. The treatment fields were planned with a 2 cm setup margin defined radiographically. The first group was treated without any immobilization and the second group was treated with the “kneefix and feetfix”TM immobilization device. Both groups of patients were selected from the patients treated on one of two linear accelerators (linac), which had weekly mechanical quality control (QC). All patients had pre-treatment verifications on the treatment machine in which a megavoltage Xray film was taken to compare with the planning simulation film. Both films were approved by the radiation oncologist managing the patient. In this study the position of the treatment couch as at the approved machine film was taken as the intended or planned position for the immobilized patients. The digital readouts of the daily treatment position of the couch were recorded for each patient as the absolute X (lateral), Y (longitudinal), and Z (vertical) position of the couch from the record and verify system interfaced to the treatment machine. A total of 1241 (582 for the immobilized and 659 for the non-immobilized patient group) daily treatment setup positions were recorded in terms of the X, Y and Z coordinates of the couch corresponding to the Medio-lateral (ML), Supero-inferior (SI) and Antero-posterior (AP) directions of the patient, respectively. The daily translational setup deviation of the patient was calculated by taking the difference between the planned (approved) and daily treatment setup positions in each direction. Each patient’s systematic setup error (mi) and the population mean setup deviation (M), was calculated. Random ( ) and systematic ( ) setup errors were then calculated for each group in each direction. The translational setup variations found in the AP, iii ML, SI directions were compared with the 2 cm x 2 cm x 2 cm Planning Target Volume (PTV). Couch tolerance limits with the immobilization device were suggested based on the ± 2SD (standard deviation) obtained for each translational movement of the treatment couch. Result: The random and systematic errors for the immobilized patient group were less than those for the non-immobilized patient group. For the immobilized patient group, the systematic setup error was greater than the random error in the ML and SI direction as shown in Table I. Table I: The random and systematic errors in the setup in the Antero-posterior (AP), Medio-lateral (ML) and Supero-inferior (SI) directions and the suggested couch tolerance limits for both patient groups. Almost all treatment setup positions had less than 2 cm variation in the AP setup for both patient groups however; one third of the immobilized positions had more than 2 cm variation in the setup in the ML and SI directions. Conclusion: The “kneefix and feetfix”TM immobilizing device resulted in a minor improvement in both the random and systematic setup errors. The systematic setup errors need to be investigated further. There are measurable patient rotations of more than 2 cm in the setup margin with the immobilizing device and this should be confirmed with an imaging study. The 2 cm margin in the ML and SI directions Immobilized patient group Non-immobilized patient group AP (cm) ML (cm) SI (cm) AP (cm) ML (cm) SI (cm) Random error (!) 0.30 1.35 1.26 0.37 2.74 7.83 Systematic error (") 0.19 1.55 1.64 0.33 1.70 8.11 Suggested couch tolerance limits (±2SD) 0.70 4.04 4.08 0.88 4.76 N/A iv established at simulation should not be changed for these patients. A 1 cm tolerance in the AP setup margin could be introduced at this institution.

Thesis (M.Sc. (Medical Laboratory Sciences)) -- University of the North, 1986 / Refer to the document

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences