Circadian rhythms are present in all living organisms; daily oscillations of biological process from the expression of a gene to the number of times that an animal displays a given behaviour. The light/dark cycle is the primary cue that entrains these rhythms and the suprachiasmatic nuclei, within the hypothalamus are the central pacemaker which synchronises peripheral body clocks. Mice are useful circadian biology models and two peripheral circadian outputs were studied, locomotor activity and the rhythm of body temperature in a common inbred strain, the C57BL/6 mouse. The use of individually ventilated cages to house mice increases biocontainment, enabling the maintenance of high health status colonies and reducing the risk of allergies for laboratory personnel. The effect of these sealed units on ambient light levels was examined, using locomotor activity as a marker of entrainment to the light/dark cycle. Mice housed closer to the overhead light source experienced greater levels of illumination than those at the lower levels, yet all entrained to the light/dark cycle. Mice housed lower on the rack showed more activity during light hours when they normally rest and the onset of activity was advanced in relation to the time the lights turned off. Individually ventilated cages do not therefore compromise circadian entrainment but cage position may alter the distribution of rest and activity in relation to the light cycle. Measuring the rhythm of body temperature of animals is often confounded by the stress associated with immobilisation and restraint. A novel non-invasive method, a thermal imaging camera was trialled against an indwelling intraperitoneal implant, to compare the relationship between peripheral and core body temperature under different light cycles. A stable relationship was found between the two methods (average R² value = 0.92) and this persisted in conditions of constant darkness, where lack of light cues resulted in free-running of the rhythm, assuming a shorter period length of oscillation. This novel method has potential for use in circadian phenotyping studies and to improve welfare, following experimental interventions where the mouse, a small, metabolically active animal is at risk of hypothermia.

Autophagy, a cellular recycling mechanism, and apoptosis, a regulated mode of cell death, are two fundamental cellular processes that contribute to carcinogenesis and tumour growth as well as treatment sensitivity and resistance. The protein encoded by DRAM1, a p53-responsive gene, has previously been described as an autophagy and apoptosis modulator downstream of p53 activation. Furthermore, a family of DRAM1-related proteins has been uncovered by in silico analysis. Of the 5 members of this protein family, only DRAM1 and DRAM-2 had previously been tested for their roles in cell death and autophagy. Much less was known about the remaining three DRAM-family members TMEM150A/B/C (termed DRAM-5/-3/-4 by us for ‘DRAM-related/associated member-5/-3/-4’) and their potential roles in autophagy, cell death or cell survival in cancer cells. In this project, we therefore aimed to test whether these DRAM-family proteins could modulate autophagy, cell death or cell survival in in vitro cancer cell line systems. We used both retroviral, constitutive overexpression systems and CRISPR/Cas9-mediated gene disruption systems to study the effect of TMEM150 overexpression or TMEM150 ablation on these processes. In summary, we found that none of the TMEM150 genes were induced by p53, but starvation conditions increased TMEM150A and C transcript levels in some conditions. Moderate changes in TMEM150 protein levels showed no dramatic effect on cell growth and survival. Of the three TMEM150 proteins, only TMEM150B affected autophagy, while TMEM150A and C did not modulate autophagic processes in any of the assays performed. Moreover, we show that TMEM150B overexpression can improve cellular survival under glucose deprived conditions, while none of the other DRAM-family proteins seems capable of doing so. Additionally, serum or amino acid starvation did not show parallel effects. Lastly, we show that the influence of TMEM150B on autophagic processes is uncoupled from its ability to modulate survival in glucose-starved cells. Taken all together, with this work we provide an initial characterisation of the TMEM150 proteins, which may lay a foundation for future, expanded studies on the cellular functions of the DRAM-family.

Background: Diagnostic imaging plays an expanding and central role in patients' medical care. Radiographer clinical reporting is being increasingly used in patient focused services. There is a paucity of research that has examined radiographer chest X-ray reporting. Aim: To determine the diagnostic accuracy of reporting radiographer chest X-ray (CXR) reporting and the influence that CXR reports have on clinicians' diagnostic decision-making. Method: A quasi-experimental study determined the diagnostic accuracy of a cohort of reporting radiographers in CXR interpretation, using a free-response methodology. The influence of CXR reports on clinicians' diagnostic decision-making was determined with a cohort study. A non-inferiority approach was used, in line with Royal College of Radiologists and College of Radiographers guidance that reporting radiographers must be comparable to consultant radiologists. Results: The diagnostic accuracy of reporting radiographers (RR) was non-inferior to consultant radiologists (CR) for all measures, all p < 0.0001; unweighted JAFROC (RR Figure of Merit [FoM]=0.828, 95%CI 0.808-0.847; CR FoM=0.788, 95%CI 0.766-0.811), weighted JAFROC (RR FoM=0.830, 95%CI 0.811-0.849; CR FoM=0.786, 95%CI 0.764-0.808) and inferred ROC (RR Area Under the Curve [AUC]=0.909, 95%CI 0.887-0.931; CR AUC=0.903, 95%CI 0.882-0.924). No difference was found in the number of CXR reports that produced a correct most likely and/or most serious diagnosis (RR 876 of 1337 cases; CR 810 of 1368; p=0.103). Uncorrected most likely diagnostic confidence (RR 72.5 to 80.2; CR 71.0 to 80.4) and uncorrected most serious diagnostic confidence (RR 34.0 to 41.9; CR 33.5 to 39.2) of reporting radiographer CXR reports was non-inferior to consultant radiologists (p < 0.001). Corrected most likely diagnostic confidence, calculated using the Tsushima methodology, was lower (RR 4.61; CR 5.02) with no apparent difference, but noninferiority was not confirmed (p > 0.05). Conclusion: With appropriate postgraduate education, reporting radiographers are able to interpret chest X-rays at a level comparable to consultant radiologists.

It is estimated that over half of colorectal cancer (CRC) cases in the UK are preventable through lifestyle changes. Perhaps unsurprisingly, bowel cancer is strongly linked to dietary choices. Diets that are high in fat and low in fibre are associated with increased risk of cancer while diets rich in fruit, vegetables and fibre have a reduced risk. Several studies have investigated the effect of certain dietary components in CRC initiation and development. Previous work, in humans and animals, has demonstrated that the polyphenols found in black raspberries (BRBs) have chemopreventative and therapeutic effects. However, the exact mechanism for these effects remain unknown. As CRC can originate from an intestinal stem cell (ISC) it is possible that the chemopreventative role is due to the impact BRBs have on the normal and/or malignant ISCs. This thesis aimed to investigate the chemopreventative effect of BRBS on normal intestinal tissue and on the initial and later stages of intestinal tumourigenesis, in the context of ISC dynamics and activated Wnt signalling. This was achieved using a Cre-loxP based approach to conditionally delete Apc (the negative regulator of the Wnt signalling pathway) within different compartments of the adult murine intestinal epithelium and also utilised the 3D intestinal organoid system. Exposure to a BRB enriched diet is reported here to be well tolerated in mice and have no major detrimental effects on normal intestinal homeostasis and health. Feeding of BRB diet 2 weeks prior to Apc gene ablation is shown to partially attenuate the ‘crypt-progenitor’ phenotype typical of acute Apc loss. In this context of activated Wnt signalling, BRBs altered ISC dynamics in vivo and reduced the self-renewal capacity of Apc deficient cells ex vivo. Additionally, long-term feeding of BRB diet was shown to significantly improve survival of mice which developed macroscopic stem-cell derived Wnt-driven adenomas. Together, these data are the first evidence that BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment. These findings further support the use of BRB intervention in cancer prevention in the context of Wnt-driven tumourigenesis.

The burden of non-communicable diseases, including cancer, is growing globally. Epidemiological studies have shown that lifestyle factors can increase the risk of these diseases. Colorectal cancer represents the third most common cancer for men and women in the UK. This thesis investigated the relationships between three classic lifestyle risk factors - alcohol intake, adiposity and smoking - and colorectal cancer in the UK Biobank cohort. UK Biobank is a cohort of 500,000 men and women aged 40-69 recruited between 2006-2010. Participants were followed-up for cancer and death registrations until 31st March 2014 through linkage with national datasets. Cox proportional hazards models were used to analyse these data. This thesis found that alcohol intake was associated with colorectal cancer for men but not for women. For men, there was a dose-response relationship between alcohol intake and colorectal cancer. Results were similar using non-drinkers or never drinkers as the reference group. Body mass index (BMI), waist circumference (WC) and waist to hip ratio (WHR) were each strongly associated with colon cancer for men but there was only slight evidence for an association with WC and WHR for women. Modelling BMI and WC/WHR together, the associations for WC/WHR remained while the association for BMI was attenuated, indicating that WC/WHR may be more directly associated with colorectal cancer risk than BMI. While former cigarette smokers had an increased risk of colorectal cancer compared to never smokers, there was no clear evidence of an increased risk for current cigarette smokers. Furthermore, former cigarette smokers with ≥40 years smoking duration had a higher risk than current cigarette smokers with ≥40 years duration. In conclusion, this thesis found that alcohol intake, adiposity and smoking are each associated with colorectal cancer risk. The prevalence of these risk factors should be minimised in order to prevent disease.

Improvements in treatment over the past century have greatly increased survival for retinoblastoma, a rare childhood tumour of the eye, caused by mutations of the RB1 tumour suppressor gene. However, as survival for retinoblastoma has improved, those with the hereditary form of the disease (RB1 germline mutation) have elevated risks of developing additional cancers, mostly bone and soft tissue sarcomas and melanoma. Despite advances in understanding of second cancer risks following treatment for retinoblastoma, key research questions remained including 1) risks of common adult onset cancers, some of which have somatic mutations in the RB1 pathway and are associated with ionizing radiation; 2) persistence of increased risks of bone and soft tissue sarcomas into adulthood; 3) clarification of risks of second cancers following chemotherapy to treat retinoblastoma and 4) role of genetic susceptibility to second cancers following retinoblastoma. In response to these questions, a large cohort of 1852 long-term survivors of retinoblastoma was assembled to evaluate systematically the risk of second cancers. The work described in this thesis, which comprises six major studies, that have used this cohort to identify a higher risk than previously assumed of lung cancer; confirmed the increased risk of second cancers in survivors with a RB1 germline mutation and past radiotherapy; documented for the first time that risk of soft tissue sarcomas varied by subtype; demonstrated that mortality from second cancers exceeded that from retinoblastoma; provided new information on variation in second cancer risk by family history of retinoblastoma; and clarified that chemotherapy in addition to radiotherapy for retinoblastoma confers a higher risk for second cancers compared with radiotherapy alone. These studies collectively have provided risk data that can be used to inform survivors and their health care providers to facilitate screening or surveillance and early identification of second cancers.

Ovarian cancer is the deadliest gynaecological cancer, with less than half of patients surviving five years from diagnosis. The asymptomatic nature of the early disease commonly results in diagnosis at an advanced stage where peritoneal metastases are prevalent, and the disease rapidly develops resistance to platinum-based agents. Innovative treatments are needed to combat this untreatable disease that has a poor prognosis. Adenoviruses (Ad) are versatile gene therapy vehicles that have been studied for six decades. Their wider clinical use has been limited by poor tumour-specificity, pre-existing immunity, and toxicity-inducing off-target delivery. We have generated an Ad5.3D.A20 vector that is re-targeted to an epithelial cancer-specific marker, αvβ6 integrin, and fully de-targeted from native interactions ‒ αvβ3/5 integrins, coxsackie and adenovirus receptor (CAR), and human coagulation factor 10 (FX). Ad5.3D.A20 selectively transduced αvβ6+ epithelial ovarian cancer (EOC) cells in vitro and clinical ovarian ascites-derived EOC cells ex vivo, including in the presence of neutralising anti-Ad antibodies. In vivo, Ad5.3D.A20 exhibited significantly reduced off-target accumulation and transduction of the liver, spleen and lungs, relative to Ad5. Efficacy studies are underway to investigate its oncolytic potential. Furthermore, we explored the potential use of alternative serotypes from the rare seroprevalence subgroup D. A novel Ad10 vector showed improved resistance from neutralisation and lack of FX binding. Chimaeric Ad5 vectors pseudotyped with Ad10, -15, -24, -29, -48 or -53 fiber had reduced interactions with CAR, but no binding to CD46. Our strategy for translation initially is via intraperitoneal delivery of oncolytic vectors, bypassingmany of the barriers presented by systemic delivery, but enabling transduction of disseminated metastases. These exquisitely tumour-targeted vectors may be further modulated to carry therapeutic moieties to complement their direct cell-killing activity via the stimulation of anti-cancer immunity. The generated tropism-modified vectors have significant therapeutic potential for a wide range of immuno-oncolytic applications.

3',5'-cyclic adenosine monophosphate (cAMP) is an important second messenger molecule involved in various signalling responses. cAMP signalling is balanced by the activity of adenylyl cyclase enzymes which catalyse the formation of cAMP from ATP, and the activity of phosphodiesterase enzymes (PDEs) which represent the only means by which cAMP is degraded. A complex balance of synthesis and degradation is underpinned by the expression of multiple cAMP PDE families and splice variants. This allows the cell type secific targeting of PDE isoforms to discreet intracellular signalling complexes. The outcome of this differential patterning of expression is a dynamic and compartmentalised web of cyclic nucleotide gradients that modulate responses to extra-cellular stimuli. Compartmentalization of signalling cascades and cAMP gradients allow multiple differing signal transduction events to occur simultaneously and controls cAMP signalling in time and space within the crowded milieu of intracellular molecular interaction. The PDE isoform expression pattern within human prostate tumours has never been fully described. I have addressed this by reporting a PDE profile for 10 androgen-sensitive and 9 androgen-insensitive cell lines and xenografts. I describe that the PDE4D sub-family is dysregulated in androgen independent disease. In particular I show that the PDE4D isoforms PDE4D3, PDE4D4 and PDE4D7 are significantly down-regulated. PDE4D7 was observed to contribute the largest degree of expression and most dramatic down-regulation of any PDE4 isoform and was therefore chosen for further study. Within this thesis, I describe my efforts to characterise PDE4D7 in the context of the human prostate. I describe its sub-cellular localisation and show it contributes a significant proportion of rolipram sensitive PDE activity at the plasma membrane of AS VCaP cells. The transcriptional regulation of PDE4D7 was also investigated and I found that PDE4D7 is regulated independently of the androgen signalling axis, and that it's genetic overlap with PART-1 poses intriguing possibilities for further research.

Reactive oxygen species (ROS) are known to play a significant role in cellular signalling pathways, associated with growth, differentiation and survival and in many cancers, elevated ROS levels have been linked with aberrant signalling. Previously, we showed that human haematopoietic progenitor cells expressing mutant RAS (a common abnormality in AML) had elevated NOX2 derived ROS which led to increased cell proliferation. However, it is currently unknown how ROS mediates these effects. Previous preliminary data suggests significant ROS attributable gene changes are associated with glycolysis, a feature also common in solid tumours. Using human haematopoietic progenitor cells, it is reported here for the first time that, elevated ROS leads to changes in extracellular lactate production and increases in glucose uptake. Furthermore, modulation of ROS levels in AML cell lines also generate results consistent with a ROS induced model of increased glycolysis. Gene expression profiling identified ROS related changes in specific glycolytic enzymes, whilst immunoblotting verified ROS dependent increases in protein expression of a key regulatory glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), in both human cells and AML cell lines. Metabolomic analysis using mass spectrometry, was performed on AML cell lines in which ROS levels were manipulated and AML patient samples were characterised according to ROS production. This revealed changes in concentrations of metabolites associated with glycolysis and metabolic pathways important in ROS regulation. Modulation of PFKFB3 expression generated changes in glucose uptake consistent with ROS mediated changes in this enzyme. In summary, this study establishes for the first time that increased ROS production in AML models leads to increased glycolysis and metabolic reprogramming as a consequence of modulation of PFKFB3 expression by ROS.

The overarching aim of this thesis was to gain an increased understanding of the psychological processes related to generalised anxiety disorder (GAD) and how they contribute to the development and maintenance of the disorder. Changes in the diagnostic criteria of GAD over the years, have led to increased identification of specific psychological processes that may be responsible for the disorder, all of which offer a plausible explanation. However, limited empirical research exists exploring the relative merits, or have made direct comparisons of each. Therefore, this thesis attempts to answer the following questions: Which psychological factors contribute to the severity of worry? and do the identified psychological factors explain the development of GAD? Delineation of these psychological processes may hold the key to increased efficacy of treatments, as outcomes are currently poor, which lead to increased health care utilisation and high economic costs to the public health service. Improving treatments is clearly an important factor for GAD sufferers who often experience significant impairment in overall functioning and quality of life. Chapter 2 presents an overview of the relevant literature, which provides a point of orientation for the research section that follows. This will initially offer a historical context, followed by an overview of GAD, the role of negative life events, and finally considers the implications for psychopharmacological and psychological treatments. The evolution of the development of psychological models of anxiety is outlined, which provided the foundation for the development of some of the current leading psychological models of GAD within this field. These have led to the identification of key processes that offer a clear hypothesis and explanation of the phenomena seen in worry and GAD; the most recent models included within this review are the Intolerance of Uncertainty (IU) model (Dugas, Gagnon, Ladouceur, & Freeston, 1998), the Metacognitive model (Wells, 1995, 1999), and finally the Acceptance Based model (Roemer, Salters, Raffa, & Orsillo, 2005). This leads into the final section of Chapter 2, which is a systematic review of the key processes to be explored within this thesis, the processes of interest are IU, negative metacognitive beliefs about worry, and finally experiential avoidance. Chapter 3 leads into the empirical paper, which provides a detailed account of the research and the outcomes. This research attempted to address some of the gaps in the literature by being the first to explore all three of these constructs in one study. The aim of which was to understand more about what factors are related to the prediction of worry severity and GAD status in a non-clinical sample. Additionally, this study attempted to address some of the limitations of previous literature by using a prospective design, which allowed inferences on causality to be made. Students were recruited and completed the study via a web-based design, completing measures at two time points. The findings of this research are presented with an overall discussion of how this relates to previous research. These are discussed in the context of several limitations. In the final chapter, the implications of the research are outlined, with reference to the theoretical and clinical relevance. In addition, methodological considerations are highlighted, including the relative strengths and limitations of the research. As the dissemination of research findings is an important process for any research, the next section is an article prepared for those participants who took. The final section relates to how future studies can continue to bridge the gaps within the literature, this is outlined in the form of a research proposal. Further empirical research is required within this field, specifically; replication of the current study within clinical samples would further and extend the findings presented in Chapter 3. The thesis then closes with an overall conclusion.