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Circulating tumour DNA (ctDNA) can be used to profile, detect, and monitor cancers. Technical advances in next-generation sequencing (NGS) strategies to address limitations in ctDNA detection and the precise classification of lymphoid cancers, using mutational features and bioinformatics tools, may help diagnosis and have predictive value for treatment. Despite these advances, application of ctDNA in current clinical practice is absent, with translational studies yet to be carried out in the U.K. for patient stratification. This study describes the development of an effective NGS workflow, comprising design of a pan-lymphoma, targeted panel focusing upon molecular characteristics specific to B-cell and T-cell malignancies for profiling ctDNA, coupled with custom bioinformatic workflow 'Deep-sequencing and Evaluation of Lymphoid Variant Effects' (DELVE). DELVE enables detection of aberrant somatic hyper-mutation (aSHM) sites including immunoglobulin heavy chain locus (IGH), fusions and clonal immunoglobulin repertoire detection. Clinical utility of ctDNA and mutational profiling by pan-lymphoma targeted panel/DELVE were investigated in Diffuse Large B-cell Lymphoma (DLBCL), primary cutaneous diffuse large B-cell lymphoma (PCDLBCL-LT) and peripheral T-cell lymphoma (PTCL). A DLBCL discovery cohort of six patients comparing FFPE and baseline ctDNA was followed by a validation cohort and longitudinal analysis during treatment. 14/15 DLBCL exhibited detectable ctDNA at baseline. Sequencing of tissue and plasma revealed significant aSHM, with detectable HMRN and LymphGen classifications. In a case of PCDLBCL-LT, limitations in use of ctDNA to monitor treatment response were evidenced with no detectable ctDNA throughout clinical course, including following 2 systemic relapses and ctDNA was detectable only when the patient was treated with BTKi. Finally, in PTCL (n=6), proof of principle and comparison with Ion Torrent sequencing was performed. This work demonstrated the feasibility of using a pan-lymphoma targeted panel and the DELVE pipeline to profile tumours and monitor residual disease and set the basis for larger scale studies.

Objective: To develop a novel Endometrial Cancer Patient-Derived-Explant (EC-PDE) preclinical model system that is capable of detecting patient-specific drug-responses to standard-of-care chemotherapies and immunotherapy ex vivo. Methods: Endometrial tumour was obtained from 35 patients with endometrial cancer and processed into explants. EC-PDEs were then cultured at the air-liquid interface for up to 24 h followed by a further 24 h treatment with Carboplatin and Paclitaxel or Pembrolizumab and then processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed for viability studies and for CD8, CD4 and FOX-P3 antibodies for immune profiling studies. Images were then analysed with quantitation of biomarker expression and necrosis area. Results: EC-PDEs maintained the histological architecture of the tumour and surrounding tumour microenvironment. EC-PDEs remained viable for up to 48 h irrespective of culture-media or serum supplementation. Differential drug-responses were detected to single- and dual-agent chemotherapy with the greatest drug-responses identified in high-risk tumours. Cell-death-responses were also identified following Pembrolizumab-treatment. Immune cell profiling in the tumour and stroma was suggestive of an immunosuppressive effect of T-regulatory cells whilst the greatest drug-responses were seen in tumours with a CD8hot phenotype. Conclusions: EC-PDEs represent a low-cost 3D pre-clinical model which offers the potential for rapid, personalised pre-clinical drug-response testing. EC-PDEs can be used to explore the biological effects of immunotherapy and to evaluate predictors of drug response.

Background: Cancer is a process whereby the accumulation of mutations leads to a clonal expansion of cells, forming a tumour mass with the capacity of expanding to surrounding tissues. Understanding how these mutations come to be in the first place is important from an epidemiological and evolutionary point of view. Several external - or exogenous - factors, such as UV light, tobacco smoke, or ionising radiation, have the capacity to create mutations. Moreover, the process of DNA replication which is necessary to create two cells from one is, like any other copying mechanism, not entirely faithful. Therefore, spontaneous - endogenous - mutations are created each time the genetic material of a cell is replicated. Some mechanisms of mutation lead to more mutations than others, and can create them either with varying intensity or at the same rate throughout life, or even throughout tumour development. Mutational signatures were introduced as a proxy to quantify the number of mutations created by each mutational process. This thesis focuses on statistical methods for the analysis of such mutational signatures. Crucially, mutational signatures, in the type of questions that I address, have a characteristic: they are compositional data, because we are interested in studying their relative contribution to the total mutation load. Because of this, they have to be analysed in a multivariate way and in relative terms. Approach: I introduce appropriate compositional models to analyse two types of mutational signature: single-nucleotide polymorphism signatures, and copy number signatures. The Dirichlet-multinomial mixed effects model for single-nucleotide data is shown to have good sensitivity compared to existing fixed-effects alternatives, and higher specificity than compositional models that do not allow any overdispersion. Similarly, the incorporation of random intercepts in the logistic-normal model for copy number data increases sensitivity with respect to the fixed-effects version. The models are publicly available on github and are readily applicable to other types of compositional data. Results: Firstly, I use the mixed-effects Dirichlet-multinomial model to characterise the differential abundance patterns between clonal and subclonal mutations across 23 cancer types of the PCAWG cohort. There is ubiquitous change, which can be detected already at nucleotide level. There is higher dispersion - higher variability between samples - of signatures in the subclonal group, indicating, possibly, the presence of di↵erent clones with distinct active mutational processes. The signatures of clearest differential abundance are signatures of low abundance, many of them with the tendency to be, to some extent, the result of bleeding from other signatures, and of unknown aetiology. Although we should be wary of these signatures, differential abundance persists despite excluding them, and the relative changes between clonal and subclonal mutations, in the form of ranked coeffi cients of the signatures of highest confidence, are robust to the subset of signatures used. Secondly, I explore the use of similar models for the study of copy number signatures, and to answer three questions about the mutation dynamics in high grade serous ovarian cancer: whether the relative contribution of mutational processes changes from early-stage to late-stage samples, whether it changes from diagnosis to relapse, and whether it changes from whole-genome-duplicated (WGD) to non-whole-genome-duplicated samples. The CN signature landscape differs significantly between early and late stage samples in that there are much higher rates of WGD in late samples. However, there is no noticeable coordinated difference between matched archival and relapsed samples, although a few patients experience WGD between archival and relapse. Overall, the results indicate that there are large levels of heterogeneity in copy number signatures between patients, but less so within patients, with the exception of punctual cases, and therefore suggest that, although the first response to therapy is dictated by the mechanisms of repair, the relapse occurs not at the level of the genome at 0.1x resolution. Moreover, by use of a Support Vector Machine, I show that copy number signatures can be used to categorise WGD from non-WGD samples at 95% accuracy, using two independently labeled cohorts of TCGA and ICGC samples. Outlook: This thesis introduces the use of compositional models to study the dynamics of mutational signatures in the comparison of two groups of samples, and they can be readily applied to several other regression settings in this discipline or others in which compositional data arises. From both the copy number and point mutation standpoint the models indicate that signatures are dynamic. Further work is needed to better elucidate which mutational signatures are behind the changes, and which mutational processes are behind the signatures. Besides the biological insight into DNA mutation and repair, these results have potential clinical relevance, as cancer treatment often targets or takes advantage of impaired mechanisms of repair.

Colorectal cancer (CRC) is the fourth most common cancer in the UK. Recently, the metabolism of non-essential amino acids in cancer has been identified as an area of interest for novel therapeutic targets. Of particular interest here, the metabolism of proline has been linked with cancer progression in a number of malignancies. The final step of proline biosynthesis is catalysed by pyrroline-5-carboxylate reductases (PYCRs) which utilise NAD(P)H as a cofactor. PYCR1 and PYCR2 are mitochondrial isoforms whilst PYCR3 is localised to the cytoplasm. PYCR1 has been reported to be upregulated in a number of cancers including CRC. Here, evidence is provided to confirm the role of PYCR1 in CRC using both RNA interference and CRISPR/cas9 technologies. siRNA knockdown resulted in decreased cell growth with increased cell cycle arrest and apoptosis. Supplementation with proline and nucleotides indicate that neither biosynthesis of proline, nor the production of nucleotides are limiting in PYCR1-depleted cells. Evidence is also provided to indicate an increased role of PYCR2 than previously thought with analysis of online datasets showing a correlation between PYCR2 expression and reduced survival in stage IV CRC. This suggests a role for PYCR2 in metastatic cancers. Overall, this thesis indicates that the proline metabolic pathway is a suitable target for novel CRC therapies. In particular, this thesis provides a novel role for PYCR2 in CRC which should be investigated further.

There is an urgent need for improved treatments and therapies for advanced stage lung cancer, and one promising strategy is to directly target hallmark phenotypes mediated by translational dysregulation. Stress-related dysregulation of mRNA translation occurs as a result of the phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF2). While stress-related dysregulation of mRNA translation has been well-characterised in cell-based models, there is limited understanding of the effects of phosphorylated eIF2a (p-eIF2a) in primary human tumour tissue, or of whether targeting translational dysregulation is a viable strategy. This project aims to investigate the extent to which stress-related dysregulation of mRNA translation occurs in human lung adenocarcinoma and if it contributes to the virulence of the disease. To address this, immunohistochemistry and in situ hybridisation methods were used to quantify markers of the integrated stress response pathway and translational control in 1025 primary human lung adenocarcinoma patient cases, statistical analyses were performed to investigate the associations between translational dysregulation and patient outcome, tumour hallmarks, and between various elements of the stress signalling pathway. It was found that the phosphorylation of eIF2a occurs in areas of hypoxic stress, despite the lack of known mechanism to mediate this. The phosphorylation of eIF2a was found to be strongly associated with aggressive tumour hallmarks, namely invasiveness, metastasis, and cellular proliferation, as well as poor clinical outcomes. Furthermore, the lethal phenotype associated with p-eIF2a was not significantly linked either to the previously characterised downstream mediator, ATF4, or by the abundance of subunits of eIF2B, the functional binding partner of eIF2a. Stress signalling through eIF2, likely mediated by regional hypoxia, is predictive of poor prognosis in primary lung adenocarcinoma, and that targeting mRNA translational dysregulation through the phosphorylation of eIF2a may be clinically valuable in preventing tumour invasiveness and metastasis.

CRC is the third most common cancer worldwide and is responsible for 8% of all cancer deaths. 60-80% of CRCs arise through a well-understood adenoma-carcinoma sequence characterised by APC mutations and upregulated Wnt signalling, whereas 15-30% arise through an alternative, less characterized "serrated" pathway often harbouring BRAFV600E mutations, with distinct clinical and morphological features and worse prognosis. Treatment options are limited, necessitating development of new therapeutic and preventative strategies. Genetically engineered mouse models have been developed to study BRAFV600E-mutant CRCs. However, in this context BrafV600E proved to be a poor oncogene, with few lesions developing after long latency. Most studies analysed the short-term impact of BrafV600E activation; instead, our objectives were to examine histological and transcriptomic features of intestinal tissue expressing BrafV600E for up to 6 months, for comparison with short-term BrafV600E induction (3 days) and with human data. We hypothesised that this approach could identify previously uncharacterised changes to be exploited in prevention and/or therapy. We show that long-term BrafV600E expression triggers hyperplasia and decreases gut barrier integrity. The number, distribution, and transcriptional signatures of differentiated epithelial cells were also affected without major changes to intestinal stem cells. Transcriptional analysis showed BrafV600E increased proliferation and Wnt signalling signatures, whereas inflammatory signatures were downregulated. Cholesterol homeostasis increased after BrafV600E expression, which was also reflected in human serrated adenomas. However, inflammatory pathways showed an opposite trend and were upregulated in adenomas. This suggests lack of inflammation limits tumour development in mutant mice, and that metabolic reprogramming sustains BRAFV600E CRCs. Indeed, in vivo inhibition of cholesterol synthesis using statins reduced crypt hyperplasia, suggesting cholesterol metabolism could be targeted to prevent serrated neoplasia. Overall, these results provide novel insights into the effects of BrafV600E expression on the intestinal epithelium, unmasking transcriptional reprogramming and highlighting pathways to be exploited for preventative or therapeutic interventions.

The time frame in which people recognise a bodily change as a potentially cancerous illness symptom and seek professional healthcare advice has been a topic of research from the 1970s. Exploring and understanding these processes are important especially when considering the earlier diagnosis of cancer. Previous research has examined the role of several factors which may influence these processes including differing social, psychological, and psychosocial factors. However, little is known about these factors in specific reference to bodily changes. Therefore, the current research aimed to investigate the role of symptom perception/ interpretation, emotional coping responses, and lay referral in the context of the time frame between symptom recognition and professional healthcare help-seeking behaviour. An exploratory approach was taken, with the presentation of the historical literature, and a scoping review which mapped the evolution of the theoretical conceptualisation of lay referral and demonstrated the psychosocial nature of lay referral including how professional healthcare help-seeking is potentially influenced through this process. A cross-sectional, inductive, idiographic Interpretative Phenomenological Analysis (IPA) study was conducted to explore the experiential and lived experience of bodily changes, symptom recognition, emotional coping responses, and lay referral. The findings highlighted the significant influence that people's previous interpersonal experiences with their family and close relationships had on how they make sense of any ambiguous, fleeting bodily changes, emotional regulation, relational-affect, lay referral, and help-seeking behaviour. An explanatory integrated theoretical framework was proposed to aid the interpretation of the findings. This framework encompassed The Common Sense Model, Interpersonal Cognition, and Adult Attachment Theory. A contemporary concept of lay referral was also discussed in connection to the study findings. The results of the IPA study informed the design of the mixed and multi-methods study. Next, a mixed and multi-methods study took an integrated concurrent triangulation pluralistic comparison design using an online survey, the story completion task, photo elicitation, and template analysis. This study examined the influence of illness perceptions, personal factors, and adult attachment style on people's thoughts, emotions, and coping responses; whilst also exploring how adult attachment style influences people's narratives concerning lay referral and consultation behaviour in the context of experiencing bodily changes during a world pandemic i.e. SARSCov2/ COVID19. The results supported previous literature in connection to the influence of a pre-existing health condition on illness perceptions and in finding gender differences in lay referral. However, it was through the triangulation of the abductive qualitative analysis and quantitative results that a more in-depth understanding arose. The results exemplified how all of the participants perceived a low to medium health threat with the majority engaging with lay referral in response to their bodily changes and some gaining professional healthcare advice. However, participants' perceptions of the experiential aspects of lay referral and help-seeking during the pandemic presented certain similarities whilst also showing differences when compared across the different adult attachment styles. The thesis concludes with a general discussion of the different study findings and how these have contributed to current theoretical understanding of how the perception and interpretation of bodily changes influence emotional coping responses, presents a relational conceptualisation of lay referral, and highlights implications for cancer related policy, suggested interventions, and potential future directions of research.

Introduction: Bowel cancer is the fourth most common type of cancer in the UK. Approximately 55% of patients are diagnosed with a late-stage tumour (Stage 3 and 4), where likelihood of survival is reduced: five-year survival is 93% at Stage 1 vs. 10% at stage 4. Early detection is crucial. The full blood count (FBC) blood test is commonly performed in primary care. It may help but currently there is limited evidence on how to utilise this test for early detection. Methods: I performed four research objectives. I reviewed relevant research studies in the literature to learn about the relevance of the FBC and eliminate some key uncertainties in existing approaches for early detection. I performed a data quality and validation assessment of electronic health record (EHR) patient data. I then performed longitudinal analyses to identify patterns in the FBC over time, which could help identify presence of bowel cancer. I subsequently incorporated my findings into a dynamic prediction model that could facilitate earlier detection and improve survival rates. Results: My systematic review identified many relevant FBC parameters for bowel cancer detection, such as haemoglobin, mean corpuscular volume, and platelets, and that all existing FBC-based prediction models are flawed. Using primary care data from the Clinical Practice Research Datalink, I identified relevant trajectories in many FBC parameters in patients with bowel cancer, not observed in patients without. Relevant differences in FBC levels occurred for years before diagnosis, suggesting the FBC could facilitate earlier detection. I incorporated the trajectories in haemoglobin, mean corpuscular volume, and platelets into multivariate joint models of longitudinal and time-to-event data to identify two-year risk of diagnosis. My joint models perform well based on my initial testing. Discussion: My joint models use routinely available data and would not require additional work for healthcare workers or patients. These models perform well based on my testing, but further testing is required before use in general practice. My models could facilitate earlier detection and improve survival rates.

In solid tumours, metastasis is the immediate cause of most cancer-related death and is orchestrated by epithelial-to-mesenchymal transition (EMT) of tumour cells. EMT manifests as the dedifferentiation of epithelial cells into mesenchymal cells, and is typified by loss of E-cadherin and cytokeratin and gain of TWIST, N-cadherin and vimentin expression. While EMT is well-characterised in in vitro models, classical morphological EMT evidence (i.e. morphological dedifferentiation/sarcomatoid change) is rare in primary tumours. mRNA translation is a highly regulated process which has been linked to EMT in cell-based studies but not in primary tumours. This project aims to investigate whether I can find evidence of EMT and of translational dysregulation in EMT in primary lung adenocarcinoma. To answer these questions, we applied histological techniques such as immunohistochemistry and in situ hybridisation methods to quantify markers of EMT and translational control and perform statistical analysis to 942 human primary lung adenocarcinoma cases to evaluate association of EMT with tumour growth pattern, physio-pathological factors and translation dysregulation. I have shown that partial EMT state is detected in all tumour growth patterns but there is an association between partial EMT and primary solid lung adenocarcinoma. EMT can be variable within whole tumour section. EMT markers are strongly associated with physio-pathological measures of invasiveness namely lymph nodes metastasis. Furthermore, there are significant associations between EMT markers and some translation factors, particularly with the phosphorylation of eukaryotic initiation factor (eIF) 4E and the α subunit of eIF2. These data suggest that partial EMT is related to solid tumour growth pattern, and that alterations in the translation initiation machinery may play a role in activating invasive and metastatic program simultaneously in human primary lung adenocarcinoma.

Ovarian cancer is the most lethal gynaecological malignancy. Of the four ovarian cancer subtypes - serous, mucinous, endometroid and clear cell - serous ovarian cancer is the most common, comprising around 70% of cases. The median stage of diagnosis of ovarian cancer is stage III and patients present with widespread metastasis, usually facilitated by peritoneal fluid accumulation in the abdomen (ascites). Live single cancer cells and aggregates from the ascites represent a population with the potential to become metastatic and are thought to be the main contributors to disease recurrence, displaying increased chemoresistance after traditional first line therapy, which consists of extensive tumour debulking followed by combination platinum and taxane chemotherapy. For this reason, research into potential therapeutic targets in this important cell population is critical for development of more efficacious treatments for ovarian cancer. The opportunity to re-purpose existing pharmaceuticals for use as chemotherapeutics is an idea that has gained traction recently in the medical research field. This method circumvents the requirement for lengthy target identification and validation and extensive toxicity testing. One class of drugs that are a possible candidate for use against ovarian cancer are the dihydropyridines, which are antagonists of voltage-gated calcium channels (CaV). In ovarian cancer, CaV have already been shown to play a role in malignant behaviours such as migration and proliferation, although most expression and functional data in the literature are from ovarian cancer cell lines. There has been very little research on the role of CaV in primary ovarian cancer cells. This work addresses the question of whether CaV are expressed in primary ovarian cancer cells and whether channels in ovarian cancer cells are functional, particularly in cells derived from malignant ascitic fluid. Here, the results from immunohistochemical staining of ovarian tumour sections and RT-qPCR using normal ovaries, tumours and cells derived from malignant ascitic fluid suggest that in the majority of cases, CaV1.2 and CaV1.3 become expressed in ovarian cancer cells only after cells have been shed from the primary tumour. CaV1.2 and CaV1.3 mRNA is expressed in ascites derived cells, although the highest expression of these mRNAs was seen in a sample from a patient with mucinous ovarian cancer. In serous ovarian cancer cells from ascites, CaV1.2 protein was shown with flow cytometry to be highly expressed and immunofluorescent staining confirmed that this expression is localised to the nucleus. Functionally, dihydropyridine antagonism with nifedipine was found to prevent cell migration only in a 2-dimensional wound-healing model, whereas invasion of cell lines and ascites derived primary cells into basement membrane extract was unchanged. Cell lines display differing apoptotic responses to nifedipine, which triggers apoptosis in SKOV-3 but no response in OVCAR-8 at the same concentration. To assess channel functionality, fluorescent measurement of cytosolic Ca2+ flux using Fluo4-AM was performed on cell lines in the presence of the CaV agonist Bay K8644 and patch clamp electrophysiology was performed on cell lines and malignant ascites derived cells. Both of these techniques confirmed that no detectable L-type CaV current is present in the ovarian cancer cell lines. CaV current was observed in cells derived from malignant ascites, from the mucinous sample. Transient receptor potential (TRP) currents were also detected in OVCAR-8 cells, as well as single cells and spheroids from the ascites derived mucinous sample and were most likely carried by the Ca2+-selective TRPV5 or TRV6. These results suggest that functional L-type CaV are present in cancer cells from malignant ascites of patients with mucinous ovarian cancer. Although CaV1.2 was nuclear expressed in serous samples, similar research from other cancers has shown that these channels may still function in migration, even when expression is restricted to the nucleus, although nuclear channels would not be amenable to therapeutic targeting. These results fit into a wider context of contemporary research which is placing a greater role for CaV and ion channels in cancer, although more research needs to be performed in ovarian cancer ascites derived cells to determine the possible function of these nuclear ion channels.

Accumulating delivered dose to the rectum to improve toxicity prediction in prostate radiotherapy Leila E. A. Shelley Gastrointestinal (GI) toxicity is a clinical issue suffered by up to 22% of prostate cancer radiotherapy patients. However, the relationship between radiation dose and toxicity is generally poorly understood. In prostate radiotherapy, the rectum is a dose-limiting structure to which treatment planning dose constraints are applied to minimise the risk of toxicity. Current normal tissue complication probability (NTCP) models are based on planned dose data and do not consider the effects of organ motion on true delivered dose. The VoxTox research programme has developed automated solutions for segmentation and dose calculation of the rectum for prostate cancer patients being treated with helical TomoTherapy. Daily image guidance scans, acquired primarily for the purposes of positional verification, are exploited by extracting quantitative information to facilitate the calculation of daily delivered and total accumulated dose to the rectum. Prospectively collected toxicity data at 2 years post-treatment were available for 295 patients across two separate cohorts. In this thesis, the hypothesis being tested is that delivered dose is a better predictor of rectal toxicity than planned dose in prostate radiotherapy. The research has successfully demonstrated, for the first time, that delivered dose produces stronger associations with rectal bleeding and proctitis than planned dose. Analysis was performed using dose surface maps (DSMs) of the rectal wall, allowing spatial aspects of dose to be retained during accumulation. A subsequent analysis on a separate cohort also found stronger links between delivered dose and GI toxicity, stool frequency, and bowel bother, in addition to rectal bleeding and proctitis. Biomechanical finite element (FE) modelling was introduced to provide a more anatomically plausible tool for dose accumulation and allowed more accurate tracking of dose at the voxel level. A sensitivity analysis was conducted which explored the effect of simulated rectal motion on dose, and corresponding change in NTCP. For VoxTox patient dose-toxicity analysis, further dose parameterisation approaches were explored in order to consider the increased resolution of information available. Voxel-based rectal subregions at risk (SRRs) were identified using geometric and statistical approaches. In general, discriminative power improved with FE modelling for both planned and accumulated delivered dose, and associations between accumulated dose and toxicity were strengthened by voxel-based subregion analysis. Multivariate NTCP models were constructed for 12 different toxicity endpoints based on planned and accumulated dose parameters. Model performance was compared between analysis approaches, and models were tested on a validation dataset. In general, FE-based dose models performed best, although the optimal dose parameter selected within the model varied with toxicity endpoint. Overall, results suggest that there is an advantage to incorporating delivered dose into NTCP modelling. However, the differences between planned and accumulated dose can be subtle. Meaningful parameterisation of accumulated dose needs careful consideration, as traditional methods for quantifying planned dose may not be directly transferable. Voxel-based analysis techniques are recommended in order to accurately preserve and register spatial dose information, and have been shown to improve the strength of dose-toxicity associations. Further research into quantifying voxel level dose distributions is encouraged. It is anticipated that the novel scientific contributions presented within this thesis will prove valuable for future development of clinical decision-making tools for adaptive radiotherapy, with the ultimate aim of reducing the incidence of radiation-induced toxicity for prostate cancer radiotherapy patients.

Glioblastoma Multiforme is the most common adult primary brain tumour, with median survival rates of around 15 months. The aggressive and heterogeneous nature of this disease means that despite multimodal treatment incorporating maximal surgical resection with radiotherapy and chemotherapy, the tumour inevitably recurs. Second-line therapeutic options are limited and so to address the need for novel options at recurrence, this project aimed to combine phenotypic screening with drug repurposing in patient-derived glioma stem-like cells to rationally select second-line chemotherapeutics based on the sensitivity of the patient's tumour cells. The first part of this project aimed to validate a proof-of-principle high content screen in vivo to elucidate whether cell culture format had an effect on drug-sensitivity. We determined through the evaluation of a sub-set of drugs that suggested three-dimensional culture of glioma stem-like cells as neurospheres correlated better with our in vivo results when compared with traditional two-dimensional culture. The second part of this project then built on the learnings taken from the high content screen and developed a reproducible, 384-well multicellular spheroid high throughput screen that identified actinomycin-D to be a potent cytotoxic agent in all of our patient-derived lines. We subsequently validated this effect in a patient-derived orthotopic xenograft model of recurrent glioblastoma, demonstrating that this drug may also have the potential to specifically target the stem-cell population of tumours through downregulation of SOX2. Thirdly, based on literature that suggested a class of amphiphilic cationic drugs, also known as Functional Inhibitors of Acid Sphingomyelinase (FIASMA), could specifically target cancer cells we incorporated loratadine, a typical FIASMA, into our high throughput screen. We demonstrate that loratadine induces glioma cell-specific death through lysosomal membrane permeabilisation, as well as validating the in vitro synergistic anti-tumour activity of afatinib and loratadine in our recurrent glioblastoma patient-derived orthotopic xenograft. Overall, this project has established a workflow that streamlines patient tumour processing, giving high quality drug sensitivity data on patient-derived cancer stem cells in a clinically actionable timeframe. The ability to predict synergistic combinations of chemotherapeutics in a short window of time is vital in glioblastoma, as many patients are found to have an increased resistance to therapy at recurrence. The screen developed in this project quickly enables the stratification of patients by phenotypic response. Through future screening of a larger cohort of patients to enrich the data set, we hope to strengthen its prognostic power and optimise this platform into an invaluable tool that can help clinicians as well as improving the outlook for many patients.

Cell competition leads to elimination of a viable cell population, by fitter cells. Despite over forty years of research, the molecular mechanisms of competition in mammals are poorly understood. During my PhD I have investigated the mechanisms of competition by exploring an established mammalian cell culture system, in which wild-type MDCK cells eliminate scribble-deficient cells, and I have also developed a novel cell culture system to model mammalian competition. My work contributed to the discovery that scribble-deficient cells are eliminated not by biochemical exchange among cells, but by mechanical compaction. We termed this phenomenon mechanical competition. I employed transcriptional profiling to determine the molecular signature of mechanical losers, and identified activation of p53 signalling as their hallmark. My colleagues and I then demonstrated that elevation of p53 is both necessary and sufficient to trigger mechanical competition. In further investigating the mechanisms of mechanical competition, I found that compaction activates ROCK in scribble-deficient cells, and that this is required for their elimination. Inhibition of Src signalling in mechanical losers also protected them form out-competition, and integrin signalling is another pathway likely involved in mechanical competition. While investigating p53 competition, we observed that p53-high and p53-low cells engage in directional migration, with p53-high cells always at the migrating front. As a side-project, I investigated the role of p53 in directional migration, by exploring an established model with a single leader cell and multiple followers. We established a method to generate multinucleated leaders on demand. By creating leaders from p53-deficient cells, I established that p53 signalling is required for some, but not all multinucleated cells to trigger collective migration, thus implicating p53 signalling in a type of migration involved in wound healing. Finally, I successfully modelled p53-driven mechanical competition in a differentiated primary tracheal epithelial cell culture, thereby establishing a novel system to study mammalian competition, and also proving that p53 competition is conserved between different mammalian epithelia. Considering the involvement of p53, mechanical competition may play a major role in cancer.

This thesis reports the investigation of spontaneous Raman Spectroscopy (RS) for its applicability in early drug discovery. A key focus has been to develop an understanding of the applicability of RS for the quantification and localisation of compound concentration inside mammalian cells. Further investigation into the use of Surface Enhanced Raman Spectroscopy (SERS) for research on Visceral Leishmaniasis (VL) and Leishmania donovani as well as investigating applicability for cancer research are decisive parts of this work. The key work described in this thesis is the investigation of whole cell concentration of compounds inside THP-1 and Madin Darby Canine Kidney (MDCK) cells. For true quantification the Cell Silent Region (CSR) is used to measure without interference from cellular background signal. The model compound is erlotinib, an anti-cancer drug with an alkyne group expressing a peak in the CSR. The developed RS system is calibrated using the current gold standard technique Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). However, because of the single cell nature of the RS information on inter cell variability can be extracted. The RS measurements suggest that there is a large variation of concentration within single cell populations. The RS measurements can therefore give insight in single cell behaviour within a large cell population. Findings shows that washing cycles, before fixation, alter the intra-cellular concentrations significantly. This is hypothesised to be caused by the sudden change in concentration on the outside of the cell that applies an osmotic pressure, leading to loss of substance from inside the cell wall. Localisation of erlotinib is shown within THP-1 cells and points towards an accumulation inside the cell nucleus. Later, internalised Au nano-particles in the range of 30 nm to 80 nm have been investigated for their enhancement effects and localisation inside THP-1 cells. Au nano-particles are found to be internalised easily by differentiated THP-1 cells and accumulate in lysosomes. This allows for a high local enhancement of the spontaneous Raman signal. However, no advantage for the detection of lysosomally trapped compounds (chloroquine, chlorpromazine) was achieved. The detection of substances without a signal in the CSR was achieved without enhancement. Nonetheless, compounds with intrinsic peaks in the CSR could benefit from this enhancement. Lastly the RS system is explored for alternative uses in early drug discovery. This includes the detection of toxicity as well as the discrimination of cell types. Toxicity has been detected using optically trapped THP-1 cells and doxorubicin. Utilising Principal Component Analysis (PCA) combined with Linear Discriminant Analysis (LDA) on these measured spectra, allowed for a clear discrimination of toxically influenced from healthy cells. Differences mainly show up in DNA content caused by the mode of action of doxorubicin and caused by the trapping, which generates most of the signal within the nucleus of the cell. Discriminating cancerogenic (DU145) from healthy prostate cells (PNT2) has been achieved by probing fixed cells and evaluating the acquired Raman spectra with a PCA/LDA combination. The accuracy of separation of these cells when tested with a 10-fold cross-validation technique, is above 98 %, allowing a good discrimination.

The timely activation of homologous recombination (HR) is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. In vivo, RAD51 polymerises on single-stranded DNA (ssDNA) at double-strand breaks (DSBs) to catalyse HR-dependent DSB repair. Biochemically, human RAD51 polymerises faster on ssDNA compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? Throughout this thesis, I tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By fitting detailed polymerisation models informed by the experimental datasets, I suggest that RAD51 is a mechano-sensor that polymerises faster on flexible ssDNA compared to rigid ssDNA or dsDNA. Furthermore, I suggest that the energy released during RAD51 polymerisation is sufficient to overcome any entropic penalty to binding flexible ssDNA. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in recruiting repair factors that multimerise at sites of DNA damage.

Mammographic density (MD), created predominantly by increased stromal tissue, is a major breast cancer risk factor, though little is known about the biological mechanisms mediating it. Tamoxifen prevents breast cancer in a subset of high risk women via mechanisms that appear dependent on reduction of MD. Animal models suggest tamoxifen remodels the mammary stroma to a tumour-inhibitory phenotype. This study aims to analyse the effect of tamoxifen on human breast fibroblast function and identify pro-tumourigenic pathways contributing to density-associated risk. Methods Primary human breast fibroblasts from normal, high risk or breast cancer patients were treated with hydroxytamoxifen (100nM-5μM). Fibroblast function was analysed by measuring: proliferation, expression of stromal proteins fibronectin and collagen 1; effects on TGF-β signalling and up-regulation of myofibroblast marker SMA. Genome wide analysis was performed using RNA-Seq. Significantly deregulated pathways were validated by PCR, western blotting and mass spectrometry. Results Fibroblasts from 23 patients were treated with hydroxytamoxifen. All patients showed reduced proliferation with treatment. 62% of patients showed reduced fibronectin expression. TGF- β-mediated up-regulation of SMA and fibronectin were consistently inhibited by tamoxifen. RNA-Seq analysis revealed down-regulation of Wnt signalling, an established profibrogenic and pro-tumourigenic pathway. In addition, there was significant modulation of many metabolic pathways, including components of the microsomal anti-oestrogen binding site (AEBS). Binding of tamoxifen to the AEBS inhibits cholesterol epoxide hydrolase (ChEH) enzyme activity, promoting an anti-tumourigenic phenotype. The effects of tamoxifen on fibroblasts could be partly replicated using tesmilifene, a commercially available 5 inhibitor of ChEH. Mass spectrometry analysis confirmed an altered cholesterol metabolite profile in tamoxifen treated fibroblasts. Conclusion These data indicate that tamoxifen can directly remodel the mammary stromal microenvironment, generating a less 'reactive' stroma. Thus, tamoxifen impacts on multiple pathways, many independent of the oestrogen receptor, to create a tumourinhibitory microenvironment. This offers exciting potential for patient monitoring and alternative breast cancer prevention strategies.

Fibroblasts are abundant mesenchymal cells present in all tissues in a quiescent state, which contribute to wound healing when activated. Cytokine transforming growth factor-β1 (TGF-β1) stimulates fibroblast-myofibroblast differentiation, which induces extracellular matrix secretion, tissue contraction and promotes cancer cell migration. Hence, chronic activity of stromal myofibroblasts correlates with a poor prognosis for cancer and organ fibrosis patients. Therefore, modulating myofibroblast activity may reduce the severity of these diseases. Previous research suggests blockade of transmembrane integrin receptors expressed by fibroblasts prevents TGF-β1- induced differentiation, indicating integrins are attractive therapeutic targets. However, fibroblasts derived from different organs exhibit heterogeneity, although their integrin expression and integrin-regulated differentiation has not been directly compared. The aim of my research was 1) to understand and compare how integrins regulate TGF-β1-induced activation of fibroblasts derived from normal skin, lung and breast tissue; 2) to examine the global gene expression of TGF-β1-treated lung fibroblasts; 3) to identify novel therapeutic targets that modulate TGF-β1-induced activation of lung fibroblasts using a drug library. qPCR showed skin, lung and breast fibroblasts differentially expressed TGF-β1- induced activation markers, including ACTA2, FN1, TIMP3, CTGF and SERPINE1, in addition to integrin genes for α1, α4, α11 and β3. Small-molecule inhibitors of αv integrins only reduced the invasion of TGF-β1-exposed skin fibroblasts, but not lung or breast fibroblasts. siRNA against α11, β3 and β5 decreased TGF-β1-induced collagen contraction and activation marker expression in skin and lung fibroblasts, while α1 siRNA prevented collagen contraction by breast fibroblasts only. RNA sequencing of TGF-β1-treated lung fibroblasts revealed pro-inflammatory and profibrotic pathways were significantly enriched, while screening TGF-β1-treated lung fibroblasts with a FDA-approved drug library identified 46 hits that significantly reduced α-smooth muscle actin and fibronectin expression. Overall, genes are differentially expressed in TGF-β1-treated skin, lung and breast fibroblasts, while different integrins in each fibroblast appear to regulate invasion, TGF-β1-induced collagen contraction and gene expression. RNA sequencing revealed TGF-β1 promotes the expression of a pro-tumour signature in lung fibroblasts and several novel therapeutic targets that modulate the activation of lung fibroblasts have been identified. Understanding these integrin-dependent and independent mechanisms will facilitate the generation of myofibroblast-targeted treatments for cancer and organ fibrosis.

The main goal of this project was the development of carbon nanotubes as a candidate for dual-functioning magnetic hyperthermia structure and magnetic resonance imaging contrast agent. This was achieved by filling carbon nanotubes with continuous ferromagnetic α-Fe nanowires and surface functionalized with paramagnetic Gd(III). Also, length control of both nanotube and nanowire was investigated. Firstly, a low vapour flow-rate and constant evaporation temperature chemical vapour deposition method based on the thermal decomposition of ferrocene was employed which achieved continuous α-Fe nanowires on the same scale as the nanotube for lengths >10 m without the necessity of post-synthesis heat-treatment or introduction of other precursor elements. The low vapour flow-rate regime has the advantage of sustaining the intrinsic temperature gradient at the tip of the forming structure which drives the vapour feedstock to the growth front to guarantee continuous nanowire formation. For initially mixed-phase nanowires of length less than 10 μm, the continuous α-Fe nanowires were achieved by postsynthesis heat treatment. Secondly, a simple wet chemical method involving only sonication in aqueous GdCl3 solution was used for surface functionalization of iron-filled multiwalled carbon nanotubes with gadolinium. Functional groups on the sidewalls produced by the sonication provide active nucleation sites for the loading of Gd3+ ions. Characterization by electron paramagnetic resonance, electron energy loss spectroscopy, and high-resolution transmission electron microscopy confirmed the presence of Gd3+ ions on the sidewall surface. The ferromagnetic properties of the encapsulated iron nanowire maintained after surface functionalization. At room temperature a saturation magnetization of 40 emu/g and a coercivity of 600 Oe were observed. Heating functionality in an alternating applied magnetic field was quantified through the measurement of specific absorption rate: 50 W/gFe and the intrinsic loss power: 1.12 nHm²kg⁻¹ at magnetic field strength 8 kA/m and frequency of 696 kHz. These structures exhibited an extremely high relaxivity r₁ ~ 200 mM⁻¹ s⁻¹ at high magnetic field (9.4 T).

Radiotherapy is the use of ionising radiation to induce localised DNA damage to cancerous tissues, leading to cell death and disease control. In order to maximise tumour growth control and to limit damage of the healthy surrounding tissues and the consequent side effects for the patient, molecular determinants of tumour radioresistance are investigated as potential clinical targets. A high-throughput siRNA colony formation assay screen in HeLa cervical carcinoma cells previously published by our laboratory identified modulators of radiosensitivity. From the list CSF1R, EPHB2, GAK and TOPK, were selected and validated. TOPK (T-LAK cell-originated protein kinase, also known as PDZ-binding kinase, PBK) was selected for further investigation because it is overexpressed in most malignancies but not in normal tissues, apart from testis and placenta. Knockdown of TOPK was shown to induce radiosensitisation in a panel of cancer cell lines with no significant effects on normal cells. A role for TOPK in the cell cycle response to ionising radiation (IR) was discovered in HCT116 colorectal cancer cells, with alterations in the G1/S and G2/M checkpoints. Furthermore, immunoprecipitation experiments identified a physical interaction between TOPK and CDKN1A (p21) at 8 hours after IR. Apoptosis and the number of multinucleated cells were significantly increased in TOPK depleted cells exposed to IR, suggesting the possibility of aberrant mitosis and mitotic catastrophe in these cells. High TOPK expression in early breast cancer patients was shown to be associated with poor recurrence-free survival. In addition, immunohistochemistry (IHC) analysis on samples from prostate cancer patients identified a strong correlation between high levels of TOPK and poor clinical response to radiotherapy. In order to facilitate future in vivo experiments, an HCT116 shRNA stable knockdown cell line was developed and two commercially available TOPK inhibitors were tested and optimised. Taken together, these data suggest that TOPK is a molecular determinant of radiosensitivity with a great potential for future clinical applications.

Ovarian Cancer is the 5th most common cause of cancer death in women and the second most common gynaecological cancer in the UK. Worldwide, around 152,000 women were estimated to have died from ovarian cancer in 2012. Survival rates for women with epithelial ovarian cancer have not significantly changed since platinum-based treatment was introduced over 30 years ago. This is particularly disconcerting considering the fact that there is a less than 5% five year survival rate for patients diagnosed with late stage high grade serous ovarian cancer. This thesis examines the role of BMP signalling in ovarian cancer using in vitro cancer cell models. It builds upon the initial published work by the Inman lab identifying autocrine BMP9 as a promoter of ovarian cancer cell proliferation. The findings of Chapters 3-5 provide strong evidence indicating BMP9 as a context specific modulator of ovarian cancer cell proliferation. This significantly builds upon on the sole pro-proliferative BMP9 growth response previously described. Responding cell lines were subjected to a microarray with and without BMP9 treatment In order to determine early BMP target genes which were subsequently transiently knocked down in order to determine their role in the aetiology of said growth phenotype. ID1 gene expression was found to significantly contribute to the BMP9 proproliferative phenotype. Moreover several other BMP genes identified significantly alter basal cell proliferation. It was subsequently determined that BMP9 implemented a cell growth phenotype by negating apoptosis. .Excitingly, preliminary evidence suggests a marked reduction in detectable levels of a recently described Bax isoform, Bax β that coincide with BMP9 addition and the resultant anti-apoptotic phenotype observed. This is very interesting as no prior evidence correlating the BMP family and Bax β currently exists. These findings provide an enhanced understanding of BMP9s contribution to ovarian cancer pathogenesis that may result in the development of effective and targeted therapeutic interventions upon further stratification of the contextuality of the BMP induced growth response.

It is imperative that the cancer research community has the means with which to effectively locate, access, manage, analyse and interpret the plethora of data values being generated by novel technologies. This thesis addresses this unmet requirement by using pancreatic cancer and breast cancer as prototype malignancies to develop a generic integrative transcriptomic model. The analytical workflow was initially applied to publicly available pancreatic cancer data from multiple experimental types. The transcriptomic landscape of comparative groups was examined both in isolation and relative to each other. The main observations included (i) a clear separation of profiles based on experimental type, (ii) identification of three subgroups within normal tissue samples resected adjacent to pancreatic cancer, each showing disruptions to biofunctions previously associated with pancreatic cancer (iii) and that cell lines and xenograft models are not representative of changes occurring during pancreatic tumourigenesis. Previous studies examined transcriptomic profiles across 306 biological and experimental samples, including breast cancer. The plethora of clinical and survival data readily available for breast cancer, compared to the paucity of publicly available pancreatic cancer data, allowed for expansion of the pipeline’s infrastructure to include functionalities for cross-platform and survival analysis. Application of this enhanced pipeline to multiple cohorts of triple negative and basal-like breast cancers identified differential risk groups within these breast cancer subtypes. All of the main experimental findings of this thesis are being integrated with the Pancreatic Expression Database and the Breast Cancer Campaign Tissue Bank bioinformatics portal, which enhances the sharing capacity of this information and ensures its exposure to a wider audience.

Hypoxia (low tissue oxygenation) is an important signalling cue for many cell types. The study of its effects has direct relevance to surgery since hypoxic gradients are generated with every cut. On a cellular level, changes in molecular oxygen are sensed by the Hypoxia-Inducible Factors (HIFs). The HIFs are a family of transcription factors that are master regulators of over 100 genes and can effect changes in multiple cellular processes including migration, survival and differentiation. The broad nature of the response to hypoxia means that study of the HIF system is also important in cancer; where many tumour cells have found ways of subverting the HIF response to ensure their continued growth and survival. This thesis explores the role of hypoxia and the HIF system in the regulation of migration, survival and differentiation in both cancer and stem cells. The first experimental chapter examines the role of hypoxia and the HIF system in the regulation of migration and three-dimensional organisation in several cancer cell lines. Using biochemical and functional assays, the HIF system is shown to exert a pleiotropic effect across a panel of cancer cell lines. In particular, HIF 1α is shown to activate proliferation in a prostate cancer cell line in findings that may be useful to inform future clinical strategies for the management of this disease. In the second experimental chapter, the first epigenetic mechanism involving histone modification for the specific regulation of HIF 2α expression is characterised. Here the family with sequence similarity 60, member A (Fam60a) protein is shown to repress expression of the HIF 2α gene through its association with the class 1 Sin3-HDAC co-repressor complex, achieving specificity by co-operation with the SP1 transcription factor. This novel mechanism is demonstrated to be important in the regulation of the basal expression of HIF 2α. Modification of HIF 2α expression through this mechanism is shown to alter cell migration, three dimensional organisation and angiogenesis in vitro. The clinical importance of these findings is demonstrated in a series of 45 patients suffering from colorectal cancer of known stage. In this cohort, the reciprocal relationship between Fam60a and HIF 2α is maintained, and both are identified as potential novel biomarkers for the development of this disease. In the final experimental chapter, the role of hypoxia in the regulation of differentiation is explored. These effects are documented in mesenchymal progenitors primarily derived from human fat. Here, hypoxia is shown to regulate differentiation in a context-dependent manner, promoting osteogenic and retarding adipose and neural differentiation in-vitro. The roles of Fam60a and HIF 2α are explored in this system. These data may be useful in optimising future surgical engraftment of these cells for regenerative purposes.

Hypoxia inducible factor (HIF)-1α is a heterodimerically-activated transcription factor central to the cellular response to hypoxic environments and is often upregulated in cancer. Binding of HIF-1α to the co-activator p300 is necessary for the hypoxia-induced transcription of many oncogenic proteins. The aim of this project was to develop novel small molecule inhibitors of the HIF-1α/p300 protein-protein interaction (PPI). Initial work focused on designing, validating and optimising two high-throughput competition binding assays to screen for inhibitors of the PPI (Chapter 2). Alongside these, zinc ejector assays for both p300 and KDM4A proteins were developed to probe the mechanism of action and selectivity. Analysis of hits from a natural product high-throughput screen (HTS) revealed two compound classes; benzoquinones and 2-substituted indandiones, which modulate the PPI. The potency of these series correlated with the reactivity of the core functional groups, which act as electrophiles to covalently modify reactive cysteines, ejecting structural zinc and disrupting the p300/KDM4A protein fold (Chapter 3). Conjugating electrophilic groups to putative HIF-1α/p300 inhibitors did not replicate the activity of the zinc ejecting HTS hits (Chapter 4). Further work focused on non-covalent inhibitors of the HIF-1α/p300 interaction, first with peptide truncates, and then rationally designed α-helix peptidomimetics. An 11mer truncate showed encouraging activity (IC50 ≈ 70 μM), and corresponded to a key α-helix in the HIF-1α C-terminal transactivation domain. Three distinct double-sided scaffolds were used to imitate up to five hot-spot ampiphilic residues on this α-helix (Chapter 6 and 7). Of the 35 compounds screened, only modest inhibition was observed (IC50 ≈ 200-500 μM). Future work will look to conjugate electrophilic functionality onto the 11mer peptide in an attempt to gain potency from zinc ejection, while maintaining selectivity for p300.

The chalcone motif is a privileged structure present in an extensive range of biologically active molecules. The chalcone structure can also serve as a versatile starting material for more complex molecules in medicinal chemistry. Eleutherobin, isolated from the Australian coral Eleutherobia and sarcodictyin, isolated from the Mediterranean coral Sarcodictyon roseum are natural products displaying nanomolar cytotoxicity against a range of cancer cell lines including Taxol®-resistant cell lines. Both natural products act as microtubule stabilising agents and will be valuable additions to the clinic, however their limited availability and lengthy total syntheses prevent further development. The urocanic ester side chain present in eleutherobin and sarcodictyin was identified as being critical for biological activity. We discuss the design, synthesis and biological evaluation of fourteen chalcone analogues based on this urocanic motif with the lead chalcone displaying promising antiproliferative activity in a range of cancer cell lines. Combretastatin A-4 is a promising microtubule destabiliser under clinical development. The Z configuration is vital for biological activity, however it can isomerise to the inactive E configuration. We report a library of twenty pyrazolines synthesised from chalcones as “Z restricted” combretastatin analogues with the lead pyrazoline displaying potent antiproliferative activity in cancer cell lines due to the disruption of tubulin. Tissue engineering is a diverse interdisciplinary field that applies engineering principles to the biological sciences with the aim of maintaining or replacing tissue function. Recent developments have revealed metal chelation to be a valuable tool to control the architecture of tissue engineering scaffolds. We report a library of ten novel pyrazolines and their potential as metal chelators. Maltol is a well established Fe3+ chelator with a low toxicity profile. We report a novel maltol hydrazide which can be attached to the cell surface which upon addition of Fe3+ results in cellular aggregation due to metal chelation. Further studies revealed that this process can be applied to form heterocellular aggregates composed of two different cancer types with valuable applications in tissue engineering and cancer research.

Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradation.

Our hypothesis is that the polyamine biosynthetic pathway, a pathway essential in many cellular functions, is modulated by NSAIDs and that this is, at least in part, how NSAID chemoprevention is mediated. An in vitro model of colorectal cancer was used: two cell lines one of which is COX positive and one COX negative to determine the effects of a range of selective and non-selective NSAIDs on various reactions within the polyamine pathway.  NSAIDs are cytotoxic to colorectal cancer cells regardless of their COX expression.  NSAID-mediated inhibition of cell growth is accompanied by inhibition of ODC activity, partial depletion of polyamine concentrations and up-regulation of polyamine catabolism. In order to investigate the importance of polyamine metabolism, a specific polyamine inhibitor α-difluoromethylornithine (DFMO) was used in combination with the NSAIDs.  DFMO per se is not toxic to cells and it does not enhance NSAID mediated toxicity.  DFMO in combination with the NSAIDs did cause increased catabolic activity and more sustained polyamine depletion than either alone, however no additional decrease in ODC activity was observed.  This suggests that NSAID toxicity is not enhanced by DFMO in this in vitro model. Analysis of the mode of death indicated that the NSAIDs caused apoptotic cell death, confirmed through biochemical and morphological studies and that the NSAIDs affected gene expression of key enzymes in the polyamine biosynthetic pathway. Our findings suggest that modulation of the polyamine pathway by NSAIDs is at least part of the mechanism of action involved in cancer chemoprevention. Therefore modulation of the polyamine pathway may be useful for design of new chemopreventative drugs.

In the field of pharmacogenomics and precision medicine, gene expression analysis has become a crucial tool in predicting patient drug response. My contributions to this field come primarily in the development and application of two bioinformatic packages: oncoPredict and scIDUC. oncoPredict is a tool based in the R programming language, primarily used to predict the response of various cancer samples (cell line, patient, etc.) to different drugs. This is made possible by incorporating machine learning to analyze the complex relationships between genomic features and drug response from pan-cancer cell lines. These relationships are learned from microarray or bulk RNA sequencing (RNA-seq) data and high-throughput drug screens, then applied to patient data to generate novel drug discovery hypotheses. In turn, oncoPredict aids in identifying potential drug candidates, understanding mechanisms of drug resistance, and predicting the effectiveness of drugs on specific cancer types. scIDUC (single-cell Integration and Drug Utility Computation) is a computational framework based in python that quickly and accurately generates predictions of drug response for cells derived from single-cell RNA sequencing (scRNA-seq) data. It is a transfer learning-based approach that learns relationships between drug sensitivities and relevant gene expression patterns based on cell line bulk RNA-seq data and high-throughput drug screens, similar to oncoPredict. The key difference, however, is that prior to training drug response models, scIDUC integrates bulk RNA-seq and target scRNA-seq data to denoise and extract shared gene expression patterns between bulk and single-cell data sources. The resulting bulk data is then used to train drug response models, whose coefficients are further applied to post-integration single-cell data to infer cellular drug sensitivity scores.

v-Jun, a mutated derivative of the c-Jun transcription factor, is the transforming oncoprotein of an avian sarcoma virus. v-Jun is thought to cause cell transformation and tumorigenesis by the mis-regulation of certain target gene promoters. v-Jun can both activate and repress gene transcription compared to c-Jun, however little is known about the underlying mechanisms and the identity of the critical "effector" target gene(s) responsible for cell transformation and tumorigenesis by v-Jun. To investigate the mechanisms of transcriptional regulation by v-Jun, a comparative study was undertaken of two gene promoters, bkj and collagenase, which are respectively activated or repressed by v-Jun. Promoter mutagenesis experiments were performed to investigate the effects of Jun binding site position and core promoter element sequences on transcriptional regulation by v-Jun. The primary conclusion was that these factors alone did not determine whether target promoters were activated or repressed by v-Jun. However, alterations in the level of transcriptional activation and fold induction of the variant promoters by v-Jun implied that binding site position and core promoter sequences did influence transcriptional regulation by Jun proteins. This analysis also suggested that v-Jun regulated transcription by different mechanisms at different target promoters. Further work investigated the relationship between transcriptional activation of v-Jun target promoters and cell transformation using DeltavJ-hER, an amino-terminally truncated v-Jun protein fused to the hormone-binding domain of estrogen receptor-alpha. This chimaeric protein was previously shown to induce activation of v-Jun target genes and cell transformation in an estradiol-dependent manner, despite lacking the v-Jun transcriptional activation domain. The estrogen receptor activating function-2 (AF-2) domain was proposed to substitute for this v-Jun domain, implying that estradiol-dependent transcriptional activation of v-Jun target gene promoters by AvJ-hER was required for cell transformation. To test this hypothesis, an inactivating mutation was introduced into helix 12 of the AF-2 domain, which mediates estrogen receptor binding to co-activator proteins. The mutant AvJ-hER protein was inactive in transcription and cell transformation assays, confirming that these processes required AF-2 function. Many estrogen receptor co-activator proteins have histone acetyltransferase activity, however the p300 histone acetyltransferase domain was unable to substitute for the estrogen receptor AF-2 domain function to induce either transcriptional activation or cell transformation. In conclusion, while the mechanisms responsible for transcriptional activation and repression by v-Jun remain unclear, these results support the hypothesis that transcriptional activation of positive v-Jun target gene promoters is required for cell transformation.

This study focuses on diffuse large cell lymphomas (DLCL) and follicle center lymphomas (FCL), two entities that encompass ca 70-80% of B-cell malignancies. The aim was to characterize the V_H gene usage and mutation patterns at diagnosis and to detect changes in these patterns over time. No skewing of V_H gene segment usage was detected. V_H genes showed a significant burden of somatic mutations, consistent with a germinal center (GC) origin. Analysis of the mutation patterns suggests antigen selection had occurred with selective pressure to maintain the framework regions of the V_H genes. Detailed analysis of a large number of templates from presentation biopsies revealed that commonly DLCL are still responsive to the somatic mutation mechanisms in the GC, resulting in introclonal sequence heterogeneity. Lymphoma progression however can lead to the selection of a subclone with subsequent loss of this heterogeneity. FCL and DLCL appear to commonly express RNA transcripts for multiple Ig isotypes. The sequencing data indicate the derivation of these transcripts from different cell populations and immunocytochemistry suggests that at least some of these transcripts are translated into protein and represent istoype switch events. V_H and V_L sequences also provide patient specific tumour-associated antigens, which can be used as a target for immune attack. A phase I/II study is currently being undertaken based on this concept. Vaccines consisting of DNA containing V_H and V_L genes are being used to activate immunity against the cancer in patients with FCL. In a second part of this thesis heavy and light chain variable genes from patients with FCL were identified in preparation for a DNA vaccine study in this patient group. A pilot study of expressing the complete IgM of one of these patients in the baculovirus system was performed, as a tool for measuring anti-adiotype responses in patients with FCL before and after vaccination.

The oncogene v-fos, is the transforming agent of the FBJ-MuSV and the FBR-MuSV osteosarcoma virus that elicits cellular responses through activation of the multigenic transcription factor AP-1. The Suppressive Subtractive Hybridisation (SSH), technique has been employed to identify differentially expressed downstream direct and indirect target genes of the AP-1 transcription factor in a rat fibroblast cell system by using FBJ-MuSV v-fos transformed 208F fibroblasts and the normal parental fibroblast 208F cell lines.

It has been demonstrated that the bioflavonoid quercetin can synergise with bovine papillomavirus type 4 (BPV4) DNA plus an activated ras oncogene to fully transform primary fibroblasts cells derived from bovine foetal palate (PalF cells). The aim of this thesis has been to identify a mechanism(s) by which quercetin may be contributing to this transformation process. Quercetin was shown to inhibit the growth of PalF cells in a concentration dependent maimer. A quercetin concentration of ~20?M inhibited the growth of PalF cells by approximately 50%. PalF cells were shown to arrest in the G1/G0 and G2/M phases of the cell cycle in response to quercetin treatment. These results indicated that the presence of quercetin in PalF cell culture medium is not conducive to normal cell proliferation. Investigations were undertaken to determine if quercetin could modulate the level of transcription from the viral long control region (LCR); this region of the virus contains sequences which are involved in regulating expression of viral genes. Results showed that quercetin was capable of increasing the level of viral transcription from the LCR up to 4 fold. In keeping with earlier observations, the timing of exposure to quercetin was critical; the increase in transcription from the viral LCR was only observed when cell were exposed to quercetin after cells had been transfected with LCR-containing reporter plasmids. No effect on transcription was observed when cells were treated with quercetin before transfection. The quercetin- mediated effect on LCR transcription activity was also limited to the LCR functioning in a promoter capacity. Quercetin had no effect on the LCR when present as an enhancer. Removal of nucleotides 311 to 331 from the 3'terminal end of the BPV4 LCR correlated with a drop in the basal level of transcription from the LCR and abrogation of the response of the LCR to quercetin. EMSA experiments identified a cellular factor, designated QX1, as binding to an oligonucleotide corresponding the sequence of the LCR from nucleotides 301 to 331. Although this sequence of the LCR contains a TRE-like binding motif, mutational analysis has confirmed that this motif is not the site to which QX1 specifically binds. Furthermore, AP-1 does not bind to the TRE- like element. API and QX1 display different mobilities in a polyacrylamide gel showing that QX1 and API are not the same factor. Mutational analysis has thus far not identified specific residues critical for the binding of QX1 to this 3' terminal region of the BPV4 LCR. Western blot analysis, using a phosphotyrosine antibody, identified several proteins which showed changes in their phosphotyrosine status in response to quercetin exposure. These results confirm that quercetin can alter the phosphorylation status, and possibly also the activity of a number of different proteins, in PalF cells. The observations made in the course of this thesis indicate that quercetin can induce PalF cells to growth arrest, possibly as a result of DNA damage. Quercetin also causes an increase in the level of viral transcription. Over-expression of viral oncoproteins such as E7, in response to quercetin treatment, would stimulate cells to continue proliferating. The stimulation of damaged cells to proliferate in an adverse environment induced by quercetin may partially explain the ability of quercetin to transform PalF cells in co-operation with BPV4 plus ras. Other epigenetic effects, as indicated by the changes induced in several phosphotyrosine-containing proteins, could further contribute to this synergism.

Complexes of the radionuclides (^67)Ga and (^III)In, or of the paramagnetic contrast agent Gd, used in MRI, provide a means of imaging tumours. The stability of the (^71)Ga- NOTA complex was verified by in vivo NMR spectroscopy. The novel phosphinic acid NOTA analogue, bearing an isopropyl substituent on phosphorus was prepared and its lipophilicity and (^III)In biodistribution in mice determined. The crystal structure of the yttrium complex of N,N"-bis(benzylcarbamoylmethyl)diethylenetriamine-N,N',N"-triacetic acid revealed amide carbonyl ligation in a distorted mono-capped square antiprismatic structure, with one metal-bound water. The biodistribution of the analogous Gd complex was examined. A novel series of 9N3 based ligands incorporating three further N donor atoms, carboxymethyl groups and a potentially larger cavity size were synthesised. The analogous series containing phosphinic acid groups and the 12N3 counterparts were also prepared. The former series formed complexes with Gd and the biodistribution in mice was studied. The 12N3 analogues failed to form Gd complexes.2-Nitroimidazoles are known to selectively target hypoxic tumour tissue. Two conjugates of 2-nitroimidazole for tumour imaging were prepared, the Gd complex of a DTPA-bis(2-nitroimidazole) amide and the (^III)In complex of a C-functionalised NOTA- nitroimidazole conjugate. The biodistribution in mice of each was studied and luminescence experiments on the Tb complex of the former revealed one metal bound water molecule. Novel conjugates of the tetrapeptide tuftsin and a complexing agent based on the 12N4 skeleton and an N-linked NOTA derivative were synthesised. Biodistributions of the Gd and In complexes respectively are being carried out. Acridine intercalators reversibly bind DNA, possibly enhancing the effectiveness of tumour targeting conjugates. Novel multifunctionally labelled acridines based on tris(2- aminoethyl) amine were sought. The p-nitrophenolate active ester of 9-acridine carbamoyl-2-(2-aminoethyl)-2-methyl amine was also prepared as a versatile agent for acridine labelling.

The recent discovery of the vaccinia virus protein phosphatase VH1, and its mammalian counterparts has highlighted a novel subfamily of protein phosphatases that exhibit dual specificity towards phosphotyrosine- and phosphoserine/threonine- residues. The aim of this thesis was to isolate and characterise further members of this phosphatase subfamily. Employing the technique of polymerase chain reaction (PCR), degenerate PCR primers, designed from amino acid sequences conserved between two members of this dual specificity phosphatase family, were used to amplify related sequences from poly (A)+ RNA isolated from the human squamous cell line A431. Screening cDNA libraries with probes derived from the PCR analysis resulted in the isolation of five novel genes, which show significant homology to the catalytic domain of the dual specificity phosphatases, such as the human phosphatase CL100. Two of these, TYP 1 and TYP 2 (threonine-tyrosine phosphatase), have been analysed further. Interestingly, whilst TYP 2 expression is regulated with similar kinetics to CL100, TYP 1 differs dramatically in its regulation. The TYP 1 gene is not expressed in human fibroblasts unlike other CL100-like genes. Furthermore, northern analysis has demonstrated that following mitogenic stimulation of squamous cells, induction of TYP 1 mRNA reaches its maximal levels after four hours, in contrast to the immediate-early CL100-like genes. Both TYP 1 and CL100 mRNAs are induced upon transforming growth factor- beta treatment of squamous cell lines sensitive to the growth factors antiproliferative effects. Investigations were undertaken to determine if TYP 1 demonstrated substrate specificity towards the mitogen-activated protein kinases, a function peculiar to the other CL100-like phosphatases. Indeed, transient transfection of TYP 1 into COS-1 cells resulted in the inhibition of both ERK2 and p54JNK MAP kinase isoforms. In addition, purified TYP 1 protein efficiently inactivates recombinant ERK2 in vitro by the concomitant dephosphorylation of both the phosphothreonine and -tyrosine residues. The subcellular localisation of TYP 1 was investigated, and shown to encode a nuclear protein. Interestingly, the mitogenic induction of TYP 1 results in an increase in the half-life of the protein. The results presented in this thesis suggest that the dual specificity phosphatases are an expanding family, some members of which are differentially regulated, therefore providing alternative mechanisms for the control of cellular responses to different ligands in different cell types. As discussed, TYP 1 is particularly exciting as its kinetics of induction and cell specificity of expression make it unique in terms of its possible roles.

The phenomenon of induced peroxisome proliferation in rodents has invariably been associated with upregulation of member(s) of the CYP4A subfamily of hemo- proteins. However, regulation of the members of the rat CYP4A family has not been fully examined at the molecular level. Despite the publication of the rat CYP4A1 and CYP4A2 genomic sequences (Kimura et al., 1989a), no functional assays have been performed to determine the mechanism of peroxisome proliferator modulated transcriptional activation of these genes. The objective of this study was to isolate members of the rat CYP4A subfamily of genes and investigate their inducibility by peroxisome proliferators at the molecular level. In this thesis I present results to demonstrate: 1) the isolation and partial characterisation of recombinant DNA clones corresponding to 3 different rat genomic sequences, showing a degree of similarity to the rat CYP4A1 cDNA, 2) identification, by sequencing, of one of the groups of clones (group C) to correspond to CYP4A2 and another (group B) to be, possibly, the genomic sequence of CYP4A3. The identification of the remaining group (group D) remains unknown, but most likely represents a novel member of the rat CYP4A subfamily, 3) the apparent absence of a peroxisome proliferator response element (PPRE) in the 5' flanking DNA sequence of the clones, as determined by hybridisation to the rat acyl-CoA oxidase PPRE oligonucleotide (GTOX285), 4) a lack of a response of a -5.8kb upstream sequence of the newly-isolated human CYP4A11 gene in a co-transfection assay with the mouse PPAR and in the presence of the peroxisome proliferator Wy 14643. The results further extend knowledge on the multiplicity of CYP4A genes in the rat and provide data on the transcriptional regulation of CYP4A genes in rodents and man.