Objective: The present study sought to investigate the extent and role of health anxiety, mental defeat, self-compassion and health related coping behaviours in relation to fear of cancer recurrence (FCR), psychological distress amongst breast cancer survivors, according to whether or not they had had chemotherapy as part of treatment. This comparison was carried out to build upon previous research findings that have pointed an especially adverse psychological impact of chemotherapy treatment. Methods: A total of 77 breast cancer survivors were recruited, and completed questionnaires in order to investigate whether more intensive treatment (i.e. including chemotherapy alongside radiotherapy and surgery) will be associated with higher levels of health anxiety, mental defeat, fear or recurrence, and psychological distress. Results: Group comparison demonstrated significantly higher levels of health anxiety, FCR, mental defeat and psychological distress amongst patients who have undergone more chemotherapy. Multiple regression analysis indicated that health anxiety, psychological distress and age, were the strongest predictors of fear of recurrence; while mental defeat and fear of recurrence were strongest for psychological distress. In addition, mental defeat was inversely associated with higher self-compassion. Conclusions: These results suggest that FCR, health anxiety and mental defeat is significantly elevated amongst breast cancer survivors, in particular those who have undergone chemotherapy, and is associated with increased levels of longer-term psychological distress. Cognitive-behavioural interventions, which target these variables, should be evaluated in these patients.

Mycobacterium tuberculosis is the causative organism for one of the pandemic diseases in the world, tuberculosis (TB). The length of treatment often results in multi-drug resistance (MDR) and patient non-compliance. One of the most important enzymes as a drug target for tuberculosis is dihydrofolate reductase (DHFR), which plays an important role in the folate cycle and inhibition of the enzyme stops cell growth. DHFR inhibitors are usually 2,4-diaminopyrimidines, which have high binding affinity to the enzyme but have the potential to inhibit the human enzyme. This project focuses on the development of new inhibitors with improved potency and selectivity for the M. tuberculosis enzyme. Inhibitors containing a 5-phenyl group were targeted in order to increase lipophilicity and binding to the enzyme, whilst reducing binding to the human enzyme. Condensation of diethyl phenylmalonate with guanidine followed by chlorination and amination of the carbonyl group gave 2-amino-6-chloro-4-pmethoxybenzylamino- 5-phenylpyrimidine, which was deprotected to give 6-chloro-2,4- diamino-5-phenylpyrimidine. Reaction of 2-amino-6-chloro-4-p-methoxybenzylamino- 5-phenyl-pyrimdine with different aromatic and aliphatic amines was also investigated. Amination with an amino alcohol in presence of potassium carbonate in the absence of solvents was used to synthesise a number of analogues. Deprotection of the pmethoxybenzylamine was achieved by DDQ oxidation to give the desired 2,4-diamino- 5-phenyl-6-aminoalcohol-pyrimidine products. The synthesis of the triol motif began with protection of ribonolactone and reduction of the carbonyl group to give the diol. Derivatisation of the product with various protecting goups was investigated.

The term ‘Lymphoma’ encompasses a diverse group of malignancies, which require very different approaches to ensure optimal patient outcomes. However, even within individual lymphoma types tumour behaviour and patient responses remain highly variable. To some extent this is influenced by patient specific factors (e.g. age, fitness, comorbidity), but there remains significant heterogeneity of tumour biology, which is not adequately identified by standard diagnostic techniques and expert pathology review. This fellowship investigated novel diagnostic techniques in lymphoma in an attempt to identify clinically relevant tissue- based factors, which could improve disease classification and characterise the underlying tumour biology more objectively. Two novel techniques were developed in lymphoma subtypes which best suited the technological platform. Firstly, gene expression-based classifiers were developed for aggressive B-cell lymphoma, using RNA extracted from formalin-fixed, paraffin-embedded (FFPE) biopsy samples. Secondly, multiplexed immunofluorescence and digital image analysis were leveraged to characterise immune checkpoints and the tumour microenvironment (TME) of classical Hodgkin lymphoma (CHL). In the first validation series of patient samples (n=55) a diagnostic molecular classifier was able to classify 92% of tumours correctly as molecular Burkitt lymphoma (mBL) and molecular diffuse large B-cell lymphoma (mDLBCL), with a sensitivity and specificity of 1, using pathological diagnosis as gold standard (15% of tumours classified as ‘molecular intermediate’). A classifier of ‘MYC activity’ predicted clinical outcome following R-CHOP chemotherapy in a small subgroup of patients, with a sensitivity I specificity of 0.771 0.83, using MYC IHC as gold standard.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) functions in the non- homologous endjoining (NHEJ) pathway of DNA double strand break (DSB) repair and is overexpressed and/or highly active in several cancers, including chronic lymphocytic leukaemia (CLL). CLL treatment options have improved substantially, however, prognosis remains poor for patients with mutations in TP53, or with dysfunctional ataxia telangiectasia mutated kinase (ATM), a key protein in the homologous recombination repair (HRR) pathway of DSB repair. Pharmacological inhibitors of DNA-PKcs have been shown to sensitise CLL cells to DNA damaging drugs ex vivo. In this thesis, DNA-PKcs was further explored as a therapeutic target in CLL using a novel, highly selective inhibitor of DNA-PKcs (NDD0004). NDD0004 sensitised HCT116 cells, but not DNA-PK-deficient HCT116 cells, to etoposide and mitoxantrone. Mechanistic studies showed that NDD0004 prevents NHEJ activity in vitro. Chemosensitisation to mitoxantrone was also observed in primary CLL samples ex vivo in viability and apoptosis assays. Chemosensitisation was highly variable (median 49-fold with NDD0004, range 1-2000-fold) but occurred in 80% of samples, including those from poor prognosis patients. When co-cultured with CD40L-expressing fibroblasts, CLL cells began to proliferate, but became resistant to mitoxantrone-induced apoptosis. There was little evidence of chemosensitisation by NDD0004 on healthy donor lymphocytes or bone marrow cells, indicating a potential therapeutic window for the clinical use of DNA-PKcs inhibitors. It was hypothesised that ATM-deficient CLL cells or cell lines would be sensitive to DNA-PKcs inhibition due to a synthetic lethal interaction between the loss of both DSB repair pathways, however, this was not the case in four cell line pairs tested. This work has characterised a novel DNA-PKcs inhibitor, and contributed to the understanding of the therapeutic benefits and limitations of targeting DNA-PKcs in cancer therapy.

Background - T-cell based immunotherapy is the greatest recent breakthrough in cancer treatment, and can induce complete lasting remission. T-cells are capable of responding to a vast diversity of antigens via their hypervariable T-cell receptor (TCR). However, current immunotherapies rely on αβ T-cells which are restricted to person-specific Human Leukocyte Antigen (HLA) molecules presenting peptides from cancer-specific antigens. Thus, a given αβ TCR therapy is applicable only to a minority of patients. In contrast, γδ T-cells, and some αβ T-cells, recognise diverse cancer types regardless of the HLA type. The aims of my thesis were to investigate the potential of using non-HLA restricted T-cells and their receptors for cancer immunotherapy, and to develop tools to facilitate the study of non-HLA restricted T-cells for cancer treatment. Results – Initially, I developed a CRISPR/Cas9 method for generation of superior TCR transduced cells, in terms of their anticancer reactivity and antigen sensitivity, in comparison to TCR transduced cells generated by current clinical methodologies. Using this TCR replacement method I demonstrated that the anticancer reactivity of broadly cancer-reactive γδ T-cells derived from a variety of clinically relevant sources is dependent on their TCRs. I also used CRISPR/Cas9 genome editing to generate a panel of cancer cell lines deficient in known ligands of non-HLA restricted T-cells that can be used for initial dissection of their anticancer reactivity. Using this approach, I demonstrated that one of non-HLA restricted T-cell clones I procured recognised targets via CD1a. Finally, I developed a whole genome CRISPR/Cas9 pipeline for discovery of ligands and pathways essential for cancer cell recognition by non-HLA restricted T-cells. Conclusions – My research demonstrated that TCRs from broadly cancer-reactive T-cells can be used to re-direct primary T-cells to many cancer types regardless of their HLA type, paving the way for pan-population immunotherapy. The discovery of non-HLA ligands for broadly cancer-reactive T-cells can be achieved using whole genome and targeted CRISPR/Cas9 gene editing technology.

Tuberculosis (TB) is an infectious disease mainly transmitted to humans by the inhalation of infected droplets (produced when an infected person coughs or sneezes). It is caused by bacteria within the Mycobacterium tuberculosis complex, several species of which can cause infection in humans. In the early 1990s, the World Health Organisation (WHO) declared TB a global emergency and this continues to be the case today. We seem to be further from eradicating this killer disease than we have been at any point in our past, and the increase in global travel, including migration, is thought to be exacerbating its spread. Building on previous projects that extracted and analysed ancient DNA of M. tuberculosis complex organisms from skeletons with bone changes consistent with TB from the Roman period in Britain, this research tests the hypothesis that people buried in Roman Britain who were infected with TB had been mobile at some point in their lives, by the application of stable isotopic analysis (C, N, Sr, O) to the same skeletons to establish if their childhoods were local or non-local to their burial locations. This study uses bone and dental samples from skeletons from the ancient DNA projects who were buried on chalk geology and with bone changes suggesting possible TB and/or a positive TB ancient DNA result. The sites investigated were Driffield Terrace in York, Baldock, Easington, Winchester, Cirencester and Poundbury. Collagen was successfully extracted from bone for 19 out of 21 individuals. Carbon and nitrogen isotope analysis revealed that all but three of these people ate a diet based on C3 terrestrial ecosystems with limited aquatic food intake, and they were similar to other people buried in the same or other contemporary cemeteries. Enamel from the teeth of all 21 individuals was also subject to strontium and oxygen isotope analysis, which identified six people as not having been brought up in the local area where they were buried. The remaining 15 people were possibly raised locally, although other places of origin have been considered. It was concluded that linking mobility, as identified using stable isotope analysis, with transmission of infectious disease evidence in the skeleton is very challenging, particularly because there is no way of knowing how long people had been infected with the disease before or after they were mobile. Finally, some suggestions of how to take this important work forward were made. This includes repeating the work on an available larger sample size of possible TB sufferers without constraints of only testing those individuals buried on chalk geology.

The cytoskeleton plays vital roles in many cell functions and any disorder could lead to different diseases such as cancer, which causes uncontrolled cell division, cell motility and invasion. Cell migration is important for developmental morphogenesis, tissue repair and tumour metastasis. During the migration process, microtubule organisation and dynamics play important roles, and are regulated by Plus-end Tracking (+TIP) proteins. A better understanding of cell migration mechanisms could lead to an efficient treatment for invasive cancer cells such as pancreatic cancer. Sulforaphane (SFN) is a potential treatment for different cancer types with various possible means of action. Studies have indicated that SFN has the potential to slow down the progression of cancer, promote apoptosis and suppress cell proliferation. However, how SFN affects microtubule dynamics, stability and organisation is poorly understood. This project investigated the effect of SFN on microtubule organisation and dynamics and the impact on cell migration. Certain pancreatic cancers show up-regulation of the end-binding protein EB2 and the tubulin deacetylase HDAC6 which both influence microtubule dynamics and stability and thus also cell migration. Other cancers such as breast cancer have reported an up-regulation in EB1. This project therefore focused on the effects of SFN on EB1, EB2 and HDAC6 expression and localisation and the consequences for cell migration. The impact of SFN on normal (ARPE-19) and cancerous (Panc-1) epithelial cell migration was assessed by live time-lapse imaging of sparsely seeded cells. Microtubule and actin filament organisation was assessed to observe the effects of SFN on the treated cells. The impact of SFN on EB and HDAC6 localisation and expression was determined by immuno-labelling. Experiments involving SFN treatment with or without functional inhibition of HDAC6 (with the HDAC6 inhibitor tubacin) were carried out and their effect on cell migration was investigated. The effect of SFN on microtubule dynamics and stability was assessed by analysing live time-lapse GFP-CLIP-170 dynamics. Microtubule post-translational modifications were studied by immuno-labelling in SFN treated cells. Focal adhesion area and dynamics were also assessed by FRAP and immuno-labelling in SFN treated cells. SFN treatment caused a dramatic decrease in the speed of random migration and cell area in ARPE-19 cells. A marked co-alignment between microtubules and actin filaments, increased EB1 decoration of the microtubule lattice and an apparent increase in cytoplasmic EB2 were also observed upon SFN treatment. However, SFN treated Panc-1 cells revealed no significant decrease in the speed of random cell migration. Interestingly, a combination of tubacin and SFN caused a significant decrease in the speed of cell migration. Analysis of microtubule dynamics in GFP-CLIP-170 expressing cells revealed that SFN treated cells possessed less dynamic microtubules. There was also evidence of SFN inducing microtubule stability. The results suggest that SFN or a combination of tubacin and SFN could be promising treatments for cancer. Moreover, these results can provide a better understanding of the effects of SFN on the organisation of the cell cytoskeleton. Treatment with SFN resulted in interesting changes in EB and HDAC6 localisation, which may provide potential targets for effective cancer treatments.

Precise control of proliferation and apoptosis is essential for the prevention of cancer. Pathways involving p53, p21, and RB are central to the regulation of these processes, and are dysregulated in almost all human cancers. This thesis describes an investigation into the effects of inactivating components of the p53-p21-RB pathway in order to better understand the mechanisms underlying its role in preventing cancer. Hepatocytes isolated from transgenic mice bearing mutations in p53, p21, Rb, and combinations of all three, were employed to provide insight into the pathways controlling hepatocyte proliferation and apoptosis. In the case of the Rb mutation, Cre/LoxP conditional gene targeting was used to overcome the problem of embryonic lethality of Rb knock-out. In vitro Rb gene deletion mediated by adenovirus-Cre was successfully developed and characterised. This revealed that adenovirus-Cre-mediated gene deletion is extremely efficient in hepatocytes and that adenoviral infection had no affect on cell viability or growth. Expression of Cre, however, stimulated DNA synthesis while expression of LacZ adversely affected viability at higher MOI. These findings underline the need for careful characterisation of gene deletion systems employing both adenovirus and Cre recombinase. Analysis of hepatocytes deficient in p53, Rb, and p21, revealed severe defects in proliferation accompanied by an increase in ploidy, and nuclear and mitotic abnormalities. Analysis of hepatocytes deficient in combinations of all three genes provided insight into the level of interdependency between these genes, suggesting that there are two major pathways regulating hepatocyte proliferation: a p21-RB pathway that operates independently of p53, and a p53-p21 pathway that operates independently of RB. The p53-p21-RB pathway is critically important in regulating cellular responses to stress. The role of p53, p21, and RB in regulation of hepatocyte responses to DNA damage was investigated. It was found that each of these proteins is required for the maintenance, but not initiation, of UV-induced arrest. Both p53- dependent and -independent pathways of UV-induced apoptosis exist, and loss of Rb or p21 increased susceptibility to p53-independent, but not p53-dependent, apoptosis. In conclusion, the work presented in this thesis demonstrates that adenovirus-Cremediated gene deletion is extremely efficient in hepatocytes. It also shows that p53, p21, and RB are critical in regulating proliferation and apoptosis both in the absence and presence of exogenous stress, underlining the importance of these proteins in regulation of cell growth and hence presumably in prevention of cancer.

Late normal tissue injury is dose-limiting for radiation therapy of cancer. The molecular mechanisms of this injury are unknown. However, almost all radiosensitive animals and cell lines are deficient in some aspect of DNA repair. We have derived EBV-transformed cell lines from five patients with late radiation injury to determine whether there was any evidence of reduced activity or expression of the enzymes active in non-homologous recombination, the major mammalian repair pathway for DNA double-strand breaks which cause radiation-induced cell death. Two of these cell lines exhibit post-radiation viability intermediate between normal controls and a cell line from an individual with ataxia-telangiectasia. DNA-dependent protein kinase activity in vitro was reduced 8-10- fold in these two cell lines compared to normal controls. The primary tumours from one of these patients, and a post-radiation cervix biopsy form the second, exhibited no immunoreactivity with a polyclonal antibody against the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Immunoblotting showed normal levels of Ku70, Ku80 and XRCC4, and the presence of DNA-PKcs, in both cell lines. This suggests that the DNA-dependent protein kinase might be an important factor in determining the predisposition of radiotherapy patients to late radiation injury.

Background: Cancer-induced bone pain (CIBP) is a major clinical problem and a considerable therapeutic challenge. Radiotherapy (XRT) is the gold standard treatment for CIBP, but only half of patients achieve adequate analgesia. Patients have increased morbidity, anxiety and depression and reduced performance and quality of life. Despite these issues, CIBP is a neglected area of clinical research. Animal models have increased current knowledge of the pathophysiology, but clinical research is needed to translate these findings from bench to bedside. Also lacking is a standardised, comprehensive tool to assess CIBP and clinical biomarkers to predict analgesic response to treatment. Aims: 1) To summarise current understanding of the pathophysiology, epidemiology, clinical features, assessment and management of malignant bone disease and CIBP. 2) To characterise CIBP using quantitative sensory testing as a measure of altered sensory processing. 3) To establish systematically the sensory, cognitive, affective and functional components of CIBP to develop a comprehensive assessment tool. 4) To explore whether clinical biomarkers can be developed to aid prediction of response to treatment for CIBP, in particular XRT. Results: Assessment of CIBP, characterising the multi-dimensional components, was clinically practical and acceptable to patients. Using objective measures of function, patients with CIBP were a frailer, less active population compared with healthy adults. Prior to treatment, pain was severe with relationships seen between CIBP and sensation, mood, fear avoidance, catastrophizing and function. Patients who dropped out prior to follow up were significantly less active, with higher levels of depression and fear avoidance behaviour. Sixty-nine percent of evaluable patients who completed two assessments (48% of all patients on an intention-to-treat basis), achieved an analgesic response to XRT for CIBP, as defined as an improvement of ≥ 30% in the Brief Pain Inventory worst pain score two months after treatment. All dimensions of pain, fear avoidance and catastrophizing improved significantly in responders, but not non-responders. Anxiety, depression and emotional distress fell by a greater degree in responders. No objective functional differences were seen after XRT. Clear evidence of altered sensory processing was seen at the site of CIBP with abnormalities in both mechanical and thermal parameters. XRT resulted in alterations in response to evoked stimuli in responders with a greater number of patients in whom sensation normalised after XRT compared with non-responders. Patients with a combination of altered sensation to thermal, pin prick and wind up stimuli showed the largest reduction in worst pain after XRT. Abnormal cool sensation at the site of CIBP was an independent predictor of analgesic response to treatment. Conclusion: Strong associations exist between CIBP, sensation, cognition, mood and function. Multi-dimensional assessment should be performed to improve quality of life. Translational research to provide targeted individualised treatment should be high on the research agenda. Future work should focus on thermal sensory processing as a potential clinical biomarker of response to palliative XRT for CIBP.