Therapies to limit or reverse fibrosis have thus far proved unsuccessful, highlighting the need for a greater understanding of the basic mechanisms driving fibrosis and in particular the link between fibrosis and inflammation. Obliterative Bronchiolitis (OB) is the pathological correlate of Bronchiolitis Obliterans Syndrome (BOS) a progressive disease that results in the fibrotic obliteration and blockage of the airways. Development of OB is strongly associated with elevated fibrotic, transforming growth factor beta 1 (TGF- β1), and inflammatory, tumour necrosis factor alpha (TNFα) and interleukin 1 beta, mediators; and the process of epithelial to mesenchymal transition (EMT) has been proposed as a mechanism of OB initiation and progression. Previous work in our group has demonstrated that a physiologically relevant dose of TGF-β1 is capable of driving EMT in primary human bronchial epithelial cells (PBEC) isolated from lung-transplant recipients, an effect that was accentuated by TNFα. It was hypothesized that an unknown synergistic signalling cascade may mediate this accentuation, and identifying candidates for this role is the main aim of this thesis. TGF-β1 and TNFα were used to induce EMT in PBEC cultures; the relative roles of several signalling proteins (SMAD3, IKKβ, TAK1, p38, ERK-1/2 and JNK-1/2) in the development of EMT were assessed by chemical inhibition and siRNA knockdown along with phosphorylation response, to describe a signalling cascade. The results describe a mechanism whereby TGF-β1 drives EMT through SMAD3 with a requirement for TAK1 and JNK-2. TNFα signals through TAK1, IKKβ and JNK-2 but is not capable of driving EMT alone. Upon co-stimulation, TAK1 and JNK-2 display an enhanced activation that leads to an accentuation of EMT, possibly through c-Jun activation. JNK-2 sits downstream of TAK1 therefore the results indicate that TAK1 activity plays a key role both modulating TGF-β1 SMAD3 driven EMT, and its accentuation by TNFα. TAK1 was also shown to be more strongly activated in fibrotic human airway sections compared to control, suggesting that the findings have direct disease relevance. Much further work into how TAK1 modulates SMAD3 activity after TGF-β1 stimulation and how its enhanced activation leads to accentuated EMT is required. Greater understanding of these mechanisms may lead to the discovery of novel therapeutic targets for fibrotic and inflammatory disorders.

Background Hospital admissions with acute exacerbations of chronic obstructive pulmonary disease are common and associated with high mortality rates, frequent readmission and worse quality of life. An ability to identify patients at risk of subsequent poor outcome is lacking and the longitudinal change in quality of life following discharge is uncertain. Methods The study consisted of two parts: 1) Clinical data were collected on 920 consecutive patients hospitalised with exacerbations. The ability of a novel modification of the traditional MRC dyspnoea scale (the extended MRC dyspnoea scale, eMRCD) to identify patients at risk of poor outcome was assessed. Independent predictors of important clinical outcomes were recorded and clinical prediction tools derived. 2) A subgroup of 183 patients underwent longitudinal assessment of quality of life following hospital discharge and predictors of quality of life decline were identified. Results The study population was similar to that reported in UK national audits. 96 (10.4%) patients died in-hospital and 37.3% were readmitted to hospital, or died without being readmitted, within 90-days of discharge. The eMRCD was a better predictor of outcome than the traditional scale and, compared to all clinical variables, was the single strongest predictor of mortality and readmission Strong independent predictors of many important clinical outcomes were identified and, notably, the DECAF (dyspnoea, eosinopenia, consolidation, acidaemia, atrial fibrillation) predictive tool was derived and shown to be an excellent, and internally valid, mortality predictor (area under ROC curve = 0.858). Most patients who survived to discharge reported an improvement in respiratory symptoms and quality of life during follow-up. We defined a subgroup of patients who experienced poor post-discharge quality of life and identified robust, simple-to- measure predictors of poor quality of life. Conclusions Important patient outcomes can be accurately predicted in this population. Application of our results may reduce morbidity and mortality in this common and frequently fatal condition by improving clinical decision making regarding appropriate level of care, location of care and resource allocation.

The Streptococcus pneumoniae capsule is an essential virulence factor and it is ideally situated to modulate interactions between the bacteria and host immune cells. Using isogenic unencapsulated mutants, flow cytometry assays and a mouse septicaemia model, this thesis has assessed the effects of the capsule on the interactions of S. pneumoniae serotype 2 and 4 strains with complement factors and phagocytes. Overall, these data demonstrate that the capsule inhibits complement activity but this only partially contributes to the effects of the capsule on neutrophil phagocytosis and virulence during septicaemia. Furthermore, interactions with macrophages were also found to be complement-dependent and independent, resulting in differences in both phagocytosis and inflammatory responses both in vitro cell line and in vivo. I also investigated whether capsular serotype affects S. pneumoniae interactions with the host immune response using otherwise isogenic TIGR4 strains expressing capsular serotypes 4, 6A, 7F, 23F. These data demonstrate that resistance to complement mediated immunity is associated with capsular serotype, and hence this might be one mechanism by which capsular serotype could affect relative invasiveness of S. pneumoniae strains. Noncapsular genetic background was also found to affect complement mediated immunity, and importantly, relatively invasive strains were on average more resistant to complement than weakly invasive strains. Overall the results in this thesis demonstrate that the S. pneumoniae capsule aids evasion of both complement dependent and independent immune mechanisms, and that serotypedependent differences in the effects on immunity could partially explain variations in virulence between strains.

Introduction For many decades gastrooesophageal reflux has been implicated in patients suffering from lung disease and in lung allograft injury. From the early 1970s studies have taken place investigating reflux in idiopathic pulmonary fibrosis (IPF) and cystic fibrosis (CF). However, these early studies were small and used primitive techniques to assess reflux. In addition, the role of microaspiration secondary to reflux has often been postulated as a cause of deteriorating lung function in these patients but has been under studied. It is also known that many of these patients require a lung transplant due to end-stage lung disease. Asymptomatic reflux and aspiration may be associated with allograft dysfunction post lung transplant. Early anti-reflux surgery has been suggested to improve long-term survival by treating reflux. This thesis reports a prospective assessment of reflux/aspiration in patients with IPF and CF. In addition, the study reports the largest European series of fundoplication in lung transplant patients. Methods Over a 2 year period patients with IPF and CF were recruited from specialist clinics. All patients completed objective assessment of oesophageal physiology using manometry and impedance-pH. Symptom and quality of life assessment using RSI, Demeester and GIQLI questionnaires were performed on all patients at the time of recruitment. For those patients taking proton pump inhibitor, questionnaires were done ‘on’ and ‘off’ their medication. IPF patients then had a bronchoscopy and lavage (BAL) whilst CF patients produced sputum. Cytospins of the BAL and sputum were produced and differential cell counts were performed and the cells were stained with Oil Red O and Prussian Blue (Perls). ELISA and mass spectrometry assays were also performed on the samples for pepsin and bile salts respectively. Lung transplant patients attended for impedance-pH studies over 3 years and those with symptomat ic reflux or reflux and deteriorating lung function were referred for a laparoscopic fundoplication. Lung function assessment, symptom and quality of life questionnaires were performed before surgery and at 6 weeks and 6 months after surgery Results IPF Patients: Thirty eight patients with IPF were initially approached and 29 consented to be studied. Nine patients dropped out from the study after consent. Twenty patients with IPF completed both the oesophageal physiology and BAL aspects of the investigation. In 12 patients there was objective evidence of reflux including 6 patients with proximal reflux. 60% of patients had an abnormal RSI score whilst taking a PPI and scores for the other questionnaires were not significantly different ‘on’ and ‘off’ PPI. Lung function was not related to the degree of reflux. The principal cell type identified was macrophages and both Oil Red O and haemosiderin scores were well above the normal range. Bile salts were detectable in 17/20 IPF patients but the levels were not higher than the normal range. 11/20 patients had higher than normal levels of pepsin in the BAL. CF Patients: Twenty-six patient with CF consented to the study but 15 dropped out. Eleven CF patients attended for oesophageal investigation and each provided 2 samples of sputum. 9/11 had reflux, including five with proximal reflux. All patients were taking acid- suppression medication and questionnaire assessments were abnormal whilst on their medication with 82% still having a GIQLI score below 121 despite medication for reflux. Twenty one samples of sputum were processed altogether. The principal cell type was neutrophils. Bile salts were detectible in all samples but these were at very low concentrations. Elevated pepsin was seen in 7/11 sputum samples with the median concentration ten times above the normal level. Lung Transplant Patients 16 lung transplant patients with symptomatic reflux or deteriorating lung function and reflux on impedance-pH had a laparoscopic fundoplication. Symptom questionnaire and quality of life assessment was significantly improved in all patients. Half the patients had presented with declining lung function and all showed an improvement in respiratory function after surgery. Summary We have demonstrated that reflux is present in patients with IPF, CF and in patients after lung transplant. Using impedance-pH we have identified patients with proximal reflux. The presence of reflux appears to affect the patients’ quality of life and despite PPI therapy the majority still had symptoms. High levels of haemosiderin stained macrophages in IPF indicate oxidative stress which may or may not be secondary to reflux. Pepsin levels are elevated in both IPF and CF patients, possibly indicating microaspiration. Conclusion Despite PPI therapy there is significant reflux in IPF and CF identifying a clinical gap in patient treatment that should be considered in management. Our results in the post lung- transplant group indicate there is a role for surgery in treating reflux and potentially reducing microaspiration. This has been shown to stabilise lung function in this cohort and may have implications for the treatment of reflux in patients with lung disease before transplantation.

Tuberculosis (TB) caused by bacteria of the Mycobacterium- tuberculosis complex (MTBC) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information of clinical isolates of MTBC. The objectives of this work include developing bioinformatic tools for processing and making accessible MTBC genomic data, as well as the identification of informative genetic markers, both strainOspecific and associated with drug resistance (DR), to barcode MTBC isolates in research and clinical settings. SpolPred software was developed to accurately predict the spoligotype from raw sequence reads, and used to bridge the gap between classical genotyping and highO throughput sequencing. A genome variation discovery pipeline was implemented to derive genomic polymorphisms from MTBC raw sequence data. This pipeline was applied to >1,500 publicly available isolates and the characterised genomic variation hosted in PolyTB, a webObased tool where genetic variants can be investigated using a genome browser, a world map showing their global allele distribution, and an additional phylogenetic view. An extensive repertoire of strainOspecific mutations was identified, of which a subset was proposed to accurately discriminate known MTBC circulating strains. A curated list of DR associated mutations was compiled from the literature and their diagnostic accuracy for predicting phenotypic resistance assessed. In addition, potentially novel genes involved in DR were discovered by applying genomeOwide association approaches to a global population of more than 2,500 MTBC strains. Whole genome sequencing (WGS) promises to be transformative for the practice of clinical microbiology, and the rapidly falling cost and turnaround time mean that this will become a viable technology in clinical settings. In this new paradigm, the presented work will facilitate the transition to and applications of WGS in clinical settings as an important tool for TB control.

Background: Socioeconomic deprivation is a determinant of health care access and quality of life in many diseases. The current study explored this effect in Chronic Obstructive Pulmonary Disease (COPD) and examined the role of psychosocial variables in this relationship. Aims: The primary aim of this thesis was to establish whether lower socio-economic status (SES) was associated with decreased health care access (HCA) and poorer quality of life (QoL) in patients with COPD. The secondary aim examined whether psychosocial factors mediated the relationship between socio-economic status, health care access and quality of life. Methods: Cross-sectional, interview-based survey in London involving COPD patients > 40 years recruited in primary care. Measures included socio-economic status, illness perceptions, health care access, quality of life, Medical Research Council (MRC) dyspnoea scale, general self-efficacy scale, social capital, Hospital Anxiety and Depression Scale (HADS), and spirometry. Results: COPD confirmed by spirometry in 176 (85%) participants. 38.6% female, mean age 69 years, distribution of disease severity (GOLD): Grades 1-4 (mild – very severe) = 15%; 51%; 30%; 5%. Lower SES was not associated with more impaired HCA. Lower SES in terms of income level was associated with poorer QoL. The relationship between SES and HCA was not mediated by any of the psychosocial variables but the relationship between SES in terms of income level and QoL was. Conclusion: More deprived COPD patients were as likely to get equal HCA as their more affluent counterparts. More deprived COPD patients in terms of income level were more likely to report poorer QoL. Illness perceptions were significantly associated with HCA and QoL. Findings emphasized the role of SES measures and illness perceptions in this patient group and the variability of their effect on different outcomes. Future research involving longitudinal design could increase understanding of these associations in different life and disease stages.

Chronic obstructive pulmonary disease (COPD) is defined by irreversible airflow limitation, usually caused by exposure to noxious particles or gases. COPD patients suffer from chronic daily symptoms, and may occasionally suffer acute exacerbations – episodes in which there is a worsening of symptoms above day-today levels. Exacerbation aetiology is variable and controversial, although infection and air pollution are believed to play a part. Certain bacterial pathogens, known as typical airway bacteria, are found with high prevalence in individuals at exacerbation. The burden of other airway bacteria is currently ill-defined. This study initially compared microbiological culture with quantitative PCR for detection of three commonly identified airway bacteria in COPD, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis (typical airway bacteria). Quantitative PCR was utilised to establish prevalence and load of these species at stable, exacerbation and exacerbation recovery states. Typical airway bacterial prevalence and load was assessed against a range of clinical factors in COPD. Additional quantitative PCRs examined the prevalence and load of atypical airway bacteria (Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae) and the entire bacterial microflora of the lungs. My original contribution to knowledge is that higher load of typical airway bacteria is associated with higher levels of systemic inflammation and airflow limitation in both stable and exacerbated COPD. For the first time it has also been demonstrated that airway microbiome load is not associated with airflow limitation or systemic inflammation changes, providing evidence that typical airway bacteria in particular are contributing to disease severity. Atypical airway bacteria prevalence in COPD was negligible. The investigations in this thesis highlight the need for rapid antibiotic therapy in exacerbations where typical airway bacteria presence is suspected or confirmed. Furthermore, prophylactic antibiotic therapy should be considered for stable COPD patients with confirmed typical airway bacterial presence, as a means of reducing inflammation and airflow limitation.

Persistent local activation of the coagulation cascade typifies tibroproliferative lung disorders such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. In addition to its role in coagulation, thrombin exerts potent pro inflammatory and pro-flbrotic effects via activation of its major cellular receptor proteinase-activated receptor-1 (PARi). In this thesis, the relevance of PARi activation to inflammation and fibrosis in a model of lung injury and fibrosis based on the intra-tracheal instillation of bleomycin was examined. Lung inflammation and fibrosis were substantially attenuated in PARi-deficient mice. The attenuated inflammatory response was manifest by substantial reductions in bronchoalveolar lavage protein content, total leukocyte numbers, and lung levels of the chemokine CCL2. The attenuated fibrotic response was manifest by a reduction in lung collagen accumulation of around 60%, and reduced mRNA and/or protein levels of the pro- fibrotic mediators connective tissue growth factor (CTGF) and transforming growth factor-beta-1 (TGF-beta1). These data suggest that PAR may promote inflammation and fibrosis, at least in part, via the expression of CCL2, CTGF, and TGFbeta1. Further studies demonstrated prominent immunostaining for PAR on lung epithelial cells following bleomycin injury, consistent with these cells being a potential source of PARi-dependent secondary mediators in the injured lung. Widespread epithelial injury is a prominent feature of fibroproliferative lung disorders, in which hyperplastic activated epithelial cells represent a major source of pro-inflammatory and pro-fibrotic mediators. The effect of PARi activation on lung epithelial expression of CCL2, CTGF and TGFbeta1 was therefore examined in vitro. Exposure to both thrombin and the PARi agonist peptide TFLLR-NH2 substantially increased mRNA levels of CCL2 and CTGF, but not TGFbeta1 in A549 and BEAS-2B human lung epithelial cells. Increases in CCL2 and CTGF mRNA levels in response to thrombin were attenuated with the PARi antagonist RWJ-58259. PAR 1-dependent increases in CCL2 and CTGF mRNA are apparently protein kinase C-dependent since increases in response to TFLLR-NH2 were inhibited by the protein kinase C inhibitor Ro-31-8425. Increases in CCL2 and CTGF mRNA in response to PARi activation were mirrorred by comparable increases at the protein level. Taken together, these data suggest that epithelial cells may represent a source of PARi-dependent pro-inflammatory and pro-fibrotic mediators following lung injury.

Although corticosteroids are very efficient in managing asthma and other inflammatory diseases, a small percentage of patients affected by “severe asthma” fail to respond even to high doses of oral glucocorticoids. It is therefore important to try to understand the potential mechanisms behind this insensitivity to corticosteroid therapy in order to be able to effectively control asthma in this patient subset. We have decided to focus on one particular channel called K[subscript Ca]3.1, a calcium-activated potassium channel. First, emerging evidence in the literature to date has strongly supported a significant role for K[subscript Ca]3.1 channel in the pathophysiology of asthma. K[subscript Ca]3.1 channel is expressed by several inflammatory and structural airway cells including mast cells and human bronchial smooth muscle (HBSM). Therefore these channels might serve as new targets for the treatment of lung diseases. Here we established a cellular model of corticosteroid insensitivity consisting of primary HBSM cells exposed to two cytokines TNF-α and IFN-γ. Under these conditions, HBSMC exhibit a marked production of different pro-asthmatic chemokines like CCL5, CX3CL1, CCL11 and CXCL10 that are completely resistant to corticosteroid treatment. In this model, we found that although K[subscript Ca]3.1 channel expression did not change between healthy control, asthmatic and COPD subjects, K[subscript Ca]3.1 channel blockers (ICA-17043 and TRAM-34) were able to inhibit the production of corticosteroid-resistant chemokines either directly via the suppression of gene expression or indirectly via the restoration of the anti-inflammatory action of fluticasone. We also found that K[subscript Ca]3.1 channel blockers restored cell sensitivity to corticosteroid in cytokine-treated HBSMC by re-establishing the transactivation function of fluticasone via the prevention of dephosphorylation of Glucocorticoid Receptors (GRα) at Ser[superscript 211] and induction of anti-inflammatory genes such as Glucocorticoid-induced leucine zipper (GILZ). The likely mechanism of this restoration of corticosteroid sensitivity by K[subscript Ca]3.1 channel blockers is via the inhibition of protein phosphatase 5 (PP5) expression found to be up-regulated in steroid resistant conditions.

Background: Patient attrition to Pulmonary Rehabilitation (PR) is significant. Whilst biomedical variables have been examined as potential predictors, their explanatory power is not substantial and increasingly psychological constructs have been considered as determinants of PR adherence. Aim: This thesis aimed to increase understanding of how PR can be facilitated and enhanced for patients following hospitalisation with an acute exacerbation of Chronic Obstructive Pulmonary Disease (COPD) by exploring issues around patient access, engagement and assessment of psychological appraisals. Methods: An observational mixed-methods design was utilised informed by a review of the published literature and a retrospective observation of stable patients. Participants in the prospective studies had recently been hospitalised with an acute exacerbation of COPD. Qualitative component: Patients who refused a referral to PR participated in in-depth interviews exploring appraisals of an exacerbation. Transcripts were subjected to Interpretative Phenomenological Analysis. Quantitative component: Questionnaires relating to illness perceptions, mood, health status and self-efficacy were completed soon after hospital discharge. At six months acceptance and uptake of PR was documented. Cluster analysis was conducted using the domains of the Illness Perceptions Questionnaire-Revised. Results: Qualitative component: Conceptual themes included: ‘Construction of the self’, ‘Relinquishing control’ and ‘Engagement with others’. Quantitative component: Three distinct illness profiles exist in patients following an acute exacerbation of COPD: ‘in control’, ‘disengaged’ and ‘distressed’. There were no differences between ‘clusters’ in acceptance or uptake of PR. Conclusion: Adherence to post-exacerbation PR is poor and may reflect the intense emotional distress reported by patients following an acute exacerbation of COPD. Health care professionals need to be mindful of how they address patients who, during a period of vulnerability, are sensitive of perceived dismissive behaviour. Targeted psychological strategies which enhance partnership working and trust, address intense fear and elicit control may be beneficial in reducing distress and supporting uptake of PR.