The national identification of high performing providers in surgery is of prime importance to patients, surgeons and commissioners of healthcare. This thesis explores how high performance is identified, defined and measured nationally and attempts to identify the factors that underlie high performance in colorectal cancer surgery during the peri-operative period. An introduction into the determinants of high performance in surgery as well as defining quality as it pertains to surgery is then undertaken. Identification of available national data sources and metrics for national performance are then identified. Comparison is made between voluntary and compulsory reporting systems highlighting greater capture of peri-operative mortality in compulsory reporting datasets. A novel marker that reflects outcome following complication management is developed. This marker is based on re-operations and is derived from compulsory reporting datasets. The use of non-operative re-interventions is then assessed in oesophago-gastric cancer resections as proof of concept. An appraisal of all colorectal cancer units in England is then undertaken using a panel of metrics demonstrating that analysis on a single marker alone may be too simplistic. Identifying factors that pertain to high performance beyond those available from routinely available datasets using a novel methodological approach called HiPer (High Performance) is performed. The interview based methodology identified rich qualitative factors in a group of colorectal cancer units worldwide that may be causal in their performance status. Finally, results from the interview study were related to hard outcome data from each unit which demonstrated some correlation between the HiPer methodology and the outcome data in the final section of the feasibility study. The implications of this may be that a dual approach of analysing routinely collected data with a more qualitative HiPer style methodology may help us better understand how high performing units achieve their results.

Human T-lymphotropic virus type 1 (HTLV-1) causes the debilitating neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a minority of infected individuals, while the majority are lifelong asymptomatic carriers (ACs). The effectiveness of the CD8+ T-cell response to HTLV-1 is a major determinant of the HTLV-1 proviral load (PVL) and the associated HAM/TSP risk. Host genotype, particularly of HLA class I, strongly influences CD8+ T-cell effectiveness against HTLV-1. Furthermore, possession of the KIR gene 2DL2 was recently shown to enhance protective and detrimental HLA class I-associated immunity to HTLV-1 and HCV (Seich al Basatena et al., 2011). The proposed mechanism explaining this effect is a KIR expression-induced increase in activation thresholds, thus promoting survival. I investigated KIR expression and function on T-cells and NK cells in HTLV-1 infection, focussing on CD8+ T-cells and using new anti-KIR mAbs to analyse 2DL2 expression alongside four other KIRs. This thesis presents evidence supporting the genetic findings and the KIR expression-induced CD8+ T-cell survival hypothesis. AC status was associated with expanded 2DL2+CD8+ T EM cell populations and 2DL2+ and 2DL3+CD8+ T EM cell frequencies were inversely correlated with PVL, consistent with a protective role for 2DL2 (and potentially 2DL3) expression on CD8+ T-cells in HTLV-1 infection. Rather than a generalised reduction in CD8+ T-cell function, specific KIR expression was associated with a shift in effector function profile towards a cytotoxic phenotype without cytokine production. This is consistent with raised activation thresholds and may suggest fine-tuning of CD8+ T-cell effector functions by KIRs, preserving killing without causing inflammation. I further speculate that 2DL2/3 could be a context-dependent modulator of CD8+ T-cell function, optimally tuning responses and contributing to CD8+ T-cell effectiveness. Together with the longevity of 2DL2/3+CD8+ T-cells in vivo, this hypothesis requires further testing, since it may be relevant to other persistent viral infections.

The classical view in systems neuroscience is that individual regions of the brain are specialised for the execution of specific tasks. This position is supported by a long history of lesion studies and more recently by a wealth of functional neuroimaging research, which has provided insights into how the anatomy of the brain relates to behaviour. However, in recent years this classical view has changed. Instead, new techniques have revealed intrinsic functional connectivity networks (ICNs) that reflect underlying patterns of structural connectivity, now considered to be the fundamental neural architecture supporting cognition and behaviour (Adams, 1982, Smith et al. 2009b, Honey et al. 2009). The activity of these ICNs is dependent on intrinsic fluctuations in their activity and the behavioural context, which dynamically reconfigure over time (Fox et al. 2005, Ances et al. 2009). Therefore, investigation of brain networks needs to consider not only the structural connections that constrain functional interactions, but also dynamic changes in functional interactions. An ongoing challenge, therefore, is to define a framework incorporating these neural dynamics that combines insights from structure, function and behaviour. Dynamical systems theory may provide a flexible framework that combines all of these levels. Current theory proposes that healthy neural dynamics operate in a multistable regime that promotes flexible information processing and behaviour (Kelso 2012, Tognoli and Kelso 2014, Shanahan 2012, 2010b, a, Friston 1997, Leech and Sharp 2014, Irner 2007). Large-scale multistable dynamics are likely constrained by underlying structural connections between brain regions. (Honey et al. 2009, Deco, Jirsa, McIntosh, Sporns, and Kötter 2009, Deco, Jirsa, and McIntosh 2011, Deco et al. 2008, Cabral et al. 2011, Cabral et al. 2012). However, it is unclear how such multistable dynamics may relate to behaviour, or how they are constrained by network structure. Moreover, whilst such dynamical accounts place considerable importance on the structural connectivity of the brain, it is unclear how the multistable dynamics are altered when pathological processes result in structural disconnections. In this thesis, I explore such dynamical accounts of the brain using a range of neuroimaging and computational approaches, and examine the implications of this level of description in one example of structural disconnection, namely the consequences of traumatic brain injury.

Clinical scores, serum and faecal markers, and endoscopy all have limitations in their use as instruments to monitor disease activity in Crohn's disease (CD). Recently, 18F-FDG-PET and novel MRI techniques have been proposed as sensitive and specific methods to quantify the inflammatory load. The aim of this work was to assess the reliability, responsiveness and, to an extent, the validity of outcome measures in these modalities, in monitoring inflammatory activity over a 12-week interval. In addition, two receptors, TSPO and IL-2R were assessed on tissue specimens ex-vivo for their potential to act as alternative targets for molecular imaging in CD. Three distinct groups of patients were recruited, 2 of which participated in the clinical imaging study, and one to donate tissue for the laboratory work. Dual timepoint FDG-PET and MRI scanning was performed within 1 week (Group 1) to assess the test-retest reliability of the imaging outcome measures, and before, and twelve weeks into anti-TNFα therapy (Group 2) to assess their responsiveness indices. The third group contributed tissue during scheduled intestinal resection for assessment of TSPO and IL-2R interactions with their corresponding radioligands. To support the latter study, stored tissue sections were also obtained for immunohistochemical assessment of target receptor expression. Results on 22 patients show that PET endpoints such as SUVMAX and SUVMEAN have high responsiveness and reliability indices and demonstrated significant differences in anti-TNF responders compared to non-responders. The finding of luminal FDG signal may affect the face validity of the scan. MRI modalities appeared less responsive at three months. Analysis on ex-vivo specimens showed increased abundance of TSPO in normal bowel, but a relative over-expression in inflamed specimens which was not statistically significant. IL-2R appeared more abundant in transmural sections containing severe CD, but autoradiographic corroboration was not achieved for technical reasons.

A top-down systems biology approach investigating metabolic responses to external stimuli or physiological processes requires multivariate statistical tools to identify metabolites associated with the global biochemical changes in a supra-organism. In this thesis I describe several tools I have developed to improve or supplement currently used methods in molecular epidemiology studies. First, I describe the MetaboNetworks toolbox which is able to create custom, multi-compartmental metabolic reaction networks for a supra-organism, combining both mammalian and microbial reactions. These networks are essentially a summary of the supra-organisms homeostatic signature. Second, I describe a novel statistical spectroscopy approach called STORM which aids in the elucidation of unknown biomarker signals in 1H NMR spectra. Third, I describe the Metabolome-Wide Association Study on obesity in U.S. and U.K. populations. Many novel metabolic associations with obesity are described in a systems framework, among which metabolites associated with energy, skeletal muscle, lipid, amino acid and gut microbial metabolism. Last, I describe a new multivariate approach to adjust for confounders, CA-OPLS. Correcting for confounders is an essential aspect in molecular epidemiology studies as metabolites can be related to a variety of factors such as lifestyle, diet and environmental exposures which or may not be causally related to disease risk. In developing CA-OPLS another aim was to simultaneously eliminate/minimize the effects of different types of sampling bias which are often not taken into account in modelling metabonomics data with current methods.

The mature FMS receptor has been demonstrated here as being subject to constitutive loss as shown by S35 methionine labelling, live antibody labelling surface loss, surface biotinylation and FACS analysis with a half-life estimated at between 1 and 2 hours. This receptor turnover has been shown to be independent of kinase activity by the use of kinase impaired mutants and also a highly specific FMS kinase inhibitor, GW2580 that is able to inhibit the ligandinduced downregulation of FMS. The intracellular domain of the FMS receptor is believed not to be involved in the proteins constitutive downregulation from experiments performed here using domain deletion mutations and broad spectrum inhibition of other intracellular kinases. All experiments demonstrated FMS constitutive loss at the same rate as the wild-type and untreated receptor. The constitutive turnover has been further demonstrated to be distinct from FMS downregulation occurring via shedding and RIP mediated by ADAM17 and the γ-secretase complex respectively. We have demonstrated that the mechanism of receptor downregulation is independent of clathrin, caveolin and dynamin. FMS has been demonstrated here to pass through EEA1 and Rab7 positive compartments before its delivery to the lysosome for degradation. Our preliminary data suggests that the mechanism of FMS downregulation by the constitutive pathway is equivalent to that induced by M-CSF, but dissimilar from that of shedding and RIP degradation. These results also suggest that the c-KIT and VEGFR2 receptors are also subject to a similar mechanism of constitutive downregulation and both are also committed to degraded via the lysosome. To gain further insight into a possible raison d'être for the seemingly wasteful process of constitutively degrading the FMS receptor and also possibly the c-KIT and VEGFR2 receptors the 3 synthesis rates of FMS was analysed. The rates of FMS synthesis were shown to be constant irrespective of the receptors mature levels. Inhibition of the receptors degradation by lysosomal inhibition resulted in an increase in the levels of the mature receptor within cells. This was also demonstrated for the c-KIT and VEGFR2 receptors. As the synthesis rate of FMS was shown to be indepedent of its mature levels, we suggest that the constitutive degradation pathway is part of a homoeostatic mechanism whose function is to buffer receptor levels, preventing their overexpression in a simplistic system where they are synthesised at a constant rate.

Tissue factor pathway inhibitor (TFPI) is a principal inhibitor of the initiation of coagulation through inhibition of factor (F) Xa and tissue factor (TF)/FVIIa. While TFPI on its own is a poor inhibitor of FXa, protein S acts as cofactor for TFPI through interaction with TFPI Kunitz 3 domain, greatly enhancing FXa inhibition (~10-fold). The aim of my thesis was to further characterise the molecular basis underlying the TFPI cofactor function of protein S by identifying the complementary functional interaction site on protein S required for TFPI enhancement. I have screened over 40 protein S variants comprising substitutions spanning the whole protein S molecule and have identified the C-terminal sex hormone-binding globulin (SHBG)-like domain to be important for the enhancement of TFPI-mediated inhibition of thrombin generation in plasma. This was demonstrated using a protein S/growth arrest-specific 6 (Gas6) chimera, in which the SHBG-like domain was replaced with the corresponding domain in Gas6. Further examination of this chimera using FXa inhibition assays together with direct binding studies confirmed that the protein S SHBG-like domain is essential for the interaction with TFPI and the enhancement of TFPI-mediated inhibition of FXa. TFPI has also been reported to bind to FV in circulation. In my thesis I have aimed to evaluate the effects of FV interaction with TFPI upon its inhibition of FXa. While FV did not enhance TFPI on its own, it further enhanced the inhibition of FXa by TFPI in the presence of protein S (~32-fold by comparison to TFPI alone), suggesting that FV acts together with protein S as a synergistic cofactor for TFPI. Co-immunoprecipitation experiments revealed a direct interaction between TFPI and FV which was augmented by the presence of protein S. I propose that FV, in cooperation with protein S localise TFPI to the activated membrane surface in a position favourable for the efficient inhibition of FXa. Collectively, my findings shed light on the molecular mechanisms of TFPI enhancement by its cofactors protein S and FV.

DNA damage transactivates TP53-regulated surveillance mechanisms that are crucial in maintaining cellular integrity and suppressing tumorigenesis. TP53 mediates this directly by transcriptionally modulating gene and microRNA (miRNA) expression and by regulating miRNA biogenesis through interaction with the DROSHA complex. However, the regulative mechanism of miRNA-AGO2 loading and the global change in AGO2 binding to its gene targets in response to DNA damage have not been investigated yet. In addition, the role of other non-coding RNAs, such as snoRNAs, in the TP53-mediated response to DNA damage has not yet been defined. Here we identify a novel group of TP53-regulated miRNAs and show that DNA damage induces and reduces the loading of a subset of miRNAs, including the let-7 family members onto AGO2, in a TP53-dependent manner and that this previously undescribed process is most likely the result of TP53 binding to AGO2. These findings indicate that TP53 control of AGO2 loading is a new mechanism of miRNA regulation in carcinogenesis. Using AGO2 RIP-Seq and PAR-CLIP we also show that TP53 modulates the reduction, induction and remodelling of AGO2 binding to the 3'UTR of different mRNA targets at specific RNA motifs. Furthermore, we determine on a transcriptome-wide level the miRNA-mRNA interaction networks involved in the response to DNA damage both in the presence or absence of TP53. We also show that those miRNAs whose cellular abundance or differential loading onto AGO2 is regulated by TP53, are involved in an intricate network of regulatory feedback and feedforward circuits that fine tune gene expression levels in response to DNA damage to permit DNA repair or the initiation of programmed cell death. Finally, we demonstrate a relationship between TP53 and the GAS5-derived snoRNAs both in cancer cell lines and human tissue samples which implies that this class of non-coding RNAs might also be involved in coordinating the TP53-mediated response. These findings provide a novel insight into the complexities surrounding the role of non-coding RNAs in the TP53 response to DNA damage and their relevance to carcinogenesis.

Virtual reality (VR) simulation and robotic surgery represent two focus areas for research and development in Otolaryngology-Head & Neck Surgery. This thesis was driven by a desire to deliver improvements in surgical training and patient care. The development and long-term prospective clinical evaluation of three novel robotic applications in Head & Neck surgery were investigated. The results suggest that robotic assisted thyroidectomy and robotic assisted parathyroidectomy are safe, feasible alternatives to conventional surgery. The primary advantage is the avoidance of a neck scar. The approach occupies a niche role that is justified in patients who have cultural or biological drivers to avoid a neck scar. Improvement in surgical exposure was necessary. A novel soft-tissue retractor was designed and manufactured to address this issue. Transoral robotic surgery represents a promising treatment option for patients with obstructive sleep apnoea who cannot tolerate or fail all the other treatment modalities. Biometric measures represent an important tool when assessing patient suitability for TORS. Only those who have undergone appropriate training, proctoring and licensure should perform robotic surgery. Safe implementation is essential. The studies of VR temporal bone simulation served as a preparatory to introducing VR simulation for robotic head and neck surgery. The face, content and construct validation of a novel temporal bone simulator was demonstrated. Further studies were conducted to benchmark and pilot a VR skills curriculum and assess the role of case specific surgical rehearsal. Simulation training represented a useful adjunct. This body work demonstrates that both technologies can be integrated to deliver effective robotic surgical training to enhance surgical performance and improve patient care.

Stroma and parenchyma represent the supportive and functional components in every organ of the body, respectively. Beyond their ability to produce structural support and to differentiate into tissues of mesodermal origin, mesenchymal stromal cells (MSC) exhibit potent immunomodulatory properties. Such a function requires an activation step ('licensing signal') provided by the inflammatory microenvironment to which MSC are exposed. My results have attributed immunosuppressive effects of MSC to essential amino acid (EAA) deprivation. Amongst the EAA consuming enzymes examined, blocking nitric oxide synthase 2 (NOS2) and histidine decarboxylase (HDC) both resulted in impaired anti-proliferative activity of MSC, while NOS2 appeared to be a more prominent effector. My results have also demonstrated that TNF-α and IFN-γ differently account for NOS2 and HDC up-regulation, respectively. Furthermore, MyD88 and NF-κB were identified as upstream mediators for initiating NOS2 production. The role of TNF-α and NOS2 in MSC-mediated immunosuppression was assessed in vivo using a murine model of peritonitis. MSC treatment remarkably reduced the local inflammatory response during acute peritoneal inflammation. Nevertheless, both Nos2-/- and Tnfr1/r2-/- MSC delivered similar effects compared to WT MSC, indicating the presence of other complementary mechanisms in MSC-mediated immunosuppression in vivo. In addition to their immunomodulatory properties, MSC are fundamental in regulating self-renewal and differentiation of haematopoietic stem cells (HSC). MSC protect HSC from potential damage by maintaining their quiescence. My results have revealed that the ability of MSC to enhance the quiescence of HSC was associated with cell-cycle arrest induced by NOS2. As striking parallels exist between the normal and malignant stem cell niche, I investigated the ability of MSC to protect haematopoietic malignant cells from chemotherapy-induced apoptosis. MSC were observed to confer protection from etoposide-induced necrosis of EL4 cells, possibly due to their ability to suppress EL4 proliferation. Collectively, my results have demonstrated the role of MSC across the fields of immunomodulation, niche-supporting and anti-apoptotic effects.