Despite considerable research on cancer treatments and preventatives, poor outcomes in cancer patients are common. The vital search for effective cancer drugs often begins in the laboratory, where unfortunately the effects of a drug in humans cannot be perfectly modelled. Epidemiology can play a vital role in determining the real world efficacy of a drug currently used for other purposes before clinical trials begin. This thesis therefore used primarily laboratory evidence to identify potential anti-cancer uses for existing common drugs. The drugs and cancers studied were; tricyclic antidepressants and both incidence and survival in a number of cancer types, particularly glioma; aspirin and colorectal cancer survival; and angiotensin converting enzyme (ACE) inhibitors and hepatocellular carcinoma (HCC) incidence. A series of studies using The General Practice Research Database as a data source assessed any potential associations: A case-control study for tricyclic antidepressant use and cancer incidence; cohort studies to examine mortality in colorectal cancer and glioma in relation to tricyclic use, and for colorectal cancer mortality in aspirin users; and a case-control study in relation to ACE inhibitor use and HCC. A strong, cancer type specific, dose and time dependant protective effect was found for the incidence of glioma and colorectal cancer. This led to a further study examining mortality for these cancer types in tricyclic users. While no significant protective effects in all-cause mortality of tricyclic users were found, a larger study could still find such an effect in glioma. For aspirin and colorectal cancer mortality, a small but significant reduction in mortality was observed, though these effects were not entirely consistent throughout the study. There were no significant associations found between ACE inhibitors and HCC. These findings contribute to the knowledge of the anti-cancer effectiveness of these drugs, and may assist in designing future clinical studies.

Consumption of fruits and vegetables has been shown to reduce the risk of various types of cancer. Macro- and micro-nutrients as well as non-nutritive phytochemicals present in fruits and vegetables have been associated with this effect. This study was conducted to investigate the chemopreventive and chemotherapeutic potential of two types of fruits which are endemic to Borneo Island: Mangifera pajang (bambangan) and Artocarpus odoratissimus (tarap). The first part of the project was to study the antioxidant potential of the crude extracts of the plants in vitro. The fruits were first separated into flesh, kernel and peel for M. pajang and flesh and seed for A. odoratissmus. DPPH (2,2- diphenyl-I-picrylhydrazyl) free radical scavenging and FRAP (ferric reducing / antioxidant power) assay were employed for the antioxidant study. The result showed that the kernel of M. pajang extract displayed strongest antioxidant activity as assessed using both assays, followed by M. pajang peel, A. odoratissmus seed, M. pajang flesh and A. odoratissmus flesh. The presence of selected phytochemicals in the plant extracts was determined in the next chapter. Polyphenols have been identified as major phytochemicals in the plant extracts, and in M. pajang kernel extract represents about 10% of its total weight. Gallic acid, coumaric acid, sinapic acid, caffeic acid, ferulic acid, chlorogenic acid, naringin, hesperidin, rutin, luteolin and diosmin have been identified as the key polyphenol phytochemicals present in the kernel of M. pajang which might be responsible for the superior antioxidant properties as compared to other extracts. Concern that the results for the chemical antioxidant assay do not necessarily reflect cellular activity led to the third part of the project; assessment of the cytoprotective activity of the crude extracts against oxidative stress induced by tert-butyl hydroperoxide (t-BHP). Only M. pajang kernel extract as well as the positive control (quercetin) displayed cytoprotective activity against this toxicant. It seems that non-cell based antioxidant assay does not necessarily reflect the activity in cell-based antioxidant assay. This is shown by lack of cytoprotective activity of both M. pajang peel and A. odoratissimus extracts despite their considerably high antioxidant activity in DPPH free radical scavenging and FRAP assay. In order to study which proteins might be involved in the cytoprotection mechanism, western blotting method was employed to determine the expression of various Cytoprotective proteins [i.e. quinone reductase (NQO I), glutathione peroxidase (OR), methionine sulfoxide reductase A (MSRA), heat shock protein 27 (HSP27) and heat shock protein 70 (HSP 70)]. Different cytoprotective mechanisms were observed by the kernel extracts and quercetin. In the present study, NQOI, OR, MSRA, HSP27 and HSP 70 have been shown to be involved in the cytoprotection activity of quercetin while only OR and MSRA were involved in the cytoprotection activity of M. pajang kernel extracts. Other cytoprotective proteins remain to be studied to fully understand the cytoprotection mechanism of both plant extract and quercetin. Some chemopreventive agents have been shown to suppress cancer proliferation, induce apoptosis in cancer cells as well as inhibit angiogenesis and metastasis in pre-clinical and clinical trials. Thus, the last part of the project was to determine the anti-cancer potential of plant extracts in a variety of cancer cell lines (derived from breast, colon, liver and ovarian carcinoma). The results showed that the kernel extract of M. pajang displayed strong anti proliferative activity in breast cancer cell lines (MCF-7 and MDA-MB-231). The kernel extract induced cell cycle arrest in MCF-7 cells at the sub-G1 (apoptosis) phase of the cell cycle in a time-dependent manner. For MDA-MB-231 cells, the kernel extract induced strong G2-M arrest in cell cycle progression at 24 hours, resulting in substantial sub-G1 (apoptosis) arrest after 48 and 72 hours of incubation. Staining with Annexin V -FITC and propidium iodide revealed that this apoptosis occurred early in both cell types, 36 hours for MCF-7 cells and 24 hours for MDA-MB- 231 cells, with 14.0% and 16.5% of the cells respectively undergoing apoptosis at these times. This apoptosis appeared to be dependent on caspases-2 and -3 in MCF-7 cells and on caspases-2, -3 and -9 in MDA-MB-231 cells. As a conclusion, from the two plants (M. pajang and A. odoratissimus) studied, the extract of M. pajang kernel displayed diverse health benefit properties, antioxidant, chemoprevention and chemotherapeutic potential. M. pajang could be fully utilized for pharmaceutical, nutraceutical as well as food products. Further study (i.e. animal and clinical study, isolation of pure compounds, bioavailability study) are required to determine the efficacy in human population.

This thesis focuses specifically on the transmission of bad news from doctors to patients and their families in the context of a hospital oncology department. It uses awareness context theory as a basis for exploring communication between patients and health care professionals, particularly when the information to be disclosed is sensitive and will have a significant bearing on how people perceive their future. In order to enhance clinical practice, senior health care professionals in particular, have in the past been encouraged through government policy and professional legislation to attend communication skills courses to develop the way they communicate and interact with patients. Yet, in spite of these interventions evidence suggests that doctors and other health care professionals still find it difficult to negotiate sensitive and emotionally challenging discussions, and frequently question whether or not patients are aware and understand the information disclosed to them and whether or not information provided meets the needs and expectations of patients. The premise of this research is that more attention needs to be given to how other more reflective and experiential professional development approaches and techniques might help doctors communicate better with their patients when disclosing sensitive information and bad news. In order to do this however, a better understanding is needed about what is going on in consultations and how each of the individuals involved experience and make sense of these interactions. It is proposed that in order to understand ‘resulting interactions’ more fully it is necessary to explore and compare the multiple perspectives of doctors, patients and others; including relatives and nurses. This thesis seeks to do this in an innovative way by reporting research, which involved observing and recording consultations between doctors and patients and their relatives and then conducting in-depth interviews with such people in order to explore their own insights into this process. In total, 115 episodes of data were collected and analysed from 16 patients and 16 doctors. The insights gained from this study are presented in relation to two main analytic themes; Doctors and Patients Acting their Parts, and Sharing Uncomfortable News. The data analysis highlighted a number of approaches used by patients and doctors to manage and control their interactions within the medical consultation. The implications of the study findings are discussed in relation to both wider theoretical perspectives and ideas for how doctors working in such settings could be assisted to consider alternative strategies for these aspects of their work.

The gastrointestinal (GI) hormone, gastrin, promotes cancer progression and its down-regulation has been linked to reduced cancer stem cell numbers. Gastrin acts through the cholecystokinin-2 receptor (CCK-2R) and its biological effects are blocked by CCK-2R inhibitors. We investigated the regulation of the CCK-2R and its potential role in promoting survival of cancer stem cells (CSC). A panel of cancer cell-lines, including GI, glioblastoma and lung, with CCK-2R-transfected cells as a positive control, were grown either as monolayers, or, to provide a 3D in vitro tumour model, as spheres. Linear-after-the-Exponential (LATE)-PCR was used to quantify CCK-2R gene expression and this was validated using siRNAs. Flow cytometry was used to investigate receptor protein expression. Activity of CCK-2R promoter reporters was quantified using luciferase assays. LATE-PCR for CCK-2R gene expression is 10,000-fold more sensitive than the Taqman-based assay, and provides a highly precise method for detection of genes which have important biological functions but low expression. This assay showed that primary non-small-cell lung tumours have significantly more expression than normal lung tissue, indicating a potential therapeutic marker. CCK-2R siRNAs resulted in up to 97% (p<0.05) knockdown of the receptor in cancer cells, confirming the specificity of LATE-PCR and offering a therapeutic possibility. The CCK-2R promoter constructs were active in lung, glioma and colorectal cancer cell-lines, demonstrating a potential drug target; however, transcriptional activity did not correlate with gene expression suggesting post-transcriptional or translational regulation is a factor affecting CCK-2R expression. Flow cytometry suggests the presence of a small population of cells within each of these cell-lines which expresses CCK-2R very highly, which was not correlated to CSC markers. However, CCK-2R expression was enriched when cells were grown as spheres, and inhibition caused a delay in sphere-forming, implying that the CCK-2R may play a role in tumour, and CSC, expansion. Thus, CCK2R provides a potential target for therapeutic intervention in cancer.

Alzheimer's disease (AD), the most common form of dementia, is a devastating age-related neurodegenerative disorder. There is a growing body of evidence suggesting that leading a stressful lifestyle is associated with a heightened risk of developing AD. This is supported by preclinical evidence using transgenic mice over-expressing genetic mutations leading to overt ß-amyloid protein production, a pathological marker of AD; stress in such mice has been capable of exacerbating AD-associated pathologies, including accelerating memory impairments and elevating ß-amyloid levels. In contrast, a recent study from our group demonstrated that a repeated mild stress procedure, novel cage stress, improved a short-term memory deficit and reduced the normal age-related increase in 3-amyloid levels. This thesis aimed to further characterise the beneficial effects of novel cage stress on AD-associated pathology in the TASTPM mouse model (double transgenic hAPP695swe x PS-I. M146V) which exhibits overt, age-related ß-amyloid pathology. First, age-related changes in AD- associated pathology, with or without exposure to novel cage stress, were assessed using a multidisciplinary approach incorporating measures of cognitive performance, in vivo magnetic resonance imaging and post-mortem analysis of ß-amyloid levels. The aim was to detect an age where we observe the most robust effect of stress; this time window was subsequently targeted to investigate a potential underlying mechanism, namely signalling though the glutamate alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic receptor (AMPAr). Studies described throughout this thesis, alongside previously published data, indicate novel cage stress appears to improve AD-associated pathology in TASTPM mice, but independently of AMPAr function. It is likely that novel cage stress is insufficiently severe to induce detrimental effects, but, rather, subsequent repeated stimulation and physical activity may improve pathological status. A better understanding of lifestyle risk factors of AD, such as stress, will aid in identifying those at risk of developing the disorder. Moreover, discovering the underlying mechanisms linking stress with AD may open novel therapeutic avenues to treat the disorder.

Persistent accumulation of free radicals in cells leads to oxidative stress, which plays a causative role in the induction and progression of various chronic diseases. Therapeutic focus has therefore shifted towards the use of antioxidants, with recent interest in those of plant origin. This study investigated radical scavenging and cytoprotective activities of phytochemicals (quercetin, curcumin, sulforaphane, rosmarinic acid, caffeic acid, danshensu (3,4-dihydroxyphenyllactic acid), ferulic acid and m-coumaric acid) against DPPH free radical in a non-cellular assay, and oxidative damage in hepatic (HepG2) and pancreatic (1.1B4) cells, elicited by an organic hydroperoxide (tert-butylhydroperoxide - tBHP) and a more physiologically relevant stressor (palmitate). Direct and indirect cytoprotective activities were assessed by neutral red viability assay after 5 h co-exposure and 20 h pre-exposure conditions, respectively. Radical scavenging activities of three well-known phytochemicals - quercetin, curcumin and sulforaphane - were initially validated against DPPH (non-cellular assay), where quercetin was shown to be more potent than curcumin; sulforaphane was without effect. With quercetin as positive control, radical scavenging activities of rosmarinic acid and three of its principal metabolites (caffeic acid, danshensu and ferulic acid) were comparable, while m-coumaric acid lacked antiradical activity against DPPH radical. Subsequently in HepG2 hepatoma cells, quercetin and curcumin were confirmed to possess direct and indirect cytoprotective acitivities against 0.5 mM tBHP while, sulforaphane only had indirect cytoprotective acitivities. Additionally, co-treatment of HepG2 cells with low concentrations of quercetin and curcumin (used together) exhibited direct cytoprotective activities against tBHP. However, direct cytoprotective potencies of rosmarinic acid and caffeic acid were less than quercetin. Similar pattern was observed for indirect cytoprotective activities; with danshensu, ferulic acid and m-coumaric acid lacking hepatoprotective activity in co-exposure and pre-exposure conditions. These results highlight the discrepancy between non-cellular and cellular antioxidant activities, which could be accounted to the poor lipophilicity profiles of rosmarinic acid and its principal metabolites. Cytotoxicity assay in 1.1B4 human pancreatic β-cells revealed that these cells were more vulnerable to tBHP-induced oxidative damage than HepG2 cells. An investigation of selected phytochemicals in 1.1B4 cells produced novel findings, with quercetin exhibiting direct and indirect cytoprotective activities against tBHP (0.125 mM and 0.5 mM). Curcumin and caffeic acid were also cytoprotective against 0.125mM tBHP but only exhibited direct cytoprotection against 0.5mM tBHP. Sulforaphane lacked both direct and indirect cytoprotective activities in 1.1B4 cells, exhibiting marked cytotoxic effects in both conditions. Further analysis in both HepG2 and 1.1B4 cells proved that indirect cytoprotective activities of selected phytochemicals were not dependent on pro-proliferative activities of quercetin, curcumin, caffeic acid and sulforaphane. Moreover, it was observed that high concentrations of curcumin and sulforaphane caused necrosis in both cell types, rather than apoptosis; caffeic acid also produced necrotic effect in 1.1B4 cells. Whilst prolonged exposure of HepG2 and 1.1B4 cells to high glucose concentrations failed to elicit any evidence of glucotoxicity, sodium palmitate caused concentration-dependent cytotoxicity after short-term (5 h) and long-term (20 h) exposure to both cell types. Overall, selected phytochemicals caused additive cytotoxicity in the presence of palmitate, although quercetin demonstrated direct cytoprotection alone in HepG2 cells. Using Western blot, curcumin, caffeic acid and sulforaphane did not upregulate NQO1, but 20 h exposure to 0.1 mM quercetin resulted in upregulation in HepG2 cells, amidst high basal levels of NQO1 in this cell type. However, both basal and inductive expression of NQO1 has not been observed in 1.1B4 cells. Thus, although rosmarinic acid, danshensu, caffeic acid and ferulic acid may possess good intrinsic antioxidant properties, their physicochemical properties may limit pharmacological activities at the cellular level. Moreover, the additive cytotoxicity resulting from treatment with selected phytochemicals and sodium palmitate highlights a discrepancy between mechanisms of cytotoxicities by tBHP and palmitate.

Overall paediatric high grade glioma (pHGG) has a poor prognosis, in part due to the lack of understanding of the underlying biology. We therefore used high resolution 244K oligo array comparative genomic hybridisation (oligo aCGH) (Agilent Technologies) to analyse DNA from 38 formalin-fixed paraffin embedded pHGG samples, including 13 DIPG. The pattern of gains and losses were distinct from those seen in HGG arising in adults. In particular we found 1q gain in 21% of our cohort compared to 9% in adults. Homozygous loss at 8p12 was seen in 6/38 (15%) of pHGG. This deletion has not been previously reported in adult or paediatric high grade gliomas. The minimal deleted region is of the gene ADAM3A and homozygous deletion of ADAM3A was confirmed by quantitative real time PCR (qPCR). This novel homozygous deletion of ADAM3A in pHGG merits further study. Amplification of the 4q11-13 region was detected in 8% of cases and included PDGFRA and KIT and subsequent qPCR analysis was consistent with amplification of PDGFRA. MYCN amplification was seen in 2/38 samples (5%) and was shown to be significantly associated with anaplastic astrocytomas (p=0.03). Loss of CDKN2A/B was seen in 4/38 (10%) samples by oligo a CGH, confirmed by FISH on TMAs, and was restricted to supratentorial tumours. ~50% of supratentorial tumours were positive for CDKN2B expression by IHC, whilst ~75% of brainstem gliomas were positive for CDKN2B expression (p = 0.03). Overall DIPG shared a similar spectrum of changes to supratentorial HGG with some notable differences including high frequency of 17p loss and 14q loss, low occurrence of 10q loss and lack of CDKN2A/B deletion.

The theory of metastatic cascade suggests that vasculature plays a central role in the metastatic processes by being the major route of spreading. Two main circulatory systems in the body are responsible for cancer cell dissemination; the blood vascular system and the lymphatic system. However, comparing between these circulatory systems, much less is known about lymphatic vessels, with few studies being conducted about the initial steps of metastasis. In the first part of this project, a series of 202 formalin fixed paraffin embedded (FFPE) cutaneous melanoma sections were stained with D2-40, CD34 and CD68 to identify lymphatics, blood vessels and macrophages respectively, to examine vessel distribution and the involvement of inflammatory infiltrate in mediating vascular invasion (VI). Sections were also stained by conventional haematoxylin and eosin (H&E), to assess VI, and results compared against those obtained by immunohistochemistry (IHC) that allow discrimination of lymphatic and blood vessel invasion. It was found that lymphatics are mainly located at the peritumoural area of the tumour but intratumoural lymphatics are present and appeared to be functional based on the presence of tumour emboli in the vessels. In addition, vascular invasion in melanoma is mainly lymphatic vessel invasion with H&E assessment underestimating its incidence. Lymphatic vessel invasion were significantly associated with markers of aggressive disease which suggest their importance in melanoma. Lymphatic vessel invasion was also associated with a high macrophage count, suggesting a role for macrophage in mediating the process of metastatic via lymphatic vessels. In the second part of this project, the adhesion pattern of melanoma and breast cancer cell lines to blood and lymphatic endothelial cell models; large vessel versus microvessel and primary versus immortalised cells were compared. In addition, the effect of macrophage secreted cytokines; TNF-α and IL-1β, tumour conditioned media and macrophage conditioned media on the adhesive process were also studied. Both melanoma and breast cancer cells exhibited a higher level of adhesion to blood compared to the lymphatic endothelial cells. IL-1β stimulation of endothelial cells, tumour cells or both together showed a significant increased in the percentage of adhered tumour cells to the endothelial cell models with a higher increased to the lymphatic endothelial cells. A significant increased tumour cell adhesion was also observed with macrophage conditioned media and this effect seemed to be associated with the amount of IL-1β present. Interestingly, the increased adhesion effect observed with this supernatant was removed with the use of interleukin-1 converting enzyme (ICE) inhibitor. Expression of adhesion molecules; CLEVER-1, ICAM-1 and VCAM-1 were examined to study which adhesion molecules might regulate the process of tumour-endothelial interactions. Stimulation of endothelial cell models with IL-1β did not show any significant altered CLEVER-1 expression suggesting that although CLEVER-1 is an important lymphatic specific adhesion molecule, it may not be the principle regulator of tumour-endothelial interactions. This process may be regulated by ICAM-1 and VCAM-1 in which the expression increased significantly upon stimulation with IL-1β. In the third part of this study, the effect of TNF-α, IL-1β, tumour conditioned media and macrophage conditioned media stimulation on melanoma and breast cancer cell migration were investigated using wound healing assays. Following exposure to TNF-α and IL-1β, a significant increase in the percentage of wound closure was observed and the increase was higher in IL-1β stimulated cells. Similarly, when tumour cells were exposed to macrophage conditioned media, there was an increase in the percentage of wound closure compared to control cells. The effect of IL-1β and macrophage conditioned media on breast cancer cell migration across blood and lymphatic endothelial cells were also studied using Boyden chamber transmigration assay. Significant increased in tumour cells transmigration was observed with IL-1β stimulation, with similar affinity across both endothelial cell types. However, when cells were stimulated with macrophage supernatant from lipopolysaccharide (LPS) stimulated macrophages, an increase transmigratory effect was notably observed to the lymphatic endothelial cells. Interestingly, the increased adhesion effect was removed with the used of ICE inhibitors. The last part of this study dealt with IL-1β expression in breast tissue samples. 1511 early stage breast cancer tissue microarray samples were stained with commercially available IL-1β antibody to examine the association with lymphatic vessel invasion, clinicopathological variables and clinical outcome. High IL-1β expression in tumour cells was significantly associated with the absence of both intra-tumoural and peri-tumoural lymphatic vessel invasion. A significant association was also observed between low IL-1β expression in tumour cells with breast cancer specific survival and disease free interval. In conclusion, lymphatic vessels have been found to play a significant role in breast cancer and melanoma cells progression by being the major route for vascular dissemination. In the in-vitro settings, this study has shown that IL-1β, with macrophages as the main producer, could regulate tumour cell invasion especially to the lymphatic circulation. This project has yielded some important results towards understanding of the lymphatic vasculature and modulation of lymphatic vessel invasion. However, more studies are needed to enable translation of research into clinical management of cancer.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour. Despite advances in GBM treatment, there is a high frequency of local relapse due to the acquisition of drug resistance. Investigation of glioma cell lines will help us to understand the molecular basis of this hard to treat tumour. In this study, the rat C6 glioma cell line was used as a model alongside two drug selected derivatives (C6-etoposide and C6-irinotecan) to investigate the mechanisms of chemo-resistance in glioma by identifying candidate proteins, genes, and key signalling pathways. Proteomic (2D gel electrophoresis) and genomic (gene array) analyses were performed to determine protein and gene expression changes. Integration of this data with cellular pathway analysis resulted in the prediction that cellular migration and the response to oxidative stress would be distinct in the drug selected C6 cell lines. Cell migration was subsequently assessed using wound scratch repair and transwell migration assays, whilst the response to oxidative stress produced by reactive oxygen species was determined fluorimetrically. The C6 cell line exposed to irinotecan (DNA topoisomerase I inhibitor) showed reduced migration, even under the influence of chemoattractant, compared to other cell lines, consistent with alterations in the expression of collagen genes. The C6 cell line exposed to etoposide (DNA topoisomerase II inhibitor) showed greater resistance to oxidative stress which was proposed to be due to alterations in the signalling pathways downstream of the PTEN/PI3Kinase. Future studies, investigating the effect of PI3Kinase pathway inhibitors are considered and it is proposed that further research into this signalling pathway will be able to uncover the molecular basis of distinct chemo-resistance in this important model cell system for aggressive glioma.

This thesis describes the search for a practical skin dosimetry method for cardiac catheterization procedures, and the application of an optimisation strategy in barium enema imaging. Kodak EDR2 film was characterised across the range of exposure conditions used in the cardiac catheterization laboratory. Its dose-response curve was modelled using a novel equation, and overall uncertainty in film response was estimated. The film saturated at 1 Gy, limiting its usefulness for skin dosimetry. Its performance was found to be strongly dependent on beam filtration, an aspect that had not previously been studied. The film was then used to measure skin doses to patients undergoing coronary angiograms and angioplasties. For angiograms, all skin doses were well below 1 Gy. For angioplasties, 23% of films showed localised saturation, indicating peak skin doses of at least 1 Gy. Dose-area-product was shown to be a poor predictor of high peak skin dose. A mathematical model was developed and software written, to calculate patient skin dose maps from exposure and projection data stored in the image files. This offered a practical method for assessing the magnitude and approximate location of the peak skin dose. Accuracy was limited by a lack of information regarding fluoroscopic exposures, couch position and beam limitation. After including an estimated contribution from fluoroscopy, the model successfully identified those patients whose skin doses exceeded 1 Gy. Following a baseline survey of local barium enema practice, several dose reduction methods were considered. It was decided to introduce copper filtration. 0.1 mm copper reduced mean patient DAP by 37%, without any measurable difference in contrast detail detectability. A detailed phantom study determined the optimal copper thickness as 0.3 mm. This reduced mean patient DAP by 55%, relative to the baseline survey. A visual grading analysis study showed no significant difference in clinical image quality.