There are more than 600,000 emergency general surgery admissions per year in England. These patients comprise about 50 percent of general surgical workload but make up 80-90 percent of all general surgical deaths. In recent years surgical colleges and societies in the UK have warned of significant variability in the quality of care between hospitals but, to date, little formal evaluation of the quality of care in emergency general surgery exists. This thesis uses the Structure/Process/Outcome quality assessment framework, devised by Avedis Donabedian, to examine quality of care in emergency general surgery across all three of these domains. A study of high risk emergency general surgical admissions using the administrative Hospital Episode Statistics dataset demonstrated significant variability in 30-day in-hospital mortality between NHS Trusts. Investigation of NHS Trust structure was performed using data from the Department of Health. There were significant differences in the provision of intensive care beds and in the utilization of computed tomography and ultrasound scanning between low mortality and high mortality NHS Trusts. The process of care was assessed using an explicit checklist for the admission phase of care and using ethnographic field notes for patients’ subsequent hospital stay. Across 5 London hospitals, process reliability during admissions to hospital was poor, with nearly 20% of recommended processes omitted. Failures in the process of care were also common in subsequent ward based care. Failures were considered to be highly preventable and frequently caused harm to patients or delayed their discharge. Overall, this thesis has identified significant variability in the quality of care for emergency general surgical patients in structure, process and outcomes. While the thesis does not evaluate every single aspect of patient care it demonstrates the degree of improvement required in emergency surgical care and provides some recommendations for future quality improvement.

Chronic thromboembolic pulmonary hypertension (CTEPH) is an uncommon sequela of acute pulmonary embolism and, untreated, leads to right ventricular (RV) failure and death. Despite its growing recognition, methods for the detection of early RV insufficiency and prediction of clinical deterioration, important to optimum preservation of RV function, are currently suboptimal. Furthermore, underlying genetic predisposition to CTEPH, unexplained by defective fibrinolysis, remains largely unexplored. The RV's physiological response to chronic thromboembolic obstruction is arguably best described by RV pressure volume loops which, historically, are best obtained using the conductance catheter. Although invasive, conductance has an indisputable advantage over current imaging modalities; catheters measure dynamic ventricular pressure and volume throughout the cardiac cycle. Using this technique, abnormal RV pressure volume loops are demonstrated in response to chronic thrombotic obstruction, independent of resting haemodynamic criteria diagnostic of CTEPH. Pressure volume differences and accrual of an exercise gas exchange deficit further suggest early 'subclinical' RV adaptation. The genetic architecture of CTEPH is also explored using high-throughput sequencing of unrelated patients. This shows that rare DNA variants in CTEPH that are predicted to harbour deleterious effects are not over-represented in fibrinolytic pathways. Finally, prognostication in CTEPH is evaluated using a clinical deterioration model which is shown to be predicted by patient-reported outcomes at diagnosis. In conclusion, RV and pulmonary circulatory function in chronic thromboembolic pulmonary vascular disease are inadequately characterised by existing routine methods. Links between the observed physiological deficits, risk of clinical deterioration and abnormal genetic architecture warrant further evaluation in this rare disease.

Detailed morphometric analysis of the neonatal brain is required to characterise normal brain development and investigate the neuroanatomical correlates of cognitive impairments. The segmentation of the brain in Magnetic Resonance Imaging (MRI) is a prerequisite to obtain quantitative measurements of regional brain structures. These measurements obtained at term-equivalent or early preterm age may lead to improved understanding of brain growth and may help evaluate long-term neurodevelopmental performance at an early stage. This thesis focuses on the development of an accurate segmentation algorithm for the neonatal brain MR images and its application in large cohorts of subjects. Neonatal brain segmentation is challenging due to the large anatomical variability as a result of the rapid brain development in the neonatal period. The lack of training data in the neonatal period, encoded in brain atlases, further hinders the development of automatic segmentation tools. A novel algorithm for the tissue segmentation of the neonatal brain is proposed. The algorithm is extended for the regional brain segmentation. This is the first segmentation method for the parcellation of the developing neonatal brain into multiple structures. A novel method is further proposed for the group-wise segmentation of the data that utilizes unlabelled data to complement the labelling information of brain atlases. Previous studies in the literature tended to overestimate the extent of the cortical region. A method based on the morphology of the cortex is introduced to correct for this over-segmentation. The segmentation method is applied on an extensive database of neonatal MR images. Regional volumetric, surface and diffusion tensor imaging measurements are derived from the early preterm period to term-equivalent age. These measurements allow characterisation of the regional brain development and the investigation of correlations with clinical factors. Finally, a spatio-temporal structural atlas is constructed for multiple regions of the neonatal brain.

Yir016w (named Sdm1 for deficient in Separation of Daughter and Mother cells) was found in a screen for Saccharomyces cerevisiae cell separation-defective mutants, where yir016w cells were clumped together in groups of three or four. This gene was of special interest due to its ability to interact with Ace2 and members of the RAM (Regulation of Ace2 and Morphogeneis) network, which exert control over cell separation. This study aimed to elucidate the function of this previously uncharacterized gene YIR016W. The cell separation defect of sdm1 cells was found to be milder than that of RAM mutants, although still of statistical significance. Also, sdm1 cells neither showed the lack of polarization of the RAM mutants, nor any defects in the localization of Cts1, a key chitinase required for cell separation controlled by the transcription factor Ace2. Instead of a role in the RAM network, Sdm1 may play a role in protein trafficking, as sdm1 cells have vacuolar defects. This study also aimed to localize Sdm1. The protein responsible for Sdm1 expression is Ume6, a key transcriptional regulator of meiosis. While an Sdm1-GFP fusion was not visible under normal growth conditions, under meiosis-inducing conditions the signal was strong enough to be visualized. Using co-localization, Sdm1-GFP was found to be present in the Golgi body, and analysis of sdm1 cells show that they contained defects in the division of their vacuoles. These results point at additional functions of Sdm1, such as the control of mitosis-meiosis switching and protein processing and export. In addition, a second protein, Yol036w (termed Sdm2) was investigated, as it interacted with both the RAM network and Sdm1. yol036w cells showed no significant phenotypes in our screens, however, its interactions support the idea that it might also be involved in protein trafficking, or in cell wall biogenesis.

The acute respiratory distress syndrome (ARDS) remains a major cause of morbidity and mortality in the intensive care. Despite improvements in intensive care and advances in respiratory support, mortality remains high with no active treatments. Tumour necrosis factor (TNF) is a cytokine that has been implicated in ARDS for over 30 years but its precise roles remain elusive. It signals through two main receptors - the p55 TNF receptor and p75 TNF receptor. The first aspect of this thesis investigated the roles of TNF receptors (TNFR) in the early phase of acute acid-induced lung injury. Using genetically modified mice we discovered that alveolar oedema, as a result of acid aspiration, was specifically mediated through the p55-TNFR, whereas, the p75-TNFR promoted a protective effect. Alveolar oedema formation occurred through an effect independent to the downstream inflammatory events, but instead through the activation of TNF/p55-TNFR/caspase-8 death signalling specifically in the alveolar epithelium. Furthermore, this death-signalling axis led to a reduced alveolar epithelial fluid clearance rate. Epithelial dysfunction occurred prior to epithelial cell death and pharmacological blockade of caspase-8 rescued epithelial function with improvements in gas exchange, suggesting that the activation of caspase-8 per se induced this functional deficit in the alveolar epithelium. The second part of the thesis describes the development of a longer-term model of acid aspiration aimed at extending investigation into the later, arguably more clinically relevant, phases of lung injury (0-10 days). Mice showed respiratory physiology that reached clinical ARDS criteria with significant inflammation and epithelial/endothelial injury, which importantly, resolved facilitating investigation into reparative processes. This model was further characterised using novel flow cytometry protocols to examine the compartmental location of leukocytes during the various phases of ARDS. This model provides a translational platform to allow investigation into the injurious, inflammatory, and resolution mechanisms of ARDS.

Infection with HIV-1 results in the progressive dysfunction of the immune system eventually leading to AIDS, characterised by low CD4+ T lymphocyte counts and increased susceptibility to opportunistic infections. In contrast to HIV-1, individuals infected with HIV-2 often remain asymptomatic, with lower viral loads and higher CD4+ T cell counts throughout the course of disease. Furthermore, HIV-2+ individuals display enhanced HIV-specific T cell responses. In addition, HIV-1 disease progression is slower in patients with pre-existing HIV-2 infection. Plasmacytoid dendritic cells (pDCs) are key mediators of the early innate immune response. Upon viral infection, pDCs secrete high levels of type I IFN (IFN-α/β) which limit viral replication and prime the adaptive immune response. Activated pDCs, in particular excessive IFN-α/β, have been implicated in HIV-1 immunopathogenesis. Specifically pDCs may contribute to the recruitment of target cells to the site of HIV-1 infection, increased apoptosis of immune cells and suppression of memory T cell responses. The aim of this study was to compare the abilities of HIV-1 and HIV-2 to activate pDCs in vitro. HIV-1 was a more potent inducer of type I IFN responses in PBMCs compared to HIV-2, measured at both the transcriptional level, and by measuring IFN-α/β secretion into cell culture supernatants. Furthermore, HIV-2, but not HIV-1, inhibited IFN-α production in response to synthetic stimuli. Phenotypic analysis of pDCs by flow cytometry revealed that both HIV-1 and HIV-2 were equally able to induce an up-regulation of co-stimulatory marker expression. Measurement of co-stimulatory molecule expression in conjunction with IFN-α secretion showed that HIV-1 favoured an IFN response, whereas HIV-2 preferentially maturated pDCs towards an antigen-presenting cell (APC) phenotype. The ability of HIV-2 to mature pDCs into APCs while reducing IFN-α secretion may be an important contributor to more robust T cell responses and therefore slower progressing HIV disease.

Preterm delivery occurs in 10% of all births. It is the major cause of infant death and handicap in the developed countries and accounts for 65% of neonatal deaths and 50% of childhood neurological disabilities. At the end of pregnancy, 'pro-labour' factors begin to mediate remodeling of the cervix resulting in cervical ripening and dilatation, uterine contractility and decidual/fetal membrane activation. The amnion plays an important role in the onset of human labour. It is a major site for prostaglandins (PG) and inflammatory cytokine synthesis which increases both before and during labour. Amnion derived inflammatory cytokines and prostaglandins contribute to the relaxation of the lower uterine segment and to cervical ripening. Oxytocin (OT) and oxytocin receptor (OTR) are classically considered to play a fundamental role in the mechanism of labour as OT stimulates uterine contractions and OTR antagonists are clinically used as tocolytics. The increase in OT and OTR expressions were observed in tissues other than myometrium, including the breast cells, decidua and amnion. However, amnion is not a contractile tissue and therefore the physiological role of the OT/OTR is less obvious. We hypothesised that the regulation of OT/OTR in human amnion is linked to NF-κB activity and plays an important role in the onset of labour. We have demonstrated that in human amnion, labour induces expression of both OT and OTR expression. Using specific inhibitors and siRNA target knockdown studies, we have shown that unlike the myometrial OTR, OT binding to OTR in human amnion drives the receptor to couple with Gαi2 and Gαi3, but not Gαq. This subsequently triggers the sequential activation of ERK, p38 MAPKs and NF-κB signalling cascades leading to PG and proinflammatory cytokine/chemokine synthesis. This suggests that OT not only induces uterine contractions but also plays a role in triggering the onset of labour by mediating the proinflammatory effects in the amnion. These proinflammatory effects of OT were suppressed by an OTR-specific antagonist, ornithine vasotocin (OVT), indicating that OT exerts its effects predominantly via OTR. However, the commonly used OTR antagonist, atosiban, had no effect on OT induced proinflammatory effects. Unexpectedly, atosiban treatment alone resulted in activation of inflammatory mediators such as MAPKs and NF-κB leading to downstream pro-labour gene expressions via Gαi. Activation of such inflammatory processes within the uterus initiates labor, whereas exposure to inflammation may be associated with fetal brain damage in preterm and term infants. Therefore, atosiban could exacerbate inflammation in the context of preterm birth and potentially have an effect on neonatal outcome. With this in mind the future development of OTR antagonists to prevent preterm birth will need to take into account the effects upon differential OTR G-protein coupling.

In this thesis, I use clinical assessments and neuroimaging modalities to investigate the pathophysiology underlying impulse control disorders (ICDs) and related addictive behaviours in Parkinson's disease (PD). Using PET with 11C-raclopride, I found that PD patients with a range of ICDs showed an increase in dopamine release in their ventral striatum on exposure to reward-related visual cues compared to PD controls. Further, the amount of increased dopamine release in patients with single ICD was similar to those with comorbid ICDs, suggesting the sensitisation of ventral striatum of PD ICD patients, regardless of their disease load. I also found that PD ICD patients with punding, characterised by stereotypical behaviours with similarities to obsessive compulsive disorders, had lower baseline dopamine D2 receptor binding in the dorsal, but not ventral, striatum at rest compared to controls. In the fMRI imaging study with a block design paradigm, PD patients with compulsive sexual behaviours (CSB) showed increase in sexual desire on exposure to sexual visual cues compared to controls with a corresponding change in blood oxygen level-dependent signal in brain regions corresponding to emotional, cognitive, autonomic, visual and motivational processes. When OFF medication, CSB patients showed decreases in activation during the presentation of sexual cues relative to rest, which was not seen when ON medication. This provides evidence that passive visual cues can act as motivational cues for vulnerable individuals. In the final data chapter, I show that the Internet use habits of PD ICD patients had greater interference (increased time spent, obsessive thoughts) with life functioning compared to PD and healthy subjects, suggesting that PD ICD patients have an increased tendency towards excessive Internet use, which may coexist as an addictive behaviour. These studies show that PD patients on dopaminergic medication have a propensity toward impulse control disorders and related behaviours via sensitisation of the dorsal and ventral striatum. There is also an increased tendency to excessive Internet use in PD ICD subjects. These findings contribute to our understanding of the mechanisms through which a subset of PD patients choose to pursue aberrant rewarding behaviours despite negative consequences, with direct implication for clinicians managing this patient group.

Complex II of the respiratory chain (RC) recently emerged as a prominent regulator of cell death. In both cancer as well as neurodegenerative diseases, mutations in its subunits have been found along with other genetic alterations indirectly affecting this complex. Moreover, anticancer compounds were developed that target complex II and cause cell death in a tumour-specific way. I found evidence that this protein assembly is specifically activated for cell death via the dissociation of its SDHA and SDHB subunits from the membrane-anchoring proteins through mitochondrial Ca2+ influx. The relationship between Ca2+ and cell death activation has been under intense investigation, but not much has been identified on the associated mechanisms. Ca2+ influx into mitochondria plays a significant role in cell death induction, but it was unknown how the observed massive Ca2 accumulation activates the organelle for cell destruction. The disintegration of complex II subunits is prevented in vitro when cardiolipin is substituted with a lipid devoid of Ca2+ binding. Cardiolipin is known to associate with complex II, but coalesces into tight homotypic clusters upon Ca2+ binding. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death induction. My results reveal Ca2+ binding to cardiolipin and subsequent complex II disintegration as a crucial step for oxidative stress and cell death induction. Ultimately, it emphasizes the important role that Ca2+ plays in ROS-induced cell death via the respiratory chain, which can be used for many translational investigations. For instance, future studies on Ca2+ and its binding to cardiolipin for cell death induction will further elucidate complex II as a target for cancer treatment as well as neurodegenerative diseases and reveal its role as a nexus for many diverse stimuli in cell death signalling.

Cattle have expanded the KIR gene repertoire, a polymorphic and polygenic im- munoglobulin family that encode Natural Killer cell receptors specific to MHC class I ligands. In humans, KIR are important mediators of innate immunity to viral pathogens such as HCV and HIV, and there is potential for exploiting cattle KIR diversity as a means of improving animal health. Cattle KIR expansion has occurred independently to humans, the result is a cattle KIR haplotype (CKH) with a completely different gene content. Successful sequencing and assembly of the CKH using whole genome techniques has failed. To interrogate cattle KIR, their function and comparative evolution, the content of a CKH must be estab- lished, then the extent of polymorphism and gene presence/absence variation can be studied. In this project the first CKH has been sequenced and assembled using second generation sequencing of BAC clones. This has provided a reference sequence for whole genome sequence data to be aligned revealing the KIR content of different Bovidae species, including the aurochs, the ancestor to all domesticated cattle. Furthermore genome capture and enrichment was performed to determine polymorphic and polygenic KIR variation within 24 different cattle genomes. The sheep KIR haplotype (SKH) was sequenced using PacBio of BAC clones to enable comparative analysis with cattle. The CKH has expanded through block duplications resulting in 16 discrete KIR loci. The haplotype is dominated by functional inhibitory receptor genes and the attenuated remains of activating KIR. Predicted similarity between au- rochs and modern CKH suggests KIR blocks expanded through natural selection and not artificial selection generated through centuries of domestication. Com- parative analysis of the SKH and CKH reveals that sheep have independently expanded at least five of the shared KIR that cattle have expanded. Cattle KIR are extremely polymorphic, with diversity focused within the Ig domains, regions predicted to interact with ligand.