Hepatitis C virus (HCV) infects 3% of the global population and HCV-related liver inflammation is a major cause of liver failure and hepatocellular carcinoma. Current treatments are based upon long courses of interferon-α (IFNα) injections, which have significant side effects and are only effective in 40-80% of individuals depending on viral genotype. Natural killer (NK) cells are innate lymphocytes, which can kill virally infected cells and are stimulated by IFNα. To establish a chronic infection HCV must evade immune responses. I hypothesised that NK cells are important for the successful eradication of HCV and that chronic HCV infection impinges upon NK cell function to prevent viral clearance. I found that NK cell function was reduced in chronic HCV and correlated with the proportion of NKp46+ NK cells in vitro. In keeping with these findings NKp46-rich intrahepatic NK cell populations were more activated and the proportion of these cells correlated with liver inflammation. During interferon-α treatment individuals who had the greatest increase in NK cell function in response to increasing stimulation had the fastest rate of viral clearance and were most likely to successfully clear the virus. Using a novel adenovirus vector expressing HCV proteins I have discovered that NS5B protein reduces NK cell cytotoxicity and cytokine production. Therefore, in this thesis I have described novel insights into the mechanisms of HCV immunoevasion, HCV-related disease pathogenesis with implications for viral eradication therapy.

The innate immune system has a variety of ways of recognizing infection from pathogens such as viruses and bacteria. One of its first lines of defence is to detect Pattern Associated Molecular Patterns (PAMPs) using Pattern Recognition Receptors (PRRs) such as the Toll-Like Receptors (TLRs), the RIG-Like Helicases (RLHs) and the Nod-Like Receptors (NLRs). These receptors recognize certain molecular structures from the pathogens and lead to first line of defence which includes increased cytokines and IFNs. This study delineate the human body’s innate immune responses to human respiratory viruses such as HRV (Human Rhinovirus), RSV (Respiratory Syncytial Virus) and IAV (Influenza A virus). In Vitro experiments carried out on various kinds of lung tissues suggest that respiratory disease pathogenesis is related to inflammatory mediators including interleukins and cytokines produced by the host’s innate immune system. Virus induced respiratory tract infection are known to trigger bronchiolitis, wheezing and acute asthma exacerbations, as a result of inflammation of lung tissues and excessive release of pro- inflammatory cytokines such as IL-1β. This study identifies that intracellular macromolecular complexes called inflammasomes assemble as a result of viral trigger. Inflammasomes convert the inactive forms of the pro-inflammatory cytokines to their active forms. Although the exact mechanism of activations of these complexes was unknown. This study identified that Virus encoded proteins such as the 2B protein of HRV, the SH protein from RSV and the Influenza M2 which are also termed viroporins can activate the inflammasome by causing ion imbalance (across cells membranes and organelles). Thus providing a trigger for inflammasome assemblage. v Drugs which act as Ion channel blockers have been shown to block viroporin activity and hence reduce IL-1β production. Therefore in the future the use of ion inhibitors could be a possible therapeutic intervention in order to reduce lung inflammation and prevent associated respiratory disease such as COPD and Asthma.

Objective: This research discovers patients’ experiences of the new and traditional routes of care and reveals the advantages and disadvantages of each within a chosen locality. It informs how a community service is delivering the new health agenda and most importantly how the patients feel and perceive their journeys through community-based care and hospital-based care. Therefore, its findings are crucial as feedback on how successful health plans have been to move more care to the community. It will reveal the Early Response team’s processes to ensure the service is fully utilised in intermediate care and give a better understanding about which patients are suitable for the home service. DESIGN – This qualitative research takes the form of multiple case studies encompassing semi-structured interviews to encourage discussion on the topic of care journeys. Participant information sheets and consent forms were used. The anonymity of the participant was upheld by using a pseudonym to refer to their contribution. All patients gave consent for the staff member who assessed them to be interviewed giving a total of three case study sources of the home patient, the hospital patient and the staff member as the units of measurement. Themes were searched for in the coding process derived from Kolcaba’s (2010) comfort theory and the bio-psycho-social model (Engel, 1977). These were physical, psychological, social and environmental along with two themes derived from the literature review of age discrimination and loneliness. Data generated helped ascertain the success of an alternative type of care service and formulated recommendations for practice. Setting: The patient interviews took place at the patients’ home so that they had time to experience their care pathway. Staff interviews took place in a quiet room at their place of work. Flexibility was offered regarding location, time of day and if the patients wished relatives to be present. Participants: 10 patients and 10 staff were interviewed totalling 20 participant interviews. The patient group was split into 5 patients who attended A & E and received their subsequent care in hospital and 5 patients who attended A & E and 4 were able to receive their subsequent care at home with the Early Response Service. The patients from each group were matched on the basis of same/similar injury. Each staff member who assessed the patients was matched to their patient, giving a multiple case study of the home and hospital patient and two staff members. The age range of patient participants was 72-89 years old and the staff participants 39-58 years old. There were 8 females and 2 males in the patient group and 9 females and 1 male in the staff group. Results: Data were analysed using the six theme headings and by searching through data for specific reference to answering the research questions. Key words found were burden, coping, independence or dependence, recovery, pain, equipment, finance, frailty and disorientation. Comfort took the form of pain relief, carer assistance and reassurance, not feeling a burden and having needs met in a timely fashion. Discomfort took the form of pain, cold, hunger, loneliness, finances, disorientation and needing more flexibility in carer calls. Tabular analysis revealed all hospital admissions were necessary and the reasons for admission were more serious or required medical/surgical intervention compared to the home care group. Positive home patient comments included being able to have care at home with less disruption to patients’ social networks and positive hospital patient comments included being able to receive maximum assistance over a 24 hour period as they felt they could not have coped at home. Results in relation to ageing theory, age discrimination and loneliness are discussed and interestingly care at home can be interpreted as positive discrimination of the older person by offering an alternative care option. Both hospital and home patients were satisfied with the care they received on the whole. Conclusion: The Early Response Service are correctly identifying the most suitable patients to receive care at home. An improvement in staff resources or skills such as intravenous drug administration would widen their referral criteria to be able to offer their service to more patients. There is still work to be done in respect of pulling patients out of hospital who are deemed medically stable, but are waiting for social care packages.

genes show monoallelic parent-of-origin specific expression and have an important role in mediating adult behaviour. Previous research has indicated that maternally expressed Nesp and paternally expressed Grb10, which are expressed in overlapping brain regions, may have a role in mediating risk-taking and/or impulsive behaviours. Impulsivity and risk taking are natural parts of human behaviour; however pathological levels of impulsivity and risk-taking are recognised as clinical traits of many psychiatric disorders. The aim of the current research is to explicitly test whether these two oppositely imprinted genes influence impulsivity and/or risk-taking behaviour in mice by examining mouse models that lack functional copies of paternal Grb10 (Grb10+/p) and maternal Nesp (Nespm/+) in a number of tests of impulsivity and risk-taking. Unlike previous findings in Nespm/+ mice, Grb10+/p mice had the same propensity to explore a novel environment as wild type (WT) controls. However, in a measure of delay-discounting behaviour it was discovered that Grb10+/p mice were less likely to discount delayed rewards. This is in contrast to previous work with Nespm/+ mice, which discounted delayed rewards more steeply. This is the first demonstration that oppositely expressed imprinted genes antagonistically affect behaviour. To further explore these behaviours, a novel test of risk-taking was developed. Using predator odours a perceived ‘risky’ environment was created in order to measure the decision between fear and reward seeking. Using the Predator Odour Risk-Taking (PORT) task it was demonstrated that Nespm/+ and Grb10+/p mice showed comparable levels of risk-taking behaviour to WT littermates. Finally, immunofluorescence was used to discover that Nesp55 and Grb10 are not only expressed in the same brain regions, but are co-expressed in some cells, particularly in serotonergic neurons. This suggests that these imprinted genes may be influencing delay discounting behaviour via the same integral neurotransmitter systems.

Huntington’s disease is an autosomal dominant neurological disease caused by an elongated CAG repeat in exon 1 of the huntingtin gene. There is currently no cure and treatments are limited. The genetic mutation causes selective cell death of the medium spiny neurons which reside in the striatum of the basal ganglia. Current disease models don’t necessarily recapitulate all aspects of the human disease and so alternatives are needed. The advent of induced pluripotent stem cells (iPSC), has allowed for HD patient specific pluripotent stem cells to be derived, hence differentiation of these cells in vitro could provide a disease model for drug testing and investigation of disease pathology. Current protocols for differentiation of pluripotent stem cells into medium spiny neurons (MSNs) are often inconsistent and lead to low yields of MSNs. Directing differentiation through forced expression of transcription factors has been used to differentiate neurons from fibroblasts and pluripotent stem cells, often with increased efficiency. Utilising transcription factors vital in post-mitotic MSN development, this study has aimed to produce MSNs in vitro, by transfection of transcription factors or combinations thereof in a multicistronic plasmid into ventral forebrain neural progenitors. This study has involved the cloning and expression of 5 different transcription factors important in MSN development in iPSC-derived neural progenitors. Two of these transcription factors; NOLZ1 (ZNF503) and ISL1 were further investigated for their ability to differentiate neural progentiroes into MSNs. This study showed that transfection of ISL1 enabled differentiation of neurons to produce a higher proportion of cells resembling MSNs, characterised by co-expression of the MSN markers DARPP32 and CTIP2 and expressing FOXP1. The combination of NOLZ1 and ISL1 in transfection improved functional maturation of neurons, becoming increasingly spontaneously active and increased excitability, as well as responding to GABA and NMDA, with dopamine D1 agonist enhancement of NMDA currents.

Bacterial infection is a feature of long-term peritoneal dialysis (PD) and a cause of loss of peritoneal function and treatment failure. Understanding how local inflammation is initiated and peritoneal host defence mechanisms are activated in PD patients is key to reducing the detrimental consequences of peritonitis. The capacity of human peritoneal mesothelial cells (HPMC) to ingest bacteria has been described, and the ability of the Toll-like family of innate immune receptors (TLR) to trigger inflammatory responses to pathogens has been demonstrated. However, the pathogen recognition ability, the potential role of TLRs, the specific role in the early inflammatory response, and the regulation of HPMC’ putative ability for pathogen recognition have not been fully investigated. To address these issues, the present study aimed at characterising TLR expression and responses in HPMC and evaluating the capacity of modulators of pro-inflammatory responses, namely soluble TLR2 (sTLR2) and the IL-6/sIL-6R complex, to regulate TLR-mediated HPMC and peritoneal responses in vitro and in vivo. HPMC were found to respond to an array of bacterial pathogens via expression and function of a specific set of TLRs. HPMC responses were susceptible to modulation by sTLR2 and sIL-6R, resulting in inhibition of TLR2-driven HPMC responses. In vivo, sTLR2 and IL-6/sIL- 6R reduced neutrophil influx partly by inhibiting NF-κB activation in stromal cells of the peritoneum. IL-6 signalling counteracted TLR2-mediated responses by reducing peritoneal leukocyte recruitment and chemokine production. Notably, following the establishment of a mouse model of peritoneal bacterial infection, IL-6 signalling was confirmed to be beneficial to bacterial clearance. The results of this thesis confirm and extend the knowledge of the pivotal role that HPMC play in peritoneal responses to infection. The capacity of sTLR2 and IL-6/sIL-6R to modulate peritoneal responses demonstrated in this study may inform the design of new therapeutic strategies to reduce PD-associated peritonitis and thus improve treatment outcomes.

Human T-cells play a major role in controlling and clearing Mycobacterial infections. The adaptive immune system deploys a complex network of specialised T-cell subsets in order to tailor an optimum immune response. Two categories of T-cells have been described that are characterised by the ligands they recognise: “conventional” T-cells (polymorphic, HLA-restricted, peptide-specific) and “unconventional” T-cells (non-polymorphic, restricted by HLA-like molecules, non-peptide-specific). Both T-cell categories were shown to be important for the elimination of cells infected with Mycobacterium tuberculosis (M. tuberculosis) and their role, specificities and functionalities are under active investigation in order to develop optimum vaccination strategies. A large interest in unconventional T-cells, such as MR1-restricted MAITs or CD1-specific T-cells, and their role in mycobacterial infections has recently arisen. I initiated my studies by dissecting T-cell responses generated during direct ex vivo boosting of PBMCs with antigen presenting cells that had phagocytosed Mycobacterium smegmatis (M. smegmatis). M. smegmatis is a non-pathogenic bacterium and is mainly eliminated by the innate immune system. However, T-cells might respond to M. smegmatis antigens and therefore play a role in clearing the pathogen. Using polychromatic flow cytometry, I successfully identified major CD3+ conventional and unconventional M. smegmatis-specific T-cell populations and evaluated their respective frequencies and distribution. The identification of a significant frequency of M. smegmatis-specific unconventional MAITs pushed me to further analyse the specificity of this interesting T-cell subset. At the time of my studies, the ligand(s) presented by MR1 to MAITs were still undiscovered. However, structural models of MR1 groove moiety provided evidences that MR1 could potentially present peptides to MAITs. Therefore, I attempted to identify the molecular and cellular mechanisms by which an M. tuberculosis-specific MAIT clone recognises peptide loaded on MR1 and to refold this MHC-like protein. Vaccination strategies have been mainly focusing on targeting CD8 T-cells, known to be essential for the host defence against mycobacterial infections. Therefore a huge effort is made to discover new immunodominant mycobacterial epitopes. Collaborators isolated the HLA-A*0201-restricted D454 T-cell clone specific to the LLDAHIPQL epitope derived from the highly immunogenic Esx-G protein. The LLDAHIPQL sequence is conserved across mycobacterial species thus offering potential for pan-mycobacterial vaccination. I aimed at proving that D454 TCR binds to HLA-A*0201-LLDAHIPQL. I successfully obtained an HLA-A*0201-LLDAHIPQL crystal structure, the first bacterially-derived HLA-peptide complex, and identified the key mechanisms involved in the molecular recognition of HLA-A*0201-LLDAHIPQL by a conventional TCR.

Investigations into Human Cytomegalovirus (HCMV) pathogenesis should be based on strains that closely reflect the causative agent of disease, however HCMV invariably mutates in vitro, generating phenotypically distinct laboratory-adapted strains. In particular, mutations are selected in the HCMV genome UL128 locus (UL128L) that encodes sub-units of a virion envelope pentameric complex that impedes virus propagation in fibroblasts (the cell type most commonly used in vitro), but is required for infection of several naturally targeted cell-types (e.g. epithelial, endothelial, and myeloid cells). Addressing this issue, the genome of wildtype HCMV strain Merlin was cloned as a stable bacterial artificial chromosome (BAC), however similarly to clinical isolates, viruses reconstituted from the Merlin-BAC grow poorly and are prone to de novo mutation in cell culture. Direct comparison to viruses from the Merlin-BAC revealed that viruses from the BACcloned versions of strains TR (TR-BAC), TB40E (TB40-BAC4) and VR1814 (FIX-BAC) could be propagated more efficiently in fibroblasts, despite containing intact UL128L ORFs. Unique nucleotide variations identified in TB40-BAC4 and FIX-BAC UL128L ORFs were transferred into the Merlin-BAC, generating variants that produced greater titres of cell-free virus following reconstitution. Virions from these novel Merlin variants displayed reduced pentameric complex content, but remained able to infect epithelial cells, albeit with slightly compromised efficiency. The greater fitness of viruses from these novel Merlin-BAC variants alleviated the selective pressures for the selection of de novo UL128L mutations in fibroblasts. The Merlin virion proteome was determined, with up to 30 novel components identified to provide a more comprehensive picture of wildtype HCMV virion composition. Comparison of virions from several Merlin variants demonstrated that varying pentameric complex content impacted the incorporation of other components, however virions from the novel Merlin variants produced in this work closely matched those from the parental Merlin variant containing wildtype UL128L ORFs. Thus, the novel Merlin-BAC variants produced in this work represent valuable reagents for future HCMV research.

The Chain Event Graph (CEG) is a new class of graphical model, first introduced in Smith and Anderson [2008], which is derived from a probability tree by merging vertices whose associated conditional probabilities are the same. It is proving to be a useful framework for modelling asymmetric problems and further generalises the Bayesian Network (BN), by allowing for context-specific dependence structures between the variables of the problem. This thesis provides a first demonstration of the value of using the CEG in real-world applications and the new techniques developed here are motivated by problems that arise from two health studies; the Christchurch Health and Development Study (CHDS) and the UK Cerebral Palsy (UKCP) Cohort Study. A direct comparison of the BN and CEG on the CHDS demonstrates that the CEG can lead to significantly higher scoring models than the BN and further that it enables additional conclusions to be drawn on the health study directly from the topology of its graph. An extension of the CEG, the Ordinal CEG, is developed in this thesis, which further enhances the graphical representation of the CEGs for studies with a binary outcome. Motivated by the UKCP this thesis further investigates how missing data structures can be explicitly represented by a CEG and how its graph can consequently provide a precise understanding of the influence of missingness. Finally, a dynamic version of the CEG is developed and it is demonstrated how this new class of models generalises the Dynamic BN and is further closely linked to (semi-) Markov processes. The expressiveness of this model is illustrated through a fictional example.

BACKGROUND Arrhythmia is a cardiovascular disorder which can lead to several complications. Over the past decade the introduction of many new drugs has raised concerns about their questionable benefits and cost-effectiveness. Classification of antiarrhythmic drugs has not been fully resolved. Although numerous clinical trials have been conducted, the value of antiarrhythmic drugs in many indications remains controversial. Two meta-analyses of clinical trials addressing the indication of quinidine (Class I) for maintenance of sinus rhythm after cardioversion have suggested high efficacy rates but increased mortality relative to placebo. Several overviews which were conducted to evaluate the impact of antiarrhythmic therapy on improving survival post acute myocardial infarction, have defined a turning point in the management strategy from Class I to Class III drugs, particularly amiodarone and sotalol, due to the unfavourable mortality outcome with the former Class. MAJOR AIMS This thesis was conducted with three major aims: 1) To assess both qualitatively and quantitatively the benefits and risks associated with flecainide (Class Ic), amiodarone (Class III), and sotalol (Class III & II) in treatment of chronic atrial fibrillation, acute medical or surgical supraventricular arrhythmias, and life-threatening ventricular arrhythmias developing post acute myocardial infarction; 2) To produce an overall summary estimate of effectiveness and probabilities of incidence of adverse effects, which can be useful for subsequent incorporation in cost-effectiveness analysis; 3) To validate the usefulness of various therapeutic outcomes implemented by general treatment guidelines. OVERVIEW OF THESIS A meta-analysis was carried out to compare the efficacy and safety of three antiarrhythmic agents (flecainide, sotalol, and amiodarone) in maintaining sinus rhythm after cardioversion of chronic atrial fibrillation. 42 of 119 clinical trials retrieved satisfied the predefined inclusion criteria. Data from 17 amiodarone trials (5 randomised, and 12 uncontrolled), 8 sotalol trials (6 randomised, and 2 nonrandomised), and 19 flecainide trials (8 randomised, 4 nonrandomised controlled, and 6 uncontrolled) were pooled separately after testing for homogeneity of treatment effect across the trials. Although the pooled rate difference in proportion of patients remaining in sinus rhythm between amiodarone and placebo (2 trials) was statistically nonsignificant (RD3mon = 16.1 %, 95% CI = -29.7 to 61.7, P>0.05), the pooled effect compared to Class IA drugs (3 trials) demonstrated significant differences at all time intervals (RDs were 20.5%, 31 %, and 28.8% at 3, 6, and 12 months respectively). Aggregating sotalol efficacy data in randomised or nonrandomised controlled trials has yielded highly significant effect in favour of sotalol as compared to placebo and equal effect as compared to Class IA and Class IC at all time points. Furthermore, comparison of flecainide to placebo or Class IA has revealed a highly superior effect in favour of flecainide. The calculated summary statistics (ORpeto, ORMH, RD, and RR) for the incidence of mortality and pro arrhythmia in the full-exposure group in amiodarone and sotalol trials were not significant, affirming the safety of those two drugs. In flecainide placebo-controlled trials, the ORMH for mortality and proarrhythmia were 1.8 (95% CI, 1.2-2.7, P=0.002), and (95% CI, 4.23-10.6, P<0.00l) respectively, thus indicating low benefit-risk ratio for flecainide as compared to amiodarone. The validity of this meta-analysis was examined by assessment of publication bias using funnel-plots. A funnel-plot of the amiodarone clinical trials displayed the shape of an 'inverted funnel', thus suggesting an evidence of low retrieval bias. However, due to the small sample size identified (18 trials only), a firm conclusion with regard to absence of publication bias could not be drawn. Evolving strategies for management of newly occurring supraventricular arrhythmias were reviewed. A meta-analysis was undertaken to determine the most effective agent for prompt cardioversion to sinus rhythm. Flecainide efficacy relative to placebo was confirmed by pooling data from 5 placebo-controlled trials (OR3hrs, 7.2; 95% CI, 4.7 to 11.1; Z=8.9; and OR8hrs, 5.5; 95% CI, 3.6 to 8.4; Z=7.85). However, pooling the data from three amiodarone, placebo-controlled trials at 3 and 8 hour-intervals demonstrated a nonsignificant effect (OR3hrs, 1.3; 95% CI, 0.7-2.4; Z=0.85; and OR8hrs, 1.03; 95% CI, 0.6-1.8, Z=0.12). All individual odds ratios for intravenous sotalol compared to placebo were highly significant with pooled OR at 1 hour of 8.8 (95% CI, 4.7-16.5; Z=6.8). The effect sizes of the three agents on mean ventricular response rate was estimated for both converted and unconverted patients. Whilst the effect size of flecainide versus placebo was not statistically significant at any time point, those of sotalol and amiodarone were statistically and clinically meaningful for both converted and unconverted patients. It is suggested that for acute cardioversion, intravenous flecainide or sotalol should be initially implemented. Intravenous amiodarone can be subsequently introduced for controlling the ventricular rate in persistent unconverted patients. Recent meta-analyses of randomised controlled trials of secondary prevention of myocardial infarction by antiarrhythmic agents have questioned the validity of using arrhythmia suppression as a substitutive end point for mortality. A meta-analysis examining the effect of sotalol and amiodarone for prevention of death post acute myocardial infarction was undertaken. In addition to single point estimates of pooled odds ratios of total mortality and sudden death, a meta-analysis of survival data which included censored end points was employed. An attempt was made to reconstruct the life tables in individual trials of amiodarone. The Kaplan-Meier percentages were recalculated and pooled at specific time points to reproduce the final meta-analytic survival curves of total mortality and sudden death. The meta-analysis confirmed the clinical efficacy of amiodarone for prolonging the survival in patients with congestive heart failure or myocardial infarction. The nonparametric log-rank odds ratio method was applied to raw actuarial data deduced from published Kaplan-Meier graphs as well as data generated by curve fitting. Pooling each set of data separately has yielded highly significant log-rank ORs for total mortality in the first set of four trials with censoring (log-rank OR at 102 months, 0.598; 95% CI, 0.43 to 0.83; Z = -3). However, log-rank ORs from data generated by curve fitting of data from a further three trials, were nonsignificant up to 48 months (log-rank OR, 0.87; 95% CI, 0.72 to 1.06, Z = -1.4). Merging of the two data sets has suggested strong evidence of efficacy for improving survival in terms of both total mortality and sudden death.