After entry into a target cell, HIV-1 must traverse the cytoplasm and enter the nucleus in order to integrate into host chromatin. Until recently, the viral CA core was thought to play a passive role in infection, simply delivering its contents to the cytoplasm soon after entry. However, CA has recently been found to interact with and/or determine interaction with several nuclear entry cofactors. In this study, we investigate the consequences of CA interacting with the host mRNA-processing factor CPSF6. We provide evidence that the interaction between CA and cytoplasmic CPSF6 during early infection dictates the downstream nuclear entry pathway and integration site selection of HIV-1, despite not affecting viral titre in cell lines. We identify clinically relevant CTL escape mutations that lie within the CPSF6 binding pocket of CA and alter the relationship of HIV-1 with the CPSF6-dependent nuclear entry pathway. The phenotypes of these mutants suggest that conservation of this pathway is important in vivo but also that the pathway differs between cell types. In addition, we examine the antiviral mechanisms of C-terminal truncations of CPSF6 and the small-molecule CPSF6 peptidomimetics PF74 and BI-1. We design a series of compounds predicted to interact with the CPSF6-binding pocket of CA and screen them for antiviral activity as well as the ability to alter the nuclear entry pathway of HIV-1. Of particular interest are a compound that exhibits a very different inhibition profile from PF74/BI-1 and a compound that causes the virus to use a CPSF6-independent nuclear entry pathway without reducing viral titre. Together, the findings in this study confirm and characterise the role of CPSF6 as an HIV-1 cofactor and demonstrate that drugs targeting the CA-CPSF6 interaction could inhibit the virus through multiple different mechanisms.

Host restriction factors such as Fv1 in mice and Trim5α in primates block retroviral infection through their interaction with capsids. One major form of Fv1, encoded by the Fv1b allele, restricts only N-tropic MLV (N-MLV) when expressed at the lower endogenous level, but also restricts B-tropic MLV (B-MLV) and NB-tropic MLV (NB-MLV) at the higher levels seen in cells transduced with Fv1 b-expressing retroviral vector. However, a previous pull- down study detected almost equal binding of Fv1b to all three MLV capsids. In this study, the relationships between Fv1 binding, restriction and expression level were studied in detail. To study the restriction specificity of Fv1 b at different expression levels, new Tet-On vectors were developed to allow doxycycline-inducible expression of Fv1. These vectors allowed restriction studies from a very low Fv1 b level where no restriction could be observed, to a high level where inhibitions of B-MLV and NB-MLV are also observed. Similar phenomenon is also observed in other Fv1 mutants. By contrast, Fv1 n, even when over-expressed, restricted only B-MLV but not NB-MLV or N-MLV. The binding of Fv1b to different capsids at different Fv1 concentration were compared using a new pull-down assay performed on microtitre plates. Fv1b appears to bind to all MLV capsids at the lowest concentration of Fv1 where binding could be detected. The study was extended to include other Fv1 mutants, and as expected binding could be detected for all restrictive Fv1-MLV pairs. Interestingly, a few outliers including Fv1b vs B-MLV demonstrated strong binding but either weak or low restriction, suggesting there may be other factors causing the lack of restriction. Together, these data suggest that although Fv1b have very similar apparent binding affinities to different MLV capsids, different amount of Fv1b are required for the restriction of N-, B- and NB-MLV.

Beta-blockers are avoided in asthma and chronic obstructive pulmonary disease (COPD) due to the potential risk of drug induced bronchospasm. Despite these concerns, beta-adrenoceptor antagonism has recently been associated with potential therapeutics benefits in asthma. Furthermore the use of beta-blockers in COPD patients may potentially result in improved survival due to optimisation of treatment in those with concurrent cardiovascular disease. This thesis evaluates the role of beta-blocker use in asthma and COPD. The introduction outlines the pharmacological principles associated with the human beta-adrenoceptor and its therapeutic application in the management of asthma and COPD through established treatment strategies including inhaled beta-agonists. The historical literature documenting concerns with beta-blocker use in asthma and COPD is reviewed and critiqued. Finally the hypothesis, on which this thesis is based, that betablockers may have a therapeutic role in the asthma and COPD is discussed. Proof of concept studies and preliminary work suggesting potential putative effects of betablocker use in asthma, data highlighting the burden of cardiovascular disease in COPD patients and the potential role of beta-blockers are discussed. New data from two randomised double-blind placebo controlled trials evaluating betablocker use in asthma and an observational study investigating the effects of betablocker use on mortality in COPD are presented. The first randomised controlled trial, addresses the safety of beta-blocker use in asthma. Using the non-selective beta-blocker propranolol, the study investigated the safety of acute exposure to propranolol in asthmatics, sequentially challenged with histamine to mimic an asthma exacerbation and evaluated the role of intravenous hydrocortisone in potentiating salbutamol reversibility. The results of this study showed there was no significance difference in salbutamol recovery measured by change in FEV1 (ml) post histamine challenge following intravenous hydrocortisone verses placebo (mean difference 0.04 (95%CI - 0.07 to 0.15), p=0.417). The study also investigates the degree of bronchoconstriction attributable to oral propranolol in mild-to-moderate asthmatics and uses impulse oscillometry as an alternative method of assessing pulmonary function to conventional spirometry. Following 10 or 20mg of oral propranolol, a mean fall in FEV1 of 4.7% was observed (95%CI 1.8 to 7.5), p=0.008. Impulse oscillometry showed a greater response to propranolol with an increase of 31.3% (95%CI 15.6 to 47), p= 0.04, 2 hours post propranolol dosing. The second randomised controlled trial within this thesis, describes the first placebocontrolled trial to assess the effects of chronic dosing with oral propranolol as add-on to inhaled corticosteroids in patients with stable persistent asthma. The study investigated the hypothesis of potential therapeutic benefits of chronic beta-blocker use in asthma by improvements in airway hyper-responsiveness. This study evaluates the effects of oral propranolol on both methacholine and histamine bronchial challenges, in addition to spirometry, impulse oscillometry and inflammatory surrogates including exhaled nitric oxide. Furthermore the effects on asthma control and quality of life post chronic betablockade are described. Finally the safety and tolerability of acute cardio-selective beta-blockade with esmolol is compared to acute propranolol dosing and the protective effects of tiotropium are evaluated. The main result of this study showed chronic propranolol dosing did not affect airway hyper-responsiveness, with no significant difference observed in methacholine challenge PC20 following chronic propranolol exposure compared to placebo, geometric mean mg/ml: 2.57 (95%CI 1.13 to 5.85) versus 2.50 (95%CI 1.14 to 5.50), -i.e. a mean doubling dilution difference (DDD) of 0.04 (95%CI -0.56 to 0.63), p=0.89. Furthermore following chronic beta-blocker dosing, FEV1 showed a fall with propranolol versus placebo amounting to a 4.3% (95%CI -0.6 to 9.2) p=0.08. The final study within this thesis is a large observational cohort study using a disease specific dataset of COPD patients. By means of data linkage using pharmacy prescriptions, hospital admissions and mortality data, the potential effects of betablocker use on COPD exacerbations and mortality is examined. This study suggested a potential survival benefit with beta-blocker use amounting to a 22% reduction in mortality (HR 0.78 (95%CI 0.67 to 0.92). The discussion of this thesis evaluates the results of each study and describes their relevance in the management of patients with asthma and COPD.

Arterial stiffness has been demonstrated to predict cardiovascular morbidity above and beyond traditional risk factors and can be estimated from measurements such as pulse wave velocity (PWV). Aortic PWV can be estimated from the carotid and femoral sites (CFPWV) by applanation tonometry and ultrasound. However, these methods only estimate a gross global burden, which may mitigate regional stiffness within the vasculature, and can be subjected to errors relating to uncertainties in estimating vessel length. Magnetic resonance imaging (MRI) is an established clinical cardiovascular imaging modality which like ultrasound, has the advantage of being non-invasive and non-ionising. Whilst not typically part of a clinical cardiovascular examination, MRI sequences can be implemented to provide localised aortic PWV measurements directly from aortic sites. PWV derived by MRI (MR-PWV) is mostly confined to clinical research. It has been determined with high reproducibility and validated against invasive pressure measurements. MRI, as a technique, bears the potential to evaluate vascular anatomy and cardiac function in a single examination. For instance, it can provide aortic stiffness measure with a corresponding left ventricular assessment. Therefore, techniques such as MR-PWV warrants further investigation to see if it may prove beneficial in the detection of subclinical disease or as an imaging biomarker of cardiovascular disease (CVD). In this research, MR-PWV was estimated using conventional methodology by determining the pulse wave transit time between two aortic sites, derived using MRI velocity encoded imaging of the aorta. When the technique was applied to a complicated diabetic population with and without symptomatic CVD in chapter 5, PWV yielded an important distinction between the groups (CVD with T2DM: 8.7 ± 2.8 ms-1, n =22, T2DM: 7.5 ± 2.3 ms-1, n =28, CVD without T2DM: 8.9 ± 3.6 ms-1, n =19 versus control 6.7 ± 1.8 ms-1, n =19, ANOVA P < 0.05). However, it did not show significant differences in patients with T2DM before onset of symptomatic CVD. This conventional method of PWV assessment was extended into a multi-site approach sampling several points along the aorta, with the hypothesis that aortic stiffness is a heterogeneous process that varies along the aortic length. This multi-site pulse wave velocity (MS-PWV) technique has been shown to increase the accuracy of PWV measures and was utilised in a subsequent study in chapter 6 and 7, involving healthy volunteers and patients with peripheral vascular disease (PVD) , (healthy volunteers under 40 years: 4.5 ± 0.7 ms-1, n = 22, older healthy volunteers over 40 years: 6.5 ± 1.5 ms-1, n=22 versus PVD patients 8.6 ± 3.1 ms-1, n = 26, P < 0.001). This thesis provides a novel insight into the process of arterial stiffening and subsequent cardiovascular health throughout different disease cohorts. It differs from current published work by examining MR-PWV in a heterogeneous diabetes and CVD population, while previous studies have solely examined either diabetic or CVD patients. Secondly, the use of MS-PWV has only been implemented in three previous studies to date; involving both a healthy population and cohort with Marfan syndrome. This research is the first to use this technique in PVD and the only study to derive MS-PWV in this population. In summary, PWV was shown to change with both the extent and severity of atherosclerosis and CVD, which is agreement with previous studies. This demonstrates that MR-PWV may be a useful research tool within the area of cardiovascular MRI and further work is needed to clinically define the potential impact of PWV.

No description available.

Pacemaker implantation remains the sole treatment options for patientswith bradycardia. The devices have evolved over the years, however recentstudies of implantable defibrillators suggested that increased levels ofpacing at the right ventricular apex (the conventional site) may beassociated with worse outcomes in patients with impaired left ventricularsystolic function.An initial observational database linkage study was performed to investigatethe prevalence of heart failure in patients referred for pacemaker insertion.This identified a significant portion of patients (19%) had heart failure andwere potentially at risk. Further study was undertaken in patients withoutheart failure to investigate the effects of right ventricular (RV) apical pacingon endothelial function. 22 patients with sino-atrial node disease wereexposed to high degrees of RV pacing and minimal RV pacing in a crossoverstudy for 1 week each. This demonstrated significant impairment ofendothelial function in the arm with a high degree of RV pacing.A subsequent study investigated the impact of biventricular pacingcompared to RV pacing again in a cross over design. Patients were implanted with a biventricular device and randomised to RV only or biventricularpacing. Biventricular pacing was associated with significantly enhanced 6minute hall walk distance together with improved quality of life and lessimpairment of endothelial function and reduction of hs-CRP when comparedto biventricular pacing.Finally a further retrospective data-linkage study demonstrated thatangiotensin receptor blocker or angiotensin converting enzyme inhibitor usewas associated with improved mortality and less hospitalisation for heartfailure in patients paced for complete heart block, who were likely to beexposed to high degrees of right ventricular pacing.In summary this thesis is a record of my study of the effects of pacemakerinsertion in patients with impaired left ventricular systolic function. Myresearch has demonstrated the potential detrimental effects of rightventricular apical pacing and has identified potential therapeutic strategiesof ameliorating these effects.

Assisting less able people to move in a healthcare setting is a very common occurrence but carries risks to staff and patients. The scientific study of patient handling activities and interventions to help reduce musculoskeletal disorders in the workplace is a relatively new but growing area. Recent literature reviews have identified two key factors, the lack of high quality studies and the lack of strong links between patient handling interventions and reduced musculoskeletal injury. This study has systematically reviewed the available literature and investigated the potential outcome measures used to show benefits of improved patient handling. A wide range of outcomes has been identified concentrated on the benefits to staff, patients and organisations. No methods were identified to compare different benefits, outcomes or intervention strategies. This study used mixed methods to develop a tool to compare the results of all types of interventions: a. Focus group studies in four EU countries recorded a priority list of the 12 most important outcomes from patient handling interventions b. The most suitable method for examining the 12 outcomes was identified c. The Intervention Evaluation Tool (IET) was developed as a single measurement tool d. The IET was translated and used in four EU countries to evaluate its usability and its usefulness to patient handling practitioners The EU trials and subsequent expert review have given favourable feedback for the IET. The IET creates 12 outcome evaluations with detail and differentiation, and an overall performance score to assist an organisation to target its future interventions. The method can be used to compare interventions, and the performance between organisations and countries across the EU. Though the IET needs more field trials and validity testing it is hoped that a wider application may be to create a benchmarking method that can assist in the improvement of patient handling systems across Europe.

The Pregnane X receptor (PXR) , is a nuclear receptor (NR) which heterodimerizes with RXRa (Retinoid X Receptor alpha) to regulate xenobiotic metabolism. The NR1I2 gene produces three isoforms, PXR1, PXR2 and PXR3. However, only the function of the major isoform PXRl is known. Moreover, PXR has been shown to have an anti-inflammatory function. This is interesting, since the processes of xenobiotic metabolism and inflammation antagonize each other. The mechanism by which PXR performs this anti-inflammatory function remains unclear. However, other nuclear receptors such as PPARa and PPARy repress pro-inflammatory gene induction via transrepression. This is a mechanism that facilitates transcriptional repression by the atypical association of NRs with co-regulatory and co-repressor complexes at foreign response elements. It is thought that SUMOylation allows these NRs to attain transrepressive functions. PXR is conjugated with SUM03 chains so it is believed that it too utilizes transrepression to mediate its anti-inflammatory actions. To study this, the five PXRIIPXR3 lysines were mutated. Additionally, five other so-called 'functional sites' which are critical to the transactivation role of PXR were also mutated. This was important to determine if the transactivation and repressive roles utilized similar mechanism of action. Luciferase reporter assay experiments were performed in RAW264.7 cells to investigate both the transactivation and repressive activities of the aforementioned mutants. Firstly, it was observed that both the putative SUMO sites and functional sites decreased PXREM induction and that PXR3 was transcriptionally inactive. Secondly, PXRl and PXR3 reduced the induction of the IL-8, iNOS and AP-l reporters used. However, the putative PXRl SUMO sites were less able to repress induction of the IL-8 reporter, while the functional mutants repressed induction just as well as wild type PXRl. Interestingly also, both the putative SUMO and functional PXR3 mutants repressed the IL-8 reporter as potently as the wild type.

A large number of participants is often required by association studies investigating the causal mechanisms of complex diseases because of the generally weak causal effects involved in these conditions. The large sample sizes necessary for adequately powered analyses are mainly achieved by large studies. This can be an expensive undertaking and it is important that the correct sample size is identified. But, the analysis of the statistical power of large consortia and major biobanks demands that a number of complicating issues are taken into proper account. This includes the impact of unmeasured aetiological determinants and the quality of measurement of both outcome and explanatory variables. Conventional methods to analyse power use closed-form solutions that are not flexible enough to allow for these elements to be taken easily into account and this results in a potentially substantial overestimation of the actual power. In this thesis, I describe the radical rebuilding of an existing power calculator known as ESPRESSO to develop and implement the ESPRESSO-forte algorithm. ESPRESSO-forte is intended as a comprehensive study simulation platform aimed at supporting the design of large scale association studies and biobanks. I then applied the newly developed software to two real world scientific problems: (1) to assess the power of a large multi-provincial Canadian cohort for the study of quantitative traits; and (2) to estimate the impact of the particular standard operating procedures that were applied to the collecting and processing of biosamples in UK Biobank, on the likely power of future nested case-control studies. Some analyses now explore the role of copy-number variants (CNVs) in disease. I evaluated the accuracy of CNVs genotypes measured on four SNP genotyping platforms to inform future studies that plan to use existing SNP intensity data to measure CNVs or carry de novo CNV measurements from SNP genotyping platforms.

FoxP3 expressing regulatory T-cells (Treg) are essential for preventing autoimmunity by the immune system. The dynamics and signalling requirements for Treg development in the thymus are not well understood but are thought to integrate TCR, co-stimulatory and cytokine signalling. Previous studies have been hampered by the difficulty of distinguishing peripheral homeostasis from de novo thymic generation of Treg. To circumvent this problem, we used mice bearing both a FoxP3 reporter allele (FoxP3GFP) and in which Zap70 expression is controlled by a Tet-inducible transgene (TetZap70), induced by administration of antibiotic doxycycine (dox). Zap70 deficient thymocytes are arrested at the CD⁴+CD⁸+ double positive stage of development. Induction of Zap70 expression by dox therefore restores positive selection and allows analysis of de novo Treg development independently of existing peripheral Treg. In timecourses of Zap70 induction of TetZap70 FoxP3GFP mice, we found that Treg develop after day 4 and remained in the thymus until day 10, at which time GFP+ Treg were first detected in peripheral lymphoid organs. To investigate the requirement for TCR signals for Treg development we used a pulse of the tetracycline analog methacycline, which resulted in a tight 48h window of Zap70 induction. Remarkably, confining Zap70 expression to the first two days of thymic development was sufficient for normal development 4 days later. Using the TetZap70 FoxP3GFP mice we also investigated the temporal requirement for TGFβ, IL-2 and CD40 signalling during Treg development. Neither TGFβ nor CD40 signalling were required for de novo thymic Treg development. Using blocking antibodies and the addition of cytokine-antibody complexes revealed an essential role for IL-2 as well as a semi redundant role for IL-15. Blockade of IL-2 had no effect on induction of FoxP3 or the number of Treg induced during development. However, induction of CD25 by FoxP3+ Treg was entirely IL-2 dependant. Using mixed bone marrow chimeras we show evidence supporting a hematopoietic source of thymic IL-2. We therefore propose a model of thymic Treg development in which TCR signals alone are sufficient to induce FoxP3 expression but that continued development of Treg is reinforced by IL-2.