>Magister Scientiae - MSc / Being overweight or obese are major factors contributing to the increased morbidity and mortality due to non-communicable diseases such as type 2 diabetes, cardiovascular disease and cancer. The treatment of obesity with pharmaceutical drugs is plagued by side effects. Plants and their phytochemicals possess a number of beneficial health effects including anti-oxidant,anti-mutagenic, anti-inflammatory, anti-obesity and anti-cancer effects, mediated by activation of the adenosine monophosphate protein kinase (AMPK).AMPK controls many metabolic processes including glucose uptake and utilisation, and adipogenesis, and is often referred to as the master regulator establishing cellular homeostasis.Cyclopia maculata, commonly known as honeybush, is an indigenous South Africa plant possessing anti-oxidant, anti-inflammatory and anti-cancer properties. Recently, others in our laboratory have shown that a hot water extract of fermented C. maculata inhibits adipocyte differentiation in 3T3-L1 pre-adipocytes, with some evidence of weight regulatory properties in a Wistar rat model of diet-induced obesity. In the rat study, 21 day old weanlings were fed a high fat, high sugar cafeteria diet for 3 months with (n=10) or without (n=10) C. maculata supplementation. This group of rats was referred to as the lean group (n=20). Another group of rats were fed a cafeteria diet for 4 months to induce obesity (obese group, n=20) and thereafter treated as described for the lean rats. The aim of this MSc study was to determine whether C. maculata induces AMPK activation.Proteins were extracted from the liver and muscle tissue of lean and obese Wistar rats using an optimized extraction method with a commercial lysis buffer and the TissueLyser.Treatment with the C. maculata extract had no effect on the protein yield in lean and obese rats. Interestingly, the protein yield in the liver of obese rats was significantly higher than that observed in lean rats. Although C. maculata treatment slightly increased AMPK activation (calculated as the ratio of phosphorylated AMPK to total AMPK) in the liver of lean and obese rats, the difference was not statistical significant. Conversely, C.maculata treatment decreased AMPK activity in muscle of lean and obese rats, with statistical significance observed in the lean group only (2.3-fold, p<0.05). Differences in AMPK activation between the groups were also noted, a 1.3-fold decreased activity observed in obese groups compared to their lean counterparts, although this was not statistically significant. Expression of PPARα, a downstream protein target affected by AMPK activation was reduced in the liver of lean and obese rats after C. maculata treatment. Moreover, PPARα expression was significantly higher in obese compared to lean rats (2.7-fold, p<0.001). PPARα is a transcription factor mediating fat metabolism (β-oxidation) and its expression is induced by circulating free fatty acids, which are increased in obese compared to lean rats. The expression of PPARα in muscle was too low for Western blot analysis and quantification.Cyclopia maculata treatment did not affect hepatic expression of UCP2, another protein important in establishing energy homeostasis. The expression of UCP2 was 2.9-fold higher in the liver of obese rats compared to their lean counterparts, although the difference was not statistically significant. The opposite results were observed in the muscle where C. maculata treatment decreased UCP2 expression in lean rats (2.8-fold,p<0.0001), and UCP2 expression was decreased 1.4-fold in obese rats compared to lean rats, although the difference was not statistically significant.ELISA results for AMPK activation revealed that C. maculata treatment increased AMPK activity, although not statistically significant. Histological analysis of retroperitoneal fat showed that C. maculata did not affect adipocyte size and number, although a slight decrease in adipocyte size was observed after treatment .This study has demonstrated that treatment of the cafeteria diet fed Wistar rats with 300 mg/kg of a hot water extract of fermented C. maculata does activate AMPK. This study revealed important differences between lean and obese rats. In particular, increased hepatic protein content, PPARα and UCP2 expression was observed in obese rats compared to the lean group. This suggests an adaptive response to the increased circulating free fatty acids during obesity and an increase in β-oxidation in these animals.

Radiotherapy has been shown to increase the subsequent risk of heart disease among survivors of breast cancer, but little is known about factors, other than the dose of radiation delivered to the heart, which determine the magnitude of the risk. In addition, survivors of teenage and young adult cancer are internationally acknowledged as an understudied population, and limited information is available on their late health risks. This thesis sought to utilise the largest observational datasets available to date for these populations: the Collaborative Group on Observational Studies of Breast Cancer Survivors and the Teenage and Young Adult Cancer Survivor Study. These were used to firstly characterise the radiation-related risks of heart disease and lung cancer, and secondly to provide an overview of the long-term risk of heart disease for the entire spectrum of cancers diagnosed in teenagers and young adults aged 15 to 39. Initially, a methodology study and systematic review demonstrated that selection effects and other biases can be very problematic during analyses of observational cohorts, particularly when using a radiotherapy comparison. However, in the case of heart disease and lung cancer, one can take advantage of the breast being a paired organ and use a laterality comparison, particularly when laterality played little effect in treatment selection. This comparison was used throughout the analyses of breast cancer patients. This thesis demonstrated that adjuvant radiotherapy for breast cancer significantly increased the risk of heart disease among women with left-sided breast cancer and those patients with ipsilateral lung cancer. Interestingly, younger women were at the highest risk of heart disease, and a progressive proportional decrease in risk with increasing age at diagnosis was found, which has not been shown before. It also suggested that radiotherapy and chemotherapy combined may further increase the risk of heart disease among breast cancer patients. Survivors of teenage and young adult cancer, particularly Hodgkin lymphoma, were at a significantly raised cardiac mortality risk compared to the matched general population. The findings of this thesis provide evidence to support continued follow-up for cancer patients, as survivors were found to be at a substantial risk into the second or third decade after treatment. It has permitted the detection of groups of individuals at particularly increased risks, for example younger patients and survivors of Hodgkin lymphoma diagnosed in teenagers and young adults, for whom closer monitoring for late effects or measures to reduce the risk, such as adaptations to treatment, may be appropriate. Finally, evidence was also presented to support the development of clinical follow-up guidelines specifically for survivors of teenage and young adult cancer.

<b><u>Introduction:</b></u> Some cancer survivors are known to have an elevated risk of morbidity and mortality from cardiovascular disease. An important cause of this elevated risk is recognised to be irradiation of normal tissues during radiotherapy received as part of cancer therapy. There are substantial difficulties in studying radiation-related cardiovascular disease (RRCD). The reasons for this include the complexities of measuring radiation normal tissue doses retrospectively and the prolonged latencies of many of the cardiovascular endpoints. A variety of complimentary research methodologies can help provide additional knowledge to guide the appropriate management of patients treated in the past and of new patients in the future. <b><u>Methods:</b></u> 1) A cohort study of mortality from circulatory disease in the nationwide British Childhood Cancer Survivor Study (BCCSS). 2) A case-control study of valvular heart disease (VHD) in Dutch Hodgkin lymphoma (HL) survivors, including retrospective radiation dosimetry to estimate the radiation dose to heart valves. 3) A dosimetric study of cardiovascular radiation doses in patients entered into the UK NCRI Lymphoma Study Group RAPID trial, including predictions of 15-year cardiac mortality using innovative methods. 4) A modelling study to predict mean whole heart dose (MWHD) from involved field radiotherapy (IFRT) for HL using anatomical measures. 5) A prospective study using cardiovascular magnetic resonance (CMR) imaging to characterise the heart in women receiving radiotherapy for breast cancer. <b><u>Results:</b></u> 1) The risks of all types of circulatory mortality are elevated in survivors of childhood cancer. The absolute excess risks continue to increase 40+ years following diagnosis. The risk of death from cardiomyopathy and heart failure increased substantially with the introduction of anthracycline chemotherapy. There is no evidence of a reduction in risk of circulatory mortality in more recent eras of diagnosis. 2) There is a strong relationship between estimated radiation dose to the affected heart valve and the risk of subsequent VHD (p<0.001). This effect was modelled to allow prediction of the risk of VHD. 3) A proportion of patients treated with IFRT received a substantial cardiac radiation dose (MWHD = 8.8 Gy, SD = 5.6) but, on average, the predicted 15-year cardiac mortality following treatment is low (absolute risk 0.2%, range 0.0 to 2.7%). 4) It is possible to estimate the mean whole heart dose from IFRT prior to detailed radiotherapy planning based on pre-treatment diagnostic imaging to an accuracy of 5-6% of the prescribed dose. 5) Although women received low cardiac doses (MWHD = 1.5 Gy, SD = 0.8) and have a low predicted risk of cardiac radiation-related morbidity and mortality, there is some evidence of subclinical effects on strain and strain rate imaging of the anterior portions of the left ventricle that receive the highest radiation dose. <b><u>Conclusions:</b></u> Using a variety of methods these studies have all succeeded in adding to knowledge about the nature, magnitude and timing of RRCD. This knowledge can be used to help the future management of cancer patients. In addition, each of the studies has natural and planned extensions and will continue to contribute further knowledge into the future.