This thesis examines methods of perioperative risk stratification and outcome in patients receiving multidisciplinary stage-directed treatment for oesophagogastric cancer. The hypotheses tested were: Suboptimal bioelectrical impedance analysis (BIA) body composition variables predict poor outcomes in oesophagogastric cancer (OGC) surgery; low CT-measured psoas muscle density (PMD) predicts poor outcomes in OGC surgery; suboptimal cardiopulmonary exercise (CPX) performance predicts poor outcomes following OGC surgery; the literature offers evidence in support of enhanced recovery programmes in OGC surgery; the use of an enhanced recovery programme in OGC surgery is feasible, safe and not associated with adverse outcomes. High values for BIA-derived measures of fat-free mass and muscle mass respectively predicted longer survival (p=0.047, p=0.011), but were not associated with reduced 30-day mortality, major morbidity or length of stay. CT-measured psoas muscle density greater than the median of 48.7 Hounsfield Units predicted longer survival (p=0.046), but was not associated with reduced 30-day mortality, major morbidity or length of stay (LOHS). Multivariable analysis demonstrated radiological TNM stage (p=0.015), and both left (p=0.046) and right PMD (p=0.047), as significant and independent predictors of survival. Cardiopulmonary exercise testing results materially altered the management plan in 6.8% patients. Major morbidity (p=0.049) and poor survival (p=0.048) were associated with a high ventilatory equivalent for carbon dioxide (VE/VCO2), but not with the anaerobic threshold (AT) or peak oxygen uptake (VO2peak). VE/VCO2 also emerged on multivariable analysis as an independent and significant predictor of LOHS (p=0.001). Systematic review and meta-analysis revealed enhanced recovery programmes (ERPs) in OGC surgery to be feasible, safe and costeffective, significantly shortening length of stay (LOHS, p<0.0001). In our unit, the introduction of ERPs in gastric and oesophageal cancer surgery respectively, significantly reduced LOHS (p=0.004; p=0.032), critical care stay (p<0.0001; p<0.0001) and overall cost (p=0.001; p<0.0001).

Breast Cancer Associated gene 2 (BCA2), is a little-studied E3 ligase that is overexpressed in 56% of all primary breast cancers and has been linked with increased cell proliferation and invasion in vitro. BCA2 has been implicated in EGFR degradation however there is conflicting evidence surrounding its function and effect on receptor biology. This project aimed to elucidate the role of BCA2 in EGFR endocytosis and downregulation and to determine its link with breast cancer survival. Data generated with online mRNA analysis tools indicated that high BCA2 levels were often associated with improved breast cancer prognosis. In silico studies also demonstrated that many genes coexpressed with BCA2 were regulators of membrane trafficking and suggested that BCA2 expression was repressed by HER2/EGFR/Ras signalling. Experimentally, it was shown that siRNA depletion of BCA2 led to increased EGFR protein levels while transient BCA2 overexpression reduced levels of the receptor. It was found that BCA2 overexpressing, EGF stimulated cells demonstrated reduced lysosomal degradation of both receptor and ligand. Associated with this, downstream EGFR signalling in BCA2 overexpressing cells was reduced in magnitude but prolonged in duration and ultimately cell viability was impaired. 3 These findings support a role for BCA2 in the endolysosomal system. In agreement with this it was shown that BCA2 overexpression inhibited the vesicle membrane association of Rab7, a regulator of late endocytosis and reported BCA2 interactor. Transferrin receptor levels and transferrin uptake were unaffected by BCA2 overexpression suggesting trafficking effects may be restricted to EGFR, a distinct class of receptor and/or to later (degradation) stages of endocytosis. This thesis provides a detailed exploration of BCA2 biology and presents evidence of a functional role for the protein in the endocytic regulation of EGFR. The mechanism/s underlying the complex relationship between BCA2 and breast cancer outcome have yet to be fully determined.

This thesis is concerned with the design of organometallic half sandwich complexes which can catalyse the conversion of oxidised coenzyme nicotinamide adenine dinucleotide to its reduced form. The coenzyme pair NAD+/NADH is involved in many biological processes, such as regulation of the redox balance, and DNA repair. Disturbance of the NAD+/NADH ratio can lead to cell death. In particular, cancer cells are under constant oxidative stress and therefore might be more susceptible to changes in the NAD+/NADH levels. A series of neutral Ru(II) complexes of the type [(η6-arene)Ru(N,N’)(L)] (arene = p-cymene (p-cym), hexamethylbenzene (hmb), biphenyl (bip), benzene (bn); N,N’ = N-(2-aminoethyl)-4-(trifluoromethyl)benzenesulfonamide (TfEn), N-(2-aminoethyl)-toluensulfonamide (TsEn), or N-(2-aminoethyl)-4-methylensulfonamide (MsEn)) were synthesized and fully characterized. The complexes were shown by 1H-NMR to catalyse region-selectively the transfer hydrogenation of NAD+ to 1,4-NADH. Comparison of the turnover frequencies (TOF) for the complexes show a trend in which a decrease in catalytic activity with the arene ligand follows the order bn > bip > p-cym > hmb and TfEn > TsEn > MsEn. Complex [(η6-bn)Ru(TfEn)Cl] (12) was found to be the most active with a TOF of 10.4 h-1. The catalytic cycle for the transfer hydrogenation reaction was studied for complex [(p-cym)Ru(TsEn)Cl] (2). The monotosylate ethylenediamine Ru(II) complexes were used to carry out, for the first time, transfer hydrogenation reactions in cellulo (A2780 ovarian cancer cells) using formate as a hydride source. The antiproliferative activity of six complexes on co-administration with non-toxic doses of sodium formate were studied. A significant potentiation of the antiproliferative activity by formate was observed. The concentrations of NAD+ and NADH in cells showed an important reduction in the NAD+/NADH ratio. This study demonstrates that it may be possible to use catalytic transfer hydrogenation as a strategy for multi-targeted redox mechanisms of action for anticancer drugs. In order to compare the anticancer mechanism of the monotosylate ethylenediamine Ru(II) complexes with the corresponding ethylenediamine (en) complexes, DNA binding studies were carried out. The complexes were shown to bind to nucleobases only moderately strongly and no direct coordination to calf thymus DNA was observed. The complexes can destabilize DNA, but they display low affinity towards DNA, which suggests that DNA is probably not involved in the mechanism of these family of compounds. Interaction of complex [(p-cym)Ru(TsEn)] (2) with glutathione (GSH) was also studied. The complex undergoes ligand substitution. Two Ru(II) dimers were formed as the main products of the reaction: ([((p-cym)Ru)2(μ-GS)3] and [((p-cym)Ru)2(μ-GS)2]). These dimers share structural similitudes with other reported Ru(II) arene anticancer drugs, which suggests that they could be responsible for the moderate antiproliferactive activity of complex 2. A series of CpxRh(III) (CpX = CpXPhPh, CpXPh or Cp*) complexes containing en or TfEn were synthesised and fully characterised. The complexes were shown to catalyse regioselectively the transfer hydrogenation of NAD+ to 1,4-NADH with higher TOF than their Ru(II) analogues. Rh(III)-en compounds were shown to be more active than the Rh(III)-TfEn analogues. The nature of the Cpx ring was shown to influence significantly the catalytic activity of the complexes following the trend: CpXPhPh > CpXPh > Cp*. Complex [(CpXPhPh)Rh(en)Cl]+ (19) was the most active, with a TOF of 24.2 h-1. The catalytic cycle for the transfer hydrogenation reaction was studied using complex [(Cp*)Rh(en)Cl]+ (17) and compared to that of the Ru(II) analogues, and was found to be similar. Antiproliferative activity of the complexes in combination with formate, in A2780 cells, was investigated, but the potentiation due to the transfer hydrogenation was much lower than that obtained with Ru(II) compounds.

The class of instruments considered in this thesis, scanning probe microscopes (SPM), raster scan a sensory probe over a surface to form both high resolution images and quantitative interaction measurements. Understanding and extracting information from SPM data has been considered extensively in the three decades since the first SPM. Major developments tend to be greeted with their own theory and data analysis techniques. The more gradual evolution of equipment has not, however, attracted the same level of theoretical consideration. In this thesis we consider the SPM from an instrumentation perspective, concentrating on two specific types of microscope: the scanning tunnelling microscope (STM) and the atomic force microscope (AFM). Both of these microscopes rely on a sensory probe or sensor to induce and measure the desired interaction. We have carefully considered a range of experiments from a `sensor-eye-view', both theoretically and experimentally. We first consider the effect of the geometry of AFM sensors on quantitative force measurements, identifying that the length of tips that the length of tips can induce an unwanted coupling of lateral and normal forces. We go further by developing methods to experimentally correct these force measurements along with designing a sensor which exploits symmetry to separate lateral and normal forces. We also consider the ways to automatically optimise the apex of the sensory probe of an STM to give the desired imaging resolution using a combination of prescribed routines and genetic algorithms. Image analysis techniques developed for this work have been developed into an open-source toolbox to automatically process and analyse SPM images. Finally, we use control theory to analyse the feedback controlling the SPM probe. We find that the methods used in the literature do not fully consider the method with which the control loop is implemented in SPM. We employ a modified approach which results in more realistic simulated SPM operation.

This study aims to evaluate a Scandinavian approach to outdoor learning, which is used in the UK. The approach, known as ‘Forest School’ involves children and young people spending regular time in natural woodland working on practical projects. Forest School promotes a child-led ethos, so children are encouraged to choose their own activities (Forest School Association, 2013). A Realist Synthesis (Pawson, 2006) was undertaken to develop an understanding of how Forest School works, according to existing research. Features of the context, change mechanisms and outcomes were abstracted to form a set of hypotheses. In line with a Realistic Evaluation (Pawson and Tilley, 1997), these hypotheses were tested through a case study of Forest School involving 14-16 year old pupils with special educational needs (SEN). Drawing on interview, observation, questionnaire and documentary evidence, the initial programme specification was refined through thematic analysis (Braun and Clarke, 2006) to create programme specification 2. Participants checked this in a Realist Interview (Pawson and Tilley, 1997) and a final programme specification was produced. The final programme specification presents findings through context + mechanism = outcome configurations. The study extends existing research by finding that Forest School can support confidence, social skills, language and communication, motivation and concentration, physical skills, knowledge and understanding of the world and emotional well-being and behaviour in young people aged 14-16 with SEN. The study further indicates that Forest School works differently for different pupils, depending on their individual characteristics. Strategies for best practice were illuminated which may be useful to other Forest School practitioners, such as a high level of adult practical skills. The evaluation has implications for professionals working with young people as it highlights how Forest School can promote positive outcomes for some young people aged 14-16 with SEN.

Persistent HPV infection can cause cervical intraepithelial neoplasia (CIN) and ultimately cervical cancer. Cervical cytology is currently used to screen for CIN with HPV testing recently emerging as an adjunct to cytology. Most women with HPV infection, however, will not go on to develop cancer. Therefore an additional biomarker is required to identify those women most at risk. The BD SurePath Plus™ test (SPP) is a novel immunocytochemistry assay based upon the detection of minichromosomemaintenance protein 2 (MCM2) and MCM7. Studies have shown both MCM2 and MCM7 have the potential for use as a biomarker for CIN2+ and cervical cancer. A two-­‐centre, prospective, observational study was devised to test SPP as triage test in women with persistent low-­‐grade cytology. Two commercial HPV tests were also examined in their role in triage of these women. A further study was conducted to examine viral integration and viral DNA methylation as potential biomarkers of high-­‐grade disease. An assay that determines the status of the HPV E2 gene and the Detection of Integrated Papillomavirus Sequences were used to assess integration. Bisulfite conversion followed by pyrosequencing was used to assess DNA methylation within two regions of the HPV genome (E2 and L1L2). Following the clinical study BD SurePath Plus™ was found to be inferior to HPV testing in the discrimination of high-­‐grade cervical disease. The age of women was found to significantly affect the results of all three tests. Viral integration and viral DNA methylation were both associated with high-­‐grade disease. New sites of viral integration were found in the study and the predilection of common fragile sites and repeat sequences as sites of integration was also reinforced. It was also discovered that integration could affect the accuracy of some HPV and biomarker tests.

T cell antigen receptors (TCRs) on CD8+ T cells recognise endogenously processed peptides bound to major histocompatibility complex (pMHC) antigens presented on the cell surface on almost all types of cells in the body, including tumour cells. The majority of tumour-associated peptide antigens (TAPAs) are derived from non-mutated self-proteins and are therefore subject to immunological tolerance, mainly through negative selection of high avidity T cells in the thymus. In addition, there is low presentation of pMHC on the surface of cancer cells. As a result, T cell responses tend to be weak and ineffective at killing tumour cells. ImmTACs (Immune mobilising monoclonal T cell receptors Against Cancer) are bispecific soluble biologics comprising a soluble TCR with an enhanced affinity for tumour-associated pMHCI fused to a humanised anti-CD3 single-chain antibody fragment (scFv) which redirect and activate T cells to lyse tumour cells. In this study, the potency, sensitivity, and specificity of ImmTACs was investigated for pMHCI epitopes derived from four tumour associated antigens (TAAs): (1) gp100, (2) MAGE-A3, (3) Melan-A/MART-1, and (4) NY-ESO-1/LAGE-1. A comprehensive range of assays and methodologies have been established to characterise the ImmTAC reagents. Cytokine release assays such as IFN-γ and Granzyme B ELISpot were used to evaluate the specificity and biological activity of ImmTACs. In concentration-response experiments, all four ImmTACs produced EC50 values in the range of 100 picomolar or lower demonstrating a high degree of sensitivity despite low epitope numbers. Killing assays, including LDH-release for assessing short-term lysis and IncuCyte technology to visualise long-term killing kinetics in real time, show that redirected T cells potently kill their targets. Furthermore, in vitro screening against a panel of antigen negative, primary human cell lines have shown that ImmTACs are highly specific and only activate T cells against target cells presenting their cognate pMHC. The potency of ImmTACs was also investigated using tumour infiltrating lymphocytes (TILs) extracted from tumour specimens and with tumour-derived cancer cells as targets. An HLA-A2, gp100 specific ImmTAC has received phase I clinical trial regulatory approval in the UK and in the US on the basis of this in vitro data, which has been used to determine minimal anticipated biological effect level (MABEL). The clinical trial is currently in progress.

Cisplatin has been used to treat various types of cancers for over 30 years, however, a number of serious side-effects of cisplatin have stimulated the quest for other metal-based anticancer agents. Iridium complexes are generally thought to be too inert to possess high reactivity, and therefore, there are only a few previous reports of the antitumour activity of iridium complexes. In this thesis a wide range of organometallic IrIII cyclopentadienyl complexes of the type [(η5-Cpx)Ir(XY)Cl]0/+ (where Cpx = pentamethylcyclopentadienyl (Cp*), tetramethyl(phenyl)cyclopentadienyl (Cpxph) or tetramethyl(biphenyl)cyclopentadienyl (Cpxbiph), XY = N,N-, N,O- or C N-chelating ligand) has been synthesised and characterised. All the complexes hydrolyse rapidly in aqueous solution. Complexes with N,N-chelating ligands readily form adducts with 9-ethylguanine but not 9-ethyladenine; C N- or N,O-chelated complexes bind to both purines. Guanine residues are preferential binding sites for 1,10-phenanthroline complexes on plasmid DNA. Replacement of the neutral N,N-bound chelating ligand by the negatively-charged C,N-bound analogues can improve biological activity. In addition, cytotoxic potency towards A2780 human ovarian cancer cells increases with phenyl substitution on Cp*: Cpxbiph > Cpxph > Cp*. This can be rationalised by increased hydrophobicity with more extended phenyl ring, resulting in increased cellular uptake and increased intercalative ability. Notably, several complexes exhibited submicromolar anticancer activity. The interconversion of 1,4-NADH and NAD+ through hydride-transfer reactions in the presence of cyclopentadienyl IrIII aqua complexes was studied. It is shown that the IrIII aqua complexes not only converts NAD+ to 1,4-NADH using formate as the hydride source, but can also catalyse the reverse reaction with hydride donation from 1,4-NADH to a iridium centre, recovered by protonation of bound hydride with generation of H2. This work demonstrates how the aqueous chemistry, nucleobase binding and anticancer activity of the IrIII cyclopentadienyl complexes can be controlled and fine-tuned by the modification of the chelating and cyclopentadienyl ligands. The results suggest that this new class of organometalic Ir(III) complexes is well suited for development as anticancer agents.

Mitochondria are cytoplasmic organelles that are found in almost all mammalian cells. Mitochondria contain their own genome, mitochondrial DNA (mtDNA) that encodes 13 subunits of the electron transfer chain, which is the primary generator of cellular energy. Precise regulation of mtDNA copy number is essential for normal cell function and also the differentiation of stem cells into specialized cell types. Abnormal regulation of mtDNA copy number is associated with cellular dysfunction, mitochondrial disease and more recently cancer. Glioblastoma multiforme (GBM) is a highly malignant subgroup of brain tumors that exhibit similar characteristics to human neural stem cells (hNSCs) including multipotency and the expression of the stem cell factors. It is unknown how GBM cells regulate their mtDNA copy number during differentiation and whether this differs to hNSCs. Furthermore, it is unknown what role mtDNA plays in the gene expression profiles and the tumorigenicity of GBM. To address these issues, GBM cells and hNSCs were differentiated for 28 days and their mtDNA copy number and gene expression were analyzed. In addition, GBM cells were progressively depleted of their mtDNA using the depletion agent, 2'-3'-dideoxycytidine, and their in vivo tumorigenicity assessed. hNSCs and GBM cell lines regulated their copy number in a differential manner during differentiation. hNSCs progressively expanded their mtDNA copy number and adopted a differentiated phenotype whilst GBM cells failed to mimic these processes and their differentiation was incomplete. In addition, progressive depletion of mtDNA copy number in GBM cells resulted in reduced proliferation rates and the down regulation of stem cell factors. In vivo, mtDNA depleted GBM cells formed tumors at a reduced rate and frequency relative to nondepleted cells. These outcomes demonstrate that mtDNA copy number is abnormally regulated in GBM cells and hinders their ability to complete differentiation. The failure of mtDNA-depleted GBM cells to consistently generate tumors strongly suggests that maintenance of mtDNA copy number is essential for GBM cells to be tumorigenic.

Tuberous sclerosis is an autosomal dominant genetic disorder characterised by the development of benign tumours in multiple organs. It is caused by mutations in the TSC1 or TSC2 tumour suppressor gene, leading to hyperactivation of mTOR signalling in affected tissues. Rapamycin and its analogues are mTOR inhibitors and have been used to treat tuberous sclerosis in both pre-clinical and clinical trials. However, tumours usually relapse after drug withdrawal. The aims of this project were to identify novel agents and strategies for prevention and therapy of tuberous sclerosis using mouse models. First T2 weighted MRI was evaluated for assessment of renal lesions in Tsc1+/- and Tsc2+/- mouse models. MRI identified all types of Tsc-associated renal lesions in both Tsc mouse models. The smallest detectable lesions were <0.1 mm3. Eighty five percent of all renal lesions detected in a first scan at 12 months of age were re-identified in a second scan 2 months later. Between the two scans, MRI revealed a significant increase in the total number and volume of lesions in 9 untreated mice. Compared to histological analysis, MRI detected most cysts and papillary tumours (64%) but only a minority of solid tumours (30%). Metformin is a mild inhibitor of mTOR. The therapeutic effect of metformin on renal lesions in Tsc1+/- mice was investigated using T2 weighted MRI and histological analysis. Metformin treatment for 9 months had no significant effect on renal lesions in these mice. Finally, the preventive effects on renal lesions in Tsc2+/- mice of rapamycin, metformin or both agents in combination were assessed using histological analysis. Treatment started from one month of age and continued for 7 to 9 months. Rapamycin or rapamycin plus metformin but not metformin alone effectively blocked the development of renal lesions including cysts, adenomas and carcinomas through the inhibition of mTOR signalling. These findings suggest that mTOR inhibition may be an effective strategy for preventing emergence of disease manifestations in tuberous sclerosis.