Increased levels of systemic immune activation are strongly associated with disease progression in HIV-1 infection, but the mechanisms causing it are not well understood. This work investigated the TH17 subset in the gastrointestinal tract of HIV-1 infected patients and looked for evidence that mucosal barrier breaches and microbial translocation contribute to peripheral immune activation. We found significant disruption in the expression of mucosal tight junction proteins in HIV-1 infection that only partially resolved on ART. This mucosal damage was positively correlated to plasma HIV viral load and 16s rDNA. Increased microbial translocation in viraemic infection correlated to elevated T cell activation and viral load. We found no evidence of reduced mucosal TH17-associated cytokines (IL-17a, IL-17f and IL-22) in our HIV cohort, but did observe reduced mucosal TH17 precursor cells (CD4+CD161+) in viraemic infection. Precursor TH17 loss was associated with increased tight junction breaches and T cell activation. To investigate the dual role of TH17 cells in promoting inflammation and maintaining barrier function in the gut mucosa we also studied inflammatory bowel disease. Tight junction disruption was observed in both active ulcerative colitis (UC) and Crohn's disease (CD) and occurred at a similar level as in viraemic HIV infection. Unlike HIV patients we found no evidence of microbial products in the periphery of IBD patients. In active UC there was evidence of TH17 cytokine driven pathology with increased levels of blood and mucosal IL-22 and IL-17f production by CD4 T cells. Blood and mucosal IL-17f proportions were positively correlated to the ulcerative colitis severity score (UCSS) and blood CD4+ IL-17f levels positively correlated with T cell activation. In contrast to UC we found little evidence of TH17 dysregulation in the blood or mucosa of CD patients. In conclusion this work found that there was a reduction in mucosal integrity, loss of mucosal CD4 T cells and precursor TH17 cells in HIV-1 infection. Loss of mucosal integrity in HIV was associated with microbial translocation and systemic T cell activation. We identified a correlation between reduced levels of mucosal precursor TH17 cells and increased mucosal damage in HIV infection, supporting the theory that this subset has a role in HIV-1 pathogenesis. However in UC, the inverse observation - elevated levels of precursor TH17 cells associated with increased mucosal damage - was made, reinforcing the paradoxical theory that this subset plays directly contrasting roles in different pathogenic environments.

The fetal vascular phenotype in fetal growth restriction (FGR) is significantly altered, with affected fetuses appearing to have relatively more dilated vessels with increased proportional blood flow to the head and neck arteries. This concurs with the concept of 'brain sparing' in FGR, but sets it in the context of a globally altered fetal circulatory system. The data presented in this thesis suggest that there are subtle alterations in the maternal antenatal inflammatory cytokine and chemokine profile in FGR. In FGR cord blood there appears to be a mixed inflammatory picture. Cord blood monocytes from FGR samples demonstrate priming towards anti-inflammatory profile with increased levels of IL-10 whilst in blood samples from neonates on day 1 of life there is evidence of a more pro-inflammatory set of cytokines. Subsequent to this, there is a persistent increase in IL-8 in samples from FGR infants collected at 6 weeks of age. The marked difference from cord blood to day 1 cytokines suggests a response of the FGR infant to delivery and their post-natal environment. Maternal blood samples at delivery from pregnancies with FGR show decreased pro-inflammatory chemokines and IL-10. In addition, maternal antenatal IL-2 and MIP1α correlate positively and MMP-9 negatively with fetal vessel diameter (per kg) after correction for gestation and pre-eclampsia. Although maternal post-natal samples show few significant differences, there is positive correlation of IL-17 and negative correlation of sIL-6ra with the severity of FGR in cord blood. This suggests that chronic inflammation mediated by IL-17 and altered IL-6 signaling are possibly associated with the processes which impair fetal growth. The overall findings indicate that FGR fetuses and infants have a syndrome of vascular and inflammatory perturbation which could be factors influencing the short and long-term sequelae of these individuals.

The brain is dependent on a constant supply of glucose as its primary fuel. Consequently, the brain has developed ways to tightly control intake of glucose, via glucose sensing neurones containing the enzyme glucokinase (GK). This thesis identifies a role for a subset of glucose sensing neurones in the arcuate nucleus (ARC) of the hypothalamus to promote glucose intake. These neurones form part of a pathway separate to the control of peripheral glucose homeostasis. Direct injection of a GK activator into the ARC increases glucose intake. Stereotactic injection of recombinant adeno-associated virus containing GK antisense (rAAV-GKAS) into the ARC of rats chronically decreases GK activity compared to controls. Knockdown of ARC GK reduces glucose intake in an acute and long-term setting. Direct injection of an ATP-sensitive potassium (KATP) channel inhibitor into the ARC increases glucose intake in a similar manner to that of a GK activator. In addition, pre-treatment with a KATP channel activator attenuates the orexigenic effect of a GK activator. ARC glucose sensing neurones are likely mediated by neuropeptide Y (NPY) as both activation of GK and inhibition of KATP channels stimulate NPY release in hypothalamic explants. Also, pharmacological inhibition of Y1 and Y5 receptors, and P/Q type voltage gated calcium channels, attenuate the orexigenic effect of GK activators. ARC glucose sensing neurones represent a novel pathway that may be part of a mechanism to ensure adequate glucose is constantly available to the brain. This work suggests that ARC GK has a physiological role in the homeostatic regulation of glucose intake. ARC GK responds to periods of fasting and may contribute to an increased preference for glucose. Thus, ARC glucose sensing may drive dietary sugar intake, which may be a contributing factor in obesity.

Therapeutic hypothermia confers significant benefit in term neonates with hypoxic-ischaemic encephalopathy (HIE). However, despite the treatment nearly half of the infants develop an unfavourable outcome. Intensive bench-based and early phase clinical research is focused on identifying treatments that augment hypothermic neuroprotection. Qualified biomarkers are required to test these promising therapies efficiently. This thesis aims to assess advanced magnetic resonance imaging (MRI) techniques, including diffusion tensor imaging (DTI) and resting state functional MRI (fMRI) as imaging biomarkers of outcome in infants with HIE who underwent hypothermic neuroprotection. FA values in the white matter (WM), obtained in the neonatal period and assessed by tract-based spatial statistics (TBSS), correlated with subsequent developmental quotient (DQ). However, TBSS is not suitable to study grey matter (GM), which is the primary site of injury following an acute hypoxic-ischaemic event. Therefore, a neonatal atlas-based automated tissue labelling approach was applied to segment central and cortical grey and whole brain WM. Mean diffusivity (MD) in GM structures, obtained in the neonatal period correlated with subsequent DQ. Although the central GM is the primary site of injury on conventional MRI following HIE; FA within WM tissue labels also correlated to neurodevelopmental performance scores. As DTI does not provide information on functional consequences of brain injury functional sequel of HIE was studied with resting state fMRI. Diminished functional connectivity was demonstrated in infants who suffered HIE, which associated with an unfavourable outcome. The results of this thesis suggest that MD in GM tissue labels and FA either determined within WM tissue labels or analysed with TBSS correlate to subsequent neurodevelopmental performance scores in infants who suffered HIE treated with hypothermia and may be applied as imaging biomarkers of outcome in this population. Although functional connectivity was diminished in infants with HIE, resting state fMRI needs further study to assess its utility as an imaging biomarker following a hypoxic-ischaemic brain injury.

Introduction: CF (cystic fibrosis) and PCD (primary ciliary dyskinesia) are obstructive airway diseases characterised by frequent infections and neutrophilic inflammation. However, PCD has a much milder course than CF. Pilot data showed that in PCD (n=8) the relationship between LCI (lung clearance index) derived from multiple breath washout (MBW), and FEV1 (forced expiratory volume in 1 second) differed from the established correlation in CF. This thesis sought to identify the reasons. Materials and Methods: Larger PCD (n=38) and CF cohorts (n=125), a non-CF bronchiectasis comparator group (n=28), and healthy controls (n=44) were recruited. All performed LCI and spirometry, and subgroups had more complex MBW parameters (conventional and modified phase III analysis and curvilinearity) calculated and HRCT scans scored. Results: As in the pilot data, there was no relationship between LCI and FEV1 in PCD, unlike in CF. PCD patients had fewer structural abnormalities than CF despite similar or worse spirometry and LCI, and the relationship between HRCT and spirometry or LCI in PCD was again different from that seen in CF. MBW analyses showed that Scond* is near-normal in PCD, suggesting less flow asynchrony, compared with CF. Conclusions: There are differences in the nature of distal airway disease between PCD and CF. As the non-CF bronchiectasis patients were similar to CF (rather than PCD), this likely results from the primary mucociliary clearance defect in PCD compared with secondary impairment in the other two conditions. This may be important as care of PCD patients is extrapolated from that of CF patients, which may not be appropriate. It is important not to extrapolate outcome measures uncritically between different disease groups, both clinically and when planning randomised controlled trials. Finally, a better understanding of what causes the better prognosis in PCD may help identify future new treatment avenues in CF.

This thesis represents research from Hospital Episode Statistics data and provides an insight into gastrointestinal resectional surgery in England. It examines national outcomes following major colorectal resection, oesophagectomy and gastrectomy. Having established these outcomes, I ventured to investigate the commonly used mortality measures in the literature. I also studied the timing and causes of deaths following colorectal resection in English NHS Trust hospital. I was able to demonstrate that a significant number of adverse outcomes occur beyond the initial hospital stay. I evaluated the role of two key factors - minimally invasive surgery and surgeon volume in trying to mitigate these adverse outcomes. I found that national outcome following elective or planned colorectal resection are comparable with other published cancer registry reports in England. For upper gastrointestinal resection for cancer, however, outcome are significantly worse than those from Far East, but superior to studies from the States. I derived 'medical morbidity' by studying secondary codes for medical complications. Surgical complications were quantified by using surrogates such as unplanned re-operation and re-intervention following the initial procedure. I undertook a review of the literature for published outcomes following planned colorectal resection in the elderly. This demonstrated heterogeneity in studies with regards to sample size and type of study. The most commonly used measure of post-operative risk was in-hospital or 30-day mortality. In the elderly population, we demonstrated high mortality up to one year following emergency colorectal resection. To understand this excess mortality that is not taken into account by short term metrics, we studied the causes of deaths in these patients. Significant deaths occur in the young and elderly due to cardiac causes, up to one year following major colorectal resection. This calls for further research to define a new intermediate term metric that accurately quantifies the mortality risk. The uptake of minimally invasive gastrointestinal resection in England has been promising. During the study period there has been a steady rise in number of resections undertaken laparoscopically. In colorectal surgery, laparoscopic resection has been associated with shorter length of stay, reduced morbidity and mortality. Outcome following minimally invasive oesophagectomy and gastrectomy have shown this technique to be safe and potentially beneficial in reducing pulmonary complications and length of stay. However further research is needed into this. Oesophagectomy, gastrectomy and pancreatectomy for cancer have all demonstrated a positive volume-outcome relationship. With increasing surgeon caseload, risk of 30-day mortality is lower. These structure and process measures may be utilised by policy makers to improve outcome following gastrointestinal resection in England.

The introduction of combination antiretroviral therapy (cART) has resulted in a dramatic improvement in the life-expectancy for people living with HIV-infection. Although AIDS-defining illnesses such as HIV-dementia are now rare, mild cognitive disorders are still prevalent in a substantial proportion of patients on effective cART and suppressed HIV plasma viraemia. Although some clinic risk factors for HIV-associated neurocognitive impairment (NCI) such as low nadir CD4 T-cell count and age have been described, the pathological processes underlying this cognitive impairment remain elusive. A major factor that might be contributing to the development of HIV-associated NCI is chronic activation of macrophages and astrocytes resident in the brain microglia. I hypothesise that in vivo microglial activation is both present in effectively treated HIV-infected individuals and is associated with disturbances in cognitive function and biomarkers of magnetic resonance imaging (functional, structural and chemical) and inflammation To address these hypotheses I used cerebral positron emission tomography with [11C] PBR28 to measure translocator protein 18kDa (TSPO), a putative marker for microglial activation. To examine the impact of inflammation on brain structure, function and chemistry, neurologically asymptomatic HIV-infected individuals stable on cART were evaluated using several magnetic resonance imaging methods, cognitive testing and measurements of inflammatory biomarker in plasma and cerebrospinal fluid. I identified the presence of in vivo microglial activation in several brain regions in HIV-infected individuals compared to HIV-negative controls. Microglial activation was associated with disturbances in cognitive function (verbal and learning memory), brain activation (reduced task-related brain activation) and white matter integrity (increased mean diffusivity), suggesting a deleterious effect of neuroinflammation on brain structure and function. Microglial activation, across several anatomical locations correlated with increased concentrations of plasma ribosomal 16s, a marker of microbial translocation. This relationship between a marker of impaired integrity of the gut-blood barrier, and microglial activation, suggests a communication route is present between peripheral inflammation and the CNS in HIV-infected individuals. In vivo detection of microglial activation, and systemic inflammatory markers could provide indices to guide risk assessment and treatments for HIV-associated brain disease.

Brown adipose tissue (BAT) dissipates energy, whereas white adipose tissue (WAT) is an energy storage site. The worldwide increase in obesity and its associated metabolic complications represents a growing challenge for health care systems. With the recent demonstration of significant deposits of adult human BAT, and that WAT is dynamic and can gain BAT characteristics, there is therefore a possibility for manipulative control of energy homeostasis via the induction of specific genes to enhance BAT levels. Nrg4 and Gpr120 are two genes we identified to be highly expressed in BAT and regulated in a temperature-dependent manner. Nrg4 is a member of the Neuregulin family that activates the ErbB-4 (HER4) tyrosine kinase receptor. Nrg4 was secreted from brown adipocytes and promoted neurite outgrowth, a function that may improve BAT thermogenesis by enhancing sympathetic innervation. Gpr120 is a member of the superfamily of G protein-coupled receptors that is activated by long chain fatty acids. Although Gpr120 is known to mediate anti-inflammatory and insulin-sensitizing effects in humans and mice, its role in BAT is unknown. In order to identify the signalling pathways and downstream functions activated by this receptor in brown adipocytes, distinct known and novel Gpr120 ligands, GW9508, TUG891 and TUG1096, were employed. These ligands increased expression of genes associated with glucose and fatty acid metabolism and reduced inflammatory gene markers. All of these effects were reversed in Gpr120 knockout cells. However, the levels of gene expression varied considerably with each ligand. Although Gpr120 is a known Gαq/11-coupled receptor in other cell systems, these distinct ligands exhibited various degrees of bias towards Gαq/11 and distinct signalling pathways. These findings could provide insight into the requirements for Gpr120 function in BAT, the exploitation of which could generate ligands for therapeutic benefit and represents a promising opportunity to develop safer and more efficacious drugs.

Premature cardiovascular disease (CVD) complicates chronic systemic inflammatory disease, and is initiated by endothelial dysfunction. Despite an ever-improving array of medications to treat these diseases, how to prevent CVD is not known. Studying the actions of endogenous mediators of endothelial cytoprotection and of disease-modifying drugs might establish how best to protect patients with systemic inflammatory diseases from atherosclerosis, if specific beneficial effects on vascular endothelium could be demonstrated. High flow laminar shear stress (LSS) and vascular endothelial growth factor (VEGF) are two well known endogenous drivers of endothelial cytoprotection. Both can induce expression of protective genes but it is not known how confluent endothelial cells (EC) respond to VEGF when conditioned with LSS. Methotrexate (MTX) is the most widely prescribed treatment for rheumatoid arthritis and clinical data suggests that it reduces CV mortality. Known mechanisms of action of MTX result in intracellular accumulation of activators of AMP-activated protein kinase (AMPK). AMPK regulates cytoprotective genes in EC and its activation is associated with diverse desirable effects. I hypothesised that EC conditioned with LSS would be primed to respond to VEGF, resulting in a synergistic induction of cytoprotective genes. Secondly I investigated whether MTX exerts beneficial protective effects on vascular endothelium via activation of AMPK, which enhance endothelial resistance to injury. Studies of human umbilical vein EC (HUVEC) exposed to LSS showed that responses to VEGF are not enhanced in these conditions. However, MTX phosphorylated AMPKαThr172 and induced expression of several cytoprotective genes, notably manganese superoxide dismutase (MnSOD). The addition of folic acid did not alter this; and it was also preserved when HUVEC were pre-treated with TNFα to mimic dysfunctional endothelium. siRNA depletion of AMPK attenuated MTX-mediated MnSOD induction. MTX treatment led to AMPK-dependent phosphorylation of the transcription factor CREBSer133. siRNA depletion of CREB also reduced MnSOD induction by MTX, and chromatin immunoprecipitation demonstrated binding of CREB to the MnSOD promoter in MTX-treated samples. Moreover, MTX protected HUVEC against apoptosis induced by glucose deprivation, demonstrating the functional importance of this pathway. Finally, treatment of the murine (BXSB x NZW)F1 SLE model of inflammatory vasculopathy with MTX improved the intramyocardial arterial vasculopathy and reduced end-organ damage, increased aortic MnSOD and phosphorylated AMPKαThr172, and reduced ICAM-1 expression. I have shown that MTX activates an AMPK-CREB pathway in vascular endothelium leading to enhanced expression of cytoprotective genes and protection against apoptosis in vitro and inflammatory vascular injury in vivo. This novel mechanism may explain its observed benefits in reducing CVD in chronic systemic inflammation.

Advances in reconstructive surgery are providing treatment options in the face of major trauma and cancer. Body Sensor Networks (BSN) have the potential to offer smart solutions to a range of clinical challenges. The aim of this thesis was to review the current state of the art devices, then develop and apply bespoke technologies developed by the Hamlyn Centre BSN engineering team supported by the EPSRC ESPRIT programme to deliver post-operative monitoring options for patients undergoing reconstructive surgery. A wireless optical sensor was developed to provide a continuous monitoring solution for free tissue transplants (free flaps). By recording backscattered light from 2 different source wavelengths, we were able to estimate the oxygenation of the superficial microvasculature. In a custom-made upper limb pressure cuff model, forearm deoxygenation measured by our sensor and gold standard equipment showed strong correlations, with incremental reductions in response to increased cuff inflation durations. Such a device might allow early detection of flap failure, optimising the likelihood of flap salvage. An ear-worn activity recognition sensor was utilised to provide a platform capable of facilitating objective assessment of functional mobility. This work evolved from an initial feasibility study in a knee replacement cohort, to a larger clinical trial designed to establish a novel mobility score in patients recovering from open tibial fractures (OTF). The Hamlyn Mobility Score (HMS) assesses mobility over 3 activities of daily living: walking, stair climbing, and standing from a chair. Sensor-derived parameters including variation in both temporal and force aspects of gait were validated to measure differences in performance in line with fracture severity, which also matched questionnaire-based assessments. Monitoring the OTF cohort over 12 months with the HMS allowed functional recovery to be profiled in great detail. Further, a novel finding of continued improvements in walking quality after a plateau in walking quantity was demonstrated objectively. The methods described in this thesis provide an opportunity to revamp the recovery paradigm through continuous, objective patient monitoring along with self-directed, personalised rehabilitation strategies, which has the potential to improve both the quality and cost-effectiveness of reconstructive surgery services.