An avian virus can become adapted to humans by mutating or recombining with currently circulating human viruses. These viruses have the potential to cause pandemics in an immunologically naïve human population. Host restriction involves multiple determinants, however, influenza polymerase is considered to play an important role. The heterotrimeric polymerase complex (PA, PB1 and PB2) associates with viral RNA and nucleoprotein (NP) to form a ribonucleoprotein (RNP) complex responsible for viral replication and transcription. Host specific genetic signatures have been identified on all of the polymerase subunits and on NP, but the PB2 protein arguably carries the dominant determinants of host range. Avian-origin influenza polymerase activity can be dramatically increased in human cells with the PB2 E627K substitution. This has been suggested to stabilise the interaction between the NP and PB2 components of the vRNP complex in the nuclei of infected cells. However, we demonstrate that a variety of adaptive PB2 substitutions including E627K did not enhance the stability of the vRNP in human cells, but rather increased the amount of replicated RNA, and that resulted in more PB2-NP co-precipitation. The introduction of many adaptive PB2 mutations enhances avian influenza polymerase activity in an in vitro reconstituted polymerase assay. However, only some of these mutations have been detected in viruses that are found circulating in nature. We explored whether the polymerase assay truly predicts viral growth and investigated viral selection pressures that might favour some adaptive mutations over others. We used reverse genetics to create a series of viral variants carrying mutations in the PB2 gene and carried out virological assays and also analysed the effects of the mutations in vivo. Some mutations that increased in vitro polymerase activity led to attenuated virus replication and resulted in an increase in interferon activation. These data increase our understanding of the host range barrier and why certain adaptive mutations may or may not have emerged.

Introduction: Varicose veins are a common problem with 25-50% of the population symptomatically affected, and chronic venous disease leads to significant impairments in quality of life with substantial health system cost implications. Significant variability exists in the symptoms suffered by patients, the treatment offered and the outcomes achieved. Identification of the optimal treatment pathways for patients remains difficult. Aims: i. To ascertain primary care disease knowledge. ii. To assess what affects treatment and identify which patients benefit most. iii. To generate a predictive model of varicose vein outcomes. iv. To assess the impact of altering treatment of varicosities in the context of endovenous truncal vein ablation v. To investigate the early impact of new technologies. Methods: i. Two survey studies were completed: - 21 questions assessing venous disease management pathways was disseminated to General Practitioners. - 19 questions assessing the management of superficial venous thrombosis and was distributed to General Practitioners and Vascular Surgeons. ii. A cohort of consecutive patients with symptomatic chronic venous disease were assessed and completed quality of life questionnaires pre and postintervention. iii. Uni-variable and multi-variable analysis of patient cohort data to facilitate the creation of generalised model of venous treatment outcomes. iv. A randomised clinical trial assessing the timing of varicosity avulsion in the context of local anaesthetic endovenous truncal ablation. - Ambulatory Varicosity avUlsion Later or Synchronised (AVULS) trial. v. Assessment of new technologies - The European Sapheon Closure system Observed ProspectivE (eSCOPE) study a multi-site cohort observational study of cyanoacrylate glue occlusion of truncal vein incompetence - The VNUS Versus Clarivein for Varicose Veins (VVCVV) multi-centre randomised clinical trial comparing the procedural pain profile of radiofrequency and mechanochemical ablation. Results: i. Education outcomes - 138 responses were received. The management of chronic venous disease in the primary care setting is disparate and knowledge of current techniques is poor, despite extensive guidance. - 369 responses were received, from 197 vascular specialists and 172 primary care physicians. Superficial thrombophlebitis management is shown to be diverse and does not adhere to recent evidence. ii. 461 patients were recruited. Patients suffering from chronic venous disease suffer from substantial quality of life impairment, including previously under-recognised depressive symptoms. Treatment of the underlying venous condition provides relief from venous symptoms and improves quality of life. - Patient symptoms and quality of life do not correlate with anatomical vein diameter, however clinical severity scores do. iii. Predictive modelling produces models that account for 30-41% of the variability in post-operative scores for disease specific quality of life tools, generic quality of life tools, and clinical severity scores. iv. The AVULS trial recruited 101 patients. Simultaneous treatment leads to improved clinical outcomes at up to 1 year and early quality of life improvement. Delayed treatment has a significantly increased risk of requiring further treatment (Odds Ratio 27.78, Relative Risk 18.36, p<0.0001). 95% of patients declining randomisation opted for simultaneous treatment. v. New Technology Outcomes - The eSCOPE study recruited 70 patients in Europe with good technical outcomes. - The VVCVV trial (ongoing) has recruited 85 patients, with significantly reduced procedural pain found with mechanochemical ablation. Conclusions: Varicose veins are a widespread problem with effective treatment that leads to a significant improvement in quality of life. Education and communication between community and hospital-based medicine is lacking. Predictive modelling of varicose vein symptoms remains difficult due to the multifactorial nature of the disease. Simultaneous treatment of varicosities during endovenous truncal ablation produces improved outcomes and is the option of choice for most patients. Early data on new technologies show they provide less painful procedures with similar outcomes as the established modalities.

Cancer care driven by a multidisciplinary team (MDT) meeting is mandatory in UK and has become the platform to discuss cancer cases in many countries worldwide. My aim in this research is to assess the functionality and efficacy of the MDT meeting in making and implementing decisions. Specifically, I aim to develop tools to evaluate how MDTs make clinical decisions and recommendations, understand the role of leadership within these teams and measure why MDT decisions may not get implemented in patient care. The introduction chapter presents an exploration of the evidence base available in the literature on the functionality of cancer MDTs with a focus on how to assess the efficacy of MDTs. In this chapter, I used an 'input-process-outcome' framework as a systems approach to the MDT and its working. Chapter 2 is a systematic review of the existing evidence on MDT decision making and decision implementation across surgical specialties. Chapter 3 focuses on urological cancers and retrospectively evaluates MDT decision making from the perspective of implementation of team decisions into patient care. Chapters 4 and 5 present the views of core MDT members on the efficacy of the MDT in addition to problems and shortcomings faced by the MDT and also suggestions for improvement. Chapter 6 presents analysis of a survey on how to improve the efficiency of MDT. Chapters 7 and 8 present data from studies that objectively assess MDT performance by developing and validating observational assessment tools. Finally, the discussion chapter reflects on the findings of this research and discusses their implications for future research and practice.

It has been well known that the extracellular matrix (ECM) is important to the aortic valve function and that mechanical forces and cell-cell communication can regulate ECM remodeling. However, it has not been determined which cells regulate ECM production by valve interstitial cells (VICs). Thus, this study aimed to investigate if the valve endothelial cells (VECs) can influence ECM production and whether there is a differential role of VECs from either surface of the valve. In addition, the study aimed to assess the suitability of adipose derived stem cells (ADSCs) to differentiate into VECs with side-specific characteristics, so that could be used to populate tissue engineered heart valves. Firstly, a reliable method of side-specific isolation and culture of VECs was developed. Consequently, the phenotypic characteristics of aortic VECs (aVECs) and ventricular VECs (vVECs) were investigated in vitro by immunostaining, western blots and protein array. Endothelial markers vWF, CD-31, VE-cadherin and eNOS and the release of cytokines IL-1β, IL-8, and TGF-β were assessed in cultured VECs from either side of the valve. However the distinct phenotypes between aVECs and vVECs in culture were not established. The differential ability of side-specific VECs to regulate the production of ECM components by VICs was observed when VICs were cultured with media containing molecules released from side-specific VECs, in static culture, and when aVECs or vVECs were co-cultured with VICs. The media collected from vVECs increased amount of collagen by VICs while the media from aVECs showed no significant changes in the content of ECM. Interestingly, in the co-culture system, vVECs-VIC co-culturing enhanced the amount of both collagen and glycosaminoglycans (GAGs) whereas aVECs did not affect the ECM proteins. Moreover side-specific VECs were exposed to the oscillatory and laminar shear stresses (flow patterns experienced by aortic and ventricular surface of the valve, respectively). Their distinct responses on the production of ECM by VICs were investigated. Aortic VECs exposed to oscillatory flow had higher content of collagen and GAGs while vVECs did not share this effect. The laminar shear stress on both sides of the valve maintained elastin content (as compared to fresh tissue). ADSCs were also exposed to side-specific patterns of flow to assess their ability to differentiate into side-specific VECs. With respect to these factors, the exposure of ADSCs to the oscillatory shear stress induced the differential expression of NOS III and SMαA, similar to reported differences found between porcine aVECs and vVECs. Both flow patterns also increased the endothelial function of ADSCs by up-taking low-density lipoprotein. In conclusion, this study reveals the unique functional phenotypes between the aortic and ventricular VECs, and the unique communication between VECs and VICs that is mediated by shear stress to regulate the specific production of ECM components. ADSCs appear to have the capacity to express endothelial markers, but a full functional assessment of their communication with VICs remains to be investigated. This information is important to the development of a living tissue engineered heart valve.

Pandemic influenza viruses are responsible for many deaths and much suffering as well as large costs accumulated through healthcare provision and lost productivity. The swine-origin H1N1 influenza virus that was at the root of the pandemic in 2009, although considered mild, in the UK alone caused the deaths of 361 people, subjected 1,700 to critical care and led to hospital admission of 7,879, before summer 2010. This virus is now embedded in the human population and will remain there, inducing further mortality, morbidity, and cost until it is replaced by a new pandemic influenza virus and the cycle repeats. However, if we improve our understanding of the characteristics of virus/host interactions that support transmission, we may be able to identify potentially problematic strains, improve our preparedness and ultimately prevent future pandemics. The ability of an influenza virus to transmit from host to host is multifactorial. One role proven to be important because its inhibition by drugs prevents replication is the ability of neuraminidase (NA) to cleave sialic acid (SA). NAs of recently circulating influenza viruses were analysed with the aim of identifying characteristics that might support new or previous pandemic influenza virus strains. NA stalk truncation, induced by influenza virus transmission through gallinaceous poultry, was found to hinder transmission through the air of an otherwise transmissible virus. While this genetic ablation did not directly affect sialidase activity, when measured using a small soluble substrate, it did reduce the ability of virions to release complex tethered substrates, separate from one another and penetrate through mucus. This work suggests that the next pandemic virus is unlikely to have a short stalk NA. Previously, loss of the ability of NA to haemabsorb (Hb) chicken red blood cells (CRBSs) was associated with the adaptation of an influenza virus from avian to human hosts and a single amino acid mutation was shown to reinstate the haemabsorption ability of an NA from a prototypic pandemic H2N2 virus of avian-origin. This thesis shows that the human-adapted 2009 pandemic N1 NA, which arose from the introduction of an avian influenza virus into swine, did not Hb CRBCs, as expected. However, unexpectedly, many back mutations to make the 2009 N1 resemble an NA that did Hb also did not alter this characteristic. Further investigations showed that some NAs from both avian and swine influenza viruses also did not Hb, despite the fact that NA haemabsorption increased viral growth in airway culture cells. Thus we conclude that loss of Hb occurs within birds, before a virus can cross the species barrier into mammals and this may be due to a reduction, rather than a gain, in activity.

Numerous clinical and experimental studies have implicated tumour necrosis factor (TNF) as a central mediator of acute lung injury (ALI). TNF signals through two cell surface receptors, TNFR1 and TNFR2, which initiate distinct signalling pathways and cellular responses. Using a novel, highly selective TNFR1 domain antibody GSK1995057 allied with TNF-receptor (TNFR) specific muteins, the work in this thesis investigated the roles of differential TNFR signalling in a range of human in vitro models of ALI using human lung microvascular endothelial cells (HMVEC-L), human alveolar type 2 cells (hAT2) and human neutrophils. In particular we focused on the cascade of events, which lead to neutrophil migration into the alveolar space during ALI. The TNFR1 signalling pathway mediated HMVEC-L activation (neutrophil adhesion molecule expression and chemokine/cytokine release) and injury (trans-endothelial resistance). These effects were associated with an amelioration of neutrophil migration through HMVEC-L monolayers. TNFR1 signalling also played key roles in neutrophil priming, activation, and cell fate. TNFR1 signalling mediated TNF-induced alterations in neutrophil cell surface molecule expression and reactive oxygen species production. TNFR1 antagonism also reversed TNF induced late survival (20 hours) of human neutrophils. In an attempt to generate translational data investigating the effects of differential TNFR1 signalling, we established a human ex vivo model of ALI. Using an LPS method of injury we demonstrated release of the pro-inflammatory cytokines TNF, interleukin (IL-)-8, IL-6 and IL-1β in the alveolar space. This was associated with a significant influx of healthy donor neutrophils and pulmonary oedema in LPS injured lung compared to control treated contralateral lung. These data suggest a central role for TNFR1 signalling in ALI. Translation of these in vitro data in our human ex vivo lung model may provide the basis for future clinical trials of selective TNFR1 antagonism in ALI.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases. ADMA is metabolised by dimethylarginine dimethylaminohydrolase (DDAH) of which there are two isoforms with the same catalytic activity but different tissue expression patterns. Clinically ADMA is relevant as increased levels are seen in multiple pathologies, most notably cardiovascular disease and renal disease. Recent studies have shown increased plasma levels of ADMA in patients with type 2 diabetes and obesity, although these studies did not distinguish between an associative or causal relationship. In human genetic studies, polymorphisms in both DDAH1 and DDAH2 have been associated with type 2 diabetes. Furthermore, mice overexpressing ddah1 have increased insulin sensitivity and overexpression of DDAH2 has been reported to increase insulin secretion from pancreatic β-cell lines. This thesis aims to investigate the relationship between ADMA and the development of insulin resistance and in particular establish whether there is a causal link between elevation of ADMA and the onset of insulin resistance. My work has utilised DDAH deficient mouse strains to mimic loss of DDAH activity and consequent elevation of ADMA. Thorough analysis of glucose homeostasis in global Ddah1 knockout mice by hyperinsulinemic-euglycemic insulin clamp showed no significant dysfunction in insulin sensitivity in these animals. However on a high fat diet, increased ADMA resulted in enhanced adiposity and enhanced insulin resistance compared to fat fed controls. These differences were linked to increased expression of mTOR and SREBP1c in liver tissue and white adipose tissue. Conversely study of Ddah2 knockout animals showed no obvious change in insulin secretion on a normal diet but a marked difference in response to a high fat diet. Ddah2 knockout mice have higher metabolic rates and reduced fat mass compared to fat fed controls. This increased metabolic rate was associated with striking changes in metabolic markers in the brown adipose tissue of Ddah2 knockouts indicating an increase in mitochondrial number and activity. This work suggests that although ADMA does not alone cause insulin resistance it does alter the body's response to a high fat diet that increases the pathology of the condition. This work shows, for the first time, clear and independent roles for DDAH1 and DDAH2, with respect to the control of metabolism and energy expenditure.

In COPD, small airways are the main site of airflow obstruction. Neutrophil infiltration into small airways is significantly associated with lung function decline. This thesis investigated the effects of activated neutrophil products on ex vivo small airway structure and function using precision-cut lung slices (PCLS). Peripheral blood neutrophils were isolated from non-smokers, smokers and COPD patients; stimulated with fMLP, and supernatants obtained. Supernatant protease concentration/activity was assessed. There were significant increases in protease concentration/activity upon fMLP stimulation, but no significant differences between subject groups or correlations with subject demographics. Rat PCLS were incubated overnight with neutrophil supernatants, and small airway structure, function and PCLS viability assessed. Stimulated COPD neutrophil supernatants caused significant airway dysfunction. All neutrophil supernatants significantly reduced PCLS viability. There was a significant inverse correlation between supernatant active MMP-9 concentration and maximal airway closure. Incubation of rat PCLS with H2O2 or porcine pancreatic elastase did not fully reproduce the effects of neutrophil supernatants: both reduced maximal airway closure; whilst H2O2 alone significantly reduced PCLS viability. Pre-incubation of COPD neutrophil supernatants with a NE inhibitor led to restoration of maximal airway closure only. Pre-incubation with neither an antioxidant, N-acetyl-cysteine (NAC), nor a MMP-9 inhibitor, had any effect on neutrophil supernatant-induced PCLS damage. Finally, small airways in PCLS from COPD lung tissue had greater maximal airway closure, compared with smokers and non-smokers. There were no correlations between subject demographics and small airway function. Overall, neutrophils from COPD patients induce small airway dysfunction and reduce tissue viability. Neither oxidative stress nor elastase reproduced these effects fully; and pre-incubation with neither antioxidants nor protease inhibitors restored small airway function. Thus, there is greater complexity in neutrophil mediation of this damage. Finally, rat small airways do not fully reproduce the response of small airways in COPD lung tissue, which exhibit evidence of hypercontractility.

Inflammation associated with asthma mainly affects large airways and is accompanied by extensive structural changes, termed airway remodelling. 5-10% of patients with asthma suffer from severe or refractory asthma which is difficult to control despite receiving high doses of inhaled and sometimes oral corticosteroids (CS). These patients show more prominent characteristics of airway remodelling, specifically sub-epithelial fibrosis and airway smooth muscle (ASM) thickening. My project focuses on one of the cells implicated in airway remodelling, circulating fibrocytes, which are bone marrow-derived peripheral blood mesenchymal progenitors expressing both leukocyte markers, such as CD45, and mesenchymal proteins including collagen I (Col I). Fibrocytes migrate to the sites of disease under the guidance of chemokine receptors such as CC chemokine receptor type 7 (CCR7), and differentiate into α-smooth muscle actin (α-SMA)-expressing myofibroblasts, a process that is facilitated by a variety of pro-inflammatory cytokines and growth factors. Myofibroblasts can promote subepithelial fibrosis as well as contribute to ASM thickening. Indeed, the number of fibrocytes in peripheral blood is correlated with the decline rate of forced expiratory volume in 1s (FEV1) in patients with chronic obstructive asthma. Most importantly, there is increased recruitment of fibrocytes to the airway wall of patients with severe asthma. However, the mechanisms driving the accumulation of fibrocytes in the airways of these patients are currently unclear. I hypothesised that in severe asthma there are increased numbers of circulating fibrocytes that have an increased capacity to differentiate into myofibroblasts and have differential responses to pro-inflammatory mediators and asthma therapeutic agents compared to non-severe asthma. Fibrocytes were isolated from the non-adherent non-T (NANT) cell fraction of peripheral blood mononuclear cells (PBMC) of healthy subjects and patients with non-severe or severe asthma. The number of fibrocytes (Col I+/CD45+ cells) and differentiating fibrocytes (α-SMA+ cells), as well as the expression of CCR7 and glucocorticoid receptor (GR) in fibrocytes were determined by flow cytometry. Apoptosis was determined by Annexin V/propidium iodide staining. Messenger ribonucleic acid (mRNA) expression was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Fibrocytes were also isolated from the adherent fraction of PBMC. Severe asthmatic patients had a higher number of circulating fibrocytes with a greater capacity to differentiate into myofibroblasts in culture compared to healthy subjects and patients with non-severe asthma. Severe asthmatic fibrocytes did not have a heightened responsiveness to either interleukin (IL)-4, IL-13, nerve growth factor or brain-derived neurotrophic factor. Dexamethasone induced apoptosis in NANT cells, including fibrocytes and differentiating fibrocytes, from healthy subjects and patients with non-severe asthma, but not in the cells from patients with severe asthma. Dexamethasone also reduced CCR7 expression in fibrocytes from patients with non-severe asthma but not in those from patients with severe asthma. The relative CS insensitivity in severe asthmatic fibrocytes may be related to the lower expression of the GR or the heightened c-Jun N-terminal kinase activity. Salmeterol xinafoate, a long-acting β2-adrenoceptor agonist (LABA), reduced the number, myofibroblastic differentiation and CCR7 expression of fibrocytes from healthy subjects and patients with non-severe asthma. Salmeterol did not improve the suppressive effect of dexamethasone, although it was not detrimental to dexamethasone's effect either. In contrast, tiotropium bromide, a long-acting muscarinic antagonist (LAMA), did reduce the number of fibrocytes and differentiating fibrocytes from patients with severe asthma. Increasing intracellular 3',5'-cyclic adenosine monophosphate (cAMP), the downstream signalling molecule of β2-adrenoceptor and muscarinic M2 receptor, by phosphodiesterase type IV inhibitor (rolipram) and cAMP analogue (8-bromoadenosine-3',5'-cyclic monophosphate) could reduce fibrocytes from patients with severe asthma. Patients with severe asthma have elevated numbers of circulating fibrocytes showing enhanced myofibroblastic differentiation and are less responsive to the suppressive effect of CS and LABA, but can be inhibited by LAMA. This study provides insight into a novel target for the treatment of airway remodelling in severe asthma.

Subjects with prediabetes are at much higher risk of developing diabetes than healthy subjects. Weight loss helps reduce the risk of developing diabetes in these subjects, but long-term weight loss is difficult to achieve due to increases in appetite. Consumption of fermentable carbohydrate has been shown to reduce food intake and body weight, and also improve insulin sensitivity independent of weight loss. Therefore, fermentable carbohydrates may help prevent the development of diabetes in subjects with prediabetes via a two-pronged effect. This thesis comprises three investigations which examine the effects of inulin compared to cellulose control on 1) appetite and food intake, 2) measures of glucose homeostasis and 3) a long-term randomised control trial examining the effect of inulin on weight loss maintenance. In each investigation, subjects take 30g/inulin a day following a 4-week dose-escalation period. In investigations 1 and 2 all subjects take both inulin and cellulose supplements for a 6-week period each, separated by a 4-week wash-out phase. Investigation 3 is a randomised control trial comprising a 9-week weight loss phase, during which participants aim to reach a 5% weight loss at 9 weeks, and a 9-week weight maintenance phase during which subjects are asked to maintain the weight they have lost. In this body of work I demonstrate that inulin reduces appetite, food intake and weight in subjects with prediabetes. I also demonstrate that inulin was significantly associated with an increase in early insulin secretion and GLP-1, potentially due to an improvement in the incretin effect. Inulin also appears to improve insulin sensitivity in subjects with a specific subtype of prediabetes only. Finally, I demonstrate that inulin supplementation results in significantly greater weight loss maintenance, alongside changes in body composition likely to be beneficial long-term.