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Introduction: Chronic Respiratory Diseases (CRDs) are increasing worldwide; more than half of the sufferers live in low- and middle-income countries (LMICs). People with CRDs live with troublesome symptoms, especially breathlessness and fatigue, which reduce their exercise capacity and ability to maintain activity levels. This affects quality-of-life, and overall performance, with many people developing co-morbid anxiety and depression. Pulmonary Rehabilitation (PR) aims to reverse the vicious circle of breathlessness, avoidance of activity, muscle weakness, and further increasing inactivity. There is strong evidence (mainly from high-income countries) that PR improves functional exercise capacity and quality of life, and guidelines recommend PR as an integral part of CRDs care. Despite the potential that implementation of PR could reduce the burden of CRDs, it is notably underprovided in LMICs. AIMS AND OBJECTIVES: I aimed to adapt and test the feasibility of a PR programme to be delivered in a low resource setting and initiate strategies for the implementation of this complex intervention in Bangladesh. My objectives were: • Engage relevant stakeholders, explore, and integrate their views. • Conduct a systematic review to synthesise the clinical effectiveness, components, and mode of delivery of PR in low-resource settings. • Identify core components from global PR guidelines. • Adapt PR protocol for implementation in Bangladesh. • Undertake a feasibility study using mixed-method (quantitative and qualitative) research. • With stakeholders, develop and initiate an ongoing implementation strategy for scaling up and delivering PR in Bangladesh. METHODS: The PhD work proceeded in six phases addressing these objectives: Stakeholder engagement: I selected stakeholders according to their interest and influence; conducting seven meetings across the country to engage them in this implementation research programme and to learn about the context. Systematic Review: I reviewed literature systematically following the Cochrane methodology to identify the evidence generated from LMICs on the effectiveness (improvement of functional exercise capacity and health-related quality of life), useable components, and deliverable models of PR services in a low -resource setting. I searched six databases from 1990 to 2018 with a pre-publication forward citation search in 2020. Global Guidelines Recommendations: I reviewed international PR guidelines and identified the key recommended components of PR. I also visited internationally-recognised centres to learn practical techniques. Adapting a PR programme to the Bangladesh context: I mapped each of the recommended components of PR to an approach that could be delivered in a low resource setting and tailored to the Bangladesh context. Feasibility Study: I planned a mixed-methods, before-and-after feasibility study of PR delivered to groups in my community-based clinic in Khulna. The feasibility study was interrupted by the COVID-19 pandemic. The original intention was to conduct an 8-week centre-based PR programme with face-to-face supervised sessions, including exercise and educational programmes. After completing about one-third of the study, this was suspended due to the pandemic. After a delay of three months, I resumed the feasibility study, having adapted the PR programme for home delivery (with Centre-based assessments) in line with national social distancing regulations and the Sponsor's requirements. Quantitative Analysis: I compared pre- and post-measurements of exercise capacity (ESWT: Endurance Shuttle Walking Test) and quality-of-life (CAT: COPD Assessment Test) using T-tests or non-parametric tests according to the distribution of the data. Secondary outcomes included dyspnoea and anxiety/depression. Qualitative data collection and analysis: Interviews with 15 patients, eleven professionals, two hospital/clinic owners cum managers, and three other stakeholders were recorded, transcribed verbatim, and analysed using two approaches: A grounded theory approach explored patients' views on living with CRDs and the acceptability, benefits, challenges, and enablers for PR. A framework approach, using the Normalisation Process Theory (NPT) Toolkit to understand professional/stakeholder' views about implementing PR in clinical practice. Finally, I synthesised the findings from both the quantitative and qualitative methods to answer the objectives of the feasibility study. Develop implementation strategy: I am working with stakeholders to raise awareness meetings, workshops, seminars, and symposiums on PR as a continuous process for the implementation and integration of PR services in routine clinical practice in Bangladesh. Results: Initial stakeholder meetings identified multiple challenges: lack of research evidence on clinical effectiveness in Bangladesh, poor patient health literacy, economic and cultural barriers, widespread exposure to risk factors, and lack of knowledge among health professionals. There is a need to educate professionals (and specifically train PR therapists), involve influential political and religious leaders, and provide accessible services. These broadly align with the policy statement of ATS/ERS with regard to raising awareness and generating evidence on PR in our own context. The systematic review included 13 controlled studies evaluating the effectiveness of PR in LMICs. In most studies, functional exercise capacity and quality of life improved, but 11/13 studies were at high risk of bias. One of the two studies at moderate risk of bias showed no benefit. All programmes included exercise training; most provided education, chest physiotherapy, and breathing exercises. Adapted to the setting, low-cost services used limited equipment and typically combined outpatient/centre delivery with a home/community-based service. From global PR guidelines, I developed a matrix of the practical components with a detailed description of each element and models of delivery in various settings. The components recommended in global PR guidelines are typically described for delivery in high-income settings. I, therefore, adapted the components to my local low-resource community-based context to develop a protocol for PR in Bangladesh. The feasibility study commenced as a Centre-based programme before the pandemic. Of 296 patients referred from my practice, 89 (30%) patients participated allocated to one of four unisex groups. Of the 207 (70%) who refused centre-based PR, 107 (52%) preferred home-based, 69 (33%) community-based, and only 32 (15%) declined to participate in the research, citing concern that PR might exacerbate their breathlessness, or impose an extra financial burden. The first group had completed 70% of the sessions, the second group had completed 50% of the sessions; the third and fourth groups had just started their programmes when the study was suddenly suspended due to the COVID-19 pandemic. Adapted for home delivery in the pandemic. Sixty-one patients were referred for PR; 51 participated (mean age 55 years (SD 12); M: F 33:18). Forty-four patients (86%) completed 11 (70%) of the remotely supervised sessions. Forty participants (78%) attended the post-PR assessment at eight weeks. Quantitative Analysis: Functional exercise capacity measured by Endurance Shuttle Walking Test (ESWT) improved by 345 seconds (Minimum Clinically Important Difference (MCID) is 174s). Pre: median (IQR) 291 (119, 989) vs post: 544 (60, 1200); P < 0.0001. Quality-of-life measured by the COPD Assessment Test improved by 7 (MCID is 2), Pre: median (IQR) 16.5 (4, 28) vs post: 7.5 (0, 26); p<0.0001. Patients defined their condition by the symptoms (as opposed to a disease). Some were surprised at being offered an exercise programme that triggered breathlessness (the symptom they were trying to cure). Most patients were concerned about the affordability and availability of the service. Professionals perceived PR as a novel intervention, and were aware of evidence of its effectiveness, but had no personal experience on which to base their opinions. Implementation strategy. Building on the evidence from this PhD, I am working on continuous stakeholder engagement, building awareness, and developing skilled professionals through seminars, symposiums and workshops. Conclusions: PR is an integral part of care of the increasing burden of CRDs. It is effective, deliverable, and has applicable components for our context. The feasibility study demonstrated the acceptability and potential benefits of implementing PR in Bangladesh. Stakeholder engagement, especially with influential groups, is the key to implementation. Improving awareness, developing a skilled workforce, and a cost-effective, affordable and easily accessible PR model are pre-requisites of providing patients with CRDs.

Introduction: The measurement of field-based cardiorespiratory fitness (CRF) has been identified as a feasible way to study the link between physical activity (PA) and health in children and adolescents at a population level (Lang et al., 2018a). Indeed, a former Chief Medical Officer for England called for the measurement of CRF as a surveillance tool almost a decade ago, yet no initiative has been put in place (Department of Health, 2010). Therefore, the overarching aim of this thesis was to determine whether there is a case for CRF testing of children in primary schools in England. Methods: Following institutional ethical approval, the following participants were recruited; children aged 8-11 years from 17 primary schools in the London boroughs of Camden and Islington; children aged 9-11 years from nine elementary schools in Birmingham, Alabama and Oakland, California in the US; and seven teachers from the primary schools in the London boroughs of Camden and Islington. CRF was measured by the 20m Multistage Shuttle Run (20mMSR) as part of an initiative called the My Personal Best Challenge (MPBC). Height and weight were also obtained to calculate body mass index (BMI). Testing was completed by trained coaching staff from Premier Education during a Physical Education (PE) lesson. Results: Study 1 involved performing a systematic review, meta-analysis and meta-regression to investigate whether changes in CRF, as a result of a PA intervention, had a significant impact on executive function or academic performance. Key Findings from Study 1: PA interventions can improve CRF in children and adolescents (effect size (ES) = 0.24 [95% confidence interval (CI) = 0.09, 0.40]), yet do not impact executive function (ES = 0.14 [95% CI = -0.12, 0.41]) or academic performance (ES = 0.11 [95% CI = -0.16, 0.38]). Furthermore, the improvements in CRF produced by PA interventions are not associated with changes in executive function or academic performance, including no negative effect on either executive function or academic performance. Given that most of the PA interventions included in the meta-analysis took place in time allotted for other curricular activities, this finding is particularly noteworthy given the health benefits associated with improving CRF. Study 2 aimed to compare the CRF of children who took part in the MPBC from primary schools in London with other studies in English, as well as to provide an international comparison between children who completed the MPBC in England with a convenience sample of children from the US.Key Findings from Study 2: The current data on children's CRF levels in England is limited, with a lack of raw data available, difference in reporting methods, and data restricted to certain regions. However, comparison of group statistics found that children in the MPBC cohort from London had significantly lower CRF than boys and girls from the East of England (p < 0.001) and boys from Liverpool (p < 0.001). Study 2 also demonstrated how the implementation of a CRF testing programme could be completed successfully in two different countries, with children from the MPBC London cohort having significantly higher CRF than children in the US cohort (p < 0.001). Study 3 analysed the relationship between BMI with CRF, as well as applying a CRF cut-point to identify the percentage of children at an increased risk of cardiometabolic disease (CMD) in the different BMI weight categories. Key Findings from Study 3: There was a significant, but weak, correlation between BMI and CRF in children in England (r = -0.35, p < 0.001). Furthermore, over half of children were at an increased risk of CMD due to low CRF (boys = 51.9%, girls = 55.2%), and approximately a quarter of children (boys = 24.1%, girls = 30.5%) were not being identified by current national measures based upon their BMI as they were "lean but unfit". Study 4 explored how CRF levels of children in primary schools in England change over a 3-year period. Key Findings from Study 4: Children's CRF levels improved during the final years of primary school after accounting for age, sex, socio-economic status, and BMI. Children who attended schools where a higher percentage of children were eligible for the Pupil Premium (PP) had significantly lower CRF (20mMSR z-scores mean difference = 0.64 [95% CI = 0.18, 1.1]), especially younger children, though this difference became less pronounced and disappeared by the end of primary school. Study 5 provided a retrospective examination of the perceptions of primary school teachers, through semi-structured interviews on fitness testing of children and collected feedback on the MPBC programme. Key Findings from Study 5: Teachers had positive perceptions overall about fitness testing in primary schools (n = 6) and believed that children enjoyed participating in fitness tests (n = 5). Conclusions: Overall, this body of work has considered the case for whether CRF testing should be implemented in primary schools in England. This thesis has shown that CRF testing in schools can: be used as a tool to evaluate PA interventions, identify children at an increased risk of CMD, track the CRF of children over time and observe how changes in CRF may differ between groups, and make cohort comparisons nationally and internationally. Further, this thesis has demonstrated that teachers have positive perceptions of fitness testing in schools, and that children enjoy participating. As a result of the included studies, this thesis can conclude that there is indeed evidence for the implementation of standardised CRF testing and that it could help develop policy approaches to children's PA, and be used to evaluate their success.

Background: An estimated 6.5 million people are providing unpaid care in the UK. Cardiorespiratory illnesses, including heart failure, chronic obstructive pulmonary disease and coronary artery disease are amongst the leading causes of mortality and hospitalisations globally. People living with these conditions are required to adapt their lifestyle and behavioural to manage their illness. Caregivers are increasingly relied upon to provide support in these lifestyle and behavioural changes. However, understanding the impact of including caregivers on the health outcomes of patients living with long-term cardiorespiratory illness is inconsistent. Additionally, understanding the impact of including caregivers in self-management interventions on caregivers' quality of life lacks clarity. Aim: This first aim of this research was to understand the impact of including caregivers in self-management interventions for patients and caregivers living with long-term cardiorespiratory illnesses. The second aim of this research was to understand how inclusion in a self-management intervention informed caregivers in their role and the subsequent caregiver actions which influenced patient health related quality of life. Methods: Four linked research studies were completed. First, a mixed-methods systematic review was undertaken to understand the lived experience of being a caregiver for patients living with long-term cardiorespiratory illness (chapter 3). Second, a meta-analysis was conducted to synthesise the current body of evidence of randomised controlled trials (RCTs), which included caregivers in intervention delivery for long-term cardiorespiratory illnesses (chapter 4). Third, quantitative and qualitative secondary analyses of the Rehabilitation Enablement in Chronic Heart Failure (REACH-HF) trials were undertaken. REACH-HF is a home-based self-management intervention for heart failure patients and their caregivers that formally included caregivers in its design and delivery. Quantitative analyses were completed at 4 and 6 months follow-up. These analyses examined three aspects of caregiver involvement in REACH-HF. The impact on patient health-related quality of life when formally involving caregivers in the REACH-HF intervention. The impact on caregiver quality of life following participation in the REACH-HF intervention and, lastly, whether there were factors that predicted caregiver health outcomes in response to the REACH-HF intervention (chapter 5). A qualitative secondary analysis utilised a cross-case analysis approach. The purpose of this analysis was to explain the quantitative findings and examine the nature of caregiving and how this contributed to patient self-care (chapter 6). Results: The mixed-methods systematic review synthesised 54 studies (26,453 caregivers) (chapter 3). The concepts identified in this review emphasised the complexity of caregiving for adults with long-term cardiorespiratory illnesses and demonstrated the impact of caregiving on: (1) mental health, (2) caregiver role, (3) lifestyle change, (4) support for caregivers (5) knowledge, and (6) relationships. Four concepts were explicitly identified in the qualitative literature: (7) expert by experience, (8) vigilance, (9) time, and (10) shared-care. The quantitative meta-analysis of 13 RCTs (1,701 patient-caregiver dyads) (chapter 4) demonstrated that the pooled effect of patient health-related quality of life in RCTs that included caregivers in intervention delivery compared with studies that did not include caregivers were not significantly different (p = 0.84). Quantitative analysis of the pooled REACH-HF randomised controlled trials included 266 heart failure patients and 118 caregivers. The univariate analysis demonstrated improved health-related quality of life outcomes for patients when a caregiver was included in the intervention. Mean increase in overall Minnesota Living with Heart Failure Questionnaire score of 10.6 (CI 95% 2.7 to 18.4, p = 0.008) at 4 months follow-up. The direction of effect towards caregiver presence remained at 6 months follow-up (9.6, CI 95% 1.1 to 18.2, p =0.026). Multivariate analysis demonstrated that an interaction effect on patient HRQoL in favour of patients with a caregiver remained at 4 months follow-up (9.9, 1.9 to 18.0, p = 0.015). However, this was not sustained at 6 months follow-up (2.2, -0.5 to 4.9, p = 0.113). Increased patient illness severity was the most consistent predictor of caregiver outcomes, however this was not observed across all caregiver outcomes measures. REACH-HF did not consistently demonstrate improvements across all caregiver outcomes; however, it did demonstrate potential to improve caregiver anxiety as well as social and lifestyle burden. The qualitative secondary analysis demonstrated that as a result of REACH-HF, caregivers were enabled to engage in overt and discrete caregiving tasks. Overt caregiving tasks included use of the REACH-HF progress tracker to communicate effectively about HF self-management. Discrete caregiving tasks included utilising the knowledge obtained from the patient manual and caregiver resource to make decisions about how much assistance to provide to the patient dependent on what the caregiver observed (e.g. face colour, tone of voice). Sustained change was difficult to achieve for some patient-caregiver dyads, some caregivers had difficulties motivating the patient to maintain engagement with self-management strategies. A combination of factors was identified by patient-caregiver dyads who reported difficulty maintaining change. These included, the duration of diagnosis, the severity of illness, the demands on the caregivers' time, and the concept of the meaningfulness of the task for the patient. Conclusions: Caregivers do have a key role in the self-management of patients with long-term cardiorespiratory illnesses, such as HF and COPD. Understanding the caregiver role and the nature of the patient-caregiver relationship can significantly influence how we can better engage patients in the self-management of their illness. REACH-HF facilitated patients and caregivers to communicate effectively about HF self-management, increased their HF knowledge and skills and enabled caregivers to feel supported with the inclusion of the wider family. That disease severity was the most consistent predictor of poor caregiver outcomes, and the challenges of sustaining behaviour change indicates the need for ongoing healthcare support for both patients and caregivers as disease progresses. The value of engaging in self-management tasks that are meaningful to the patient and caregiver was an important finding and is an approach that may facilitate sustained behaviour change. This thesis discusses the methodological and practical implications of these findings on the involvement of caregivers in healthcare interventions for long-term cardiorespiratory illnesses and future research.

The work presented in this thesis aims to advance the understanding the role of cellular senescence following acute lung injury, and the subsequent processes that lead to pulmonary fibrosis and ventilator dependence. Herein is described various approaches that were taken to investigate type II alveolar epithelial (AT2) cell senescence as a potential therapeutic target in acute respiratory distress syndrome (ARDS). ARDS is common within the intensive care unit, consisting of the exudative, restorative and fibrotic phases, this disease is spatially and temporally heterogeneous across areas within the lungs. There are no biological therapies routinely used in clinic for this extremely challenging syndrome meaning there is an incredibly high mortality rate of around 40% in patients that are diagnosed with moderate to severe ARDS. Therefore, greater understanding of potential avenues for therapeutics in ARDS are needed, something all the more prominent during the recent SARS-CoV-2 pandemic. The AT2 cell is the stipulated progenitor cell in the lung, as AT2 hyperplasia is required for the repopulation of the alveolar epithelium following injury. Characterisation of senescent AT2 cells in-vitro (chapter 3) investigates the features of senescent cells that can be detrimental to functional tissue repair such as proliferative arrest and a pro-inflammatory senescence-associated secretory phenotype (SASP). Chapter 4 investigates senolytics, an emerging class of compounds that exploit specific phenotypic changes to selectively deplete senescent cells, as a possible pharmacological intervention in acute lung injury. Senescent AT2 cells demonstrate susceptibility to the senolytic compound ABT263 (Navitoclax) invitro, which shows promise for further study. ARDS is classically considered to be neutrophil-mediated disease, with neutrophil elastase (NE)-null mice reported to show reduced levels of active TGF-β and pulmonary fibrosis. In-vitro and in-vivo models were used to investigate the effects of NE as an inducer of senescence in AT2 cells (chapter 5). Finally, chapter 6 describes the development and characterisation of a β-galactosidase activated fluorescent probe, called XD-βGal, aimed at specifically labelling senescent cells for imaging both in-vitro and in-situ. Optical molecular imaging of senescent cells with fluorescent probes is an appealing method as it is iii incredibly versatile, and the monitoring of senescent cell burden within tissue would be of huge advantage for diagnosis and the stratification of patient treatments.

Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are among the most common causes of respiratory infections worldwide, causing morbidity and mortality especially in young children but also elderly or immunocompromised adults. Infections result in hundreds of thousands of hospitalizations each year, leading also to a significant global health and economic burden. RSV alone infects nearly all children by the age of 2 years and is estimated to cause a higher disease burden than influenza in the adult population. Despite intensive research, there are still no licenced vaccines or effective treatments against RSV and PIV infections. Most antiviral approaches to date directly target the virus, although many host factors are involved in the viral replication cycle. MicroRNAs (miRNAs) are a class of small regulatory RNAs that canonically supress gene expression by binding to the 3' untranslated region (3' UTR) of messenger RNAs (mRNAs). Several miRNAs have been reported to be altered upon viral infection and these alterations can suppress or boost host immune responses and the viral infection progress, depending on the targets of the miRNAs. However, there is no systematic analysis of miRNA and target regulation in respiratory virus infection. Previous functional screening from our group using miRNA mimics and inhibitors demonstrated that some miRNAs including miR-744 and miR-28 have antiviral properties against a wide range of viruses. However, the targets of these miRNAs and their mechanisms of action remain largely unknown. This thesis focuses on the global characterisation of host miRNA regulation in RSV and PIV-3 infections in the A549 epithelial cell line and then further investigates the targets of key de-regulated or antiviral miRNAs in RSV infections. Dynamic changes in miRNA expression over the time course of 96 h of RSV and PIV-3 infections were determined by small RNA sequencing. In general, RSV and PIV-3 induced similar de-regulation of miRNA levels, with miR-149 and miR-744 being down-regulated and the miR-34/miR-449 cluster being upregulated. Other miRNAs that we previously identified as antiviral (such as miR-28) were not differentially expressed. In addition, several viral small RNAs (vsRNAs) with a length of 22-30 nt were identified in the small RNA sequencing data for both RSV and PIV-3. The RSV vsRNAs were found to be present not only inside the cell but also in cell culture supernatant at early time points of infection. Small RNA sequencing from RSV-infected patients confirmed their presence in bronchial alveolar lavage fluid. Inhibition of one of these RNAs (vsRNA L-1) inhibited RSV infection and replication, making this RNA an interesting candidate for future therapeutic approaches. To investigate the mechanism of action of deregulated and antiviral miRNAs in RSV infection, miRNA targets were identified by immunoprecipitation of the RNA-induced silencing complex (RISC), followed by ligation of the miRNA to its target and sequencing of these chimeric RNAs. After initial optimisation, two protocols were tested side by side to identify optimal conditions for the A549 cell line. A variation of the CLEAR-CLIP (covalent ligation of endogenous Argonaute-bound RNAs- Crosslinking and immunoprecipitation) protocol resulted in 3-6% of miRNA-target interactions. Apart from canonical interactions with 3' UTRs, high confidence targets were identified in coding sequences (CDS) and regulatory regions using a new bioinformatic approach developed by our laboratory. In general, miRNA expression correlated with the number of target counts. However, for a subset of miRNAs that were highly abundant, no high-confidence targets could be identified, including the miR-449 family. These data suggest that additional mechanisms, such as RISC association, regulate targeting of these miRNAs. In addition to the identification of host targets, this analysis also identified bindings sites in the viral genome for host miRNAs miR-26 and miR-27, which could represent a viral mechanism to suppress these host miRNAs and up-regulate host gene expression. High confidence targets were analysed in detail for a subset of lead candidate miRNAs to identify their mechanisms of action. Target network analysis suggested miR-26 and miR-27 as regulators of host genes important for viral processes. Too few targets were found to perform target network analysis for miR-28 or miR-744, but initial results confirm the Signal Peptidase Complex Subunit 3 (SPCS3) 3' UTR as a target for miR-28. This gene was reported to be essential in viral infections and could explain the antiviral effect of miR-28 mimics. In summary, this thesis provides a global overview of miRNA regulation during the time course of respiratory virus infections and provides insight into new target interaction of de-regulated or antiviral miRNAs during RSV infection. Expanding our knowledge of miRNA-mediated gene regulations in the context of infection will contribute to a better understanding of pathways that could be targeted in new therapeutic strategies.

Pulmonary arterial hypertension (PAH) is a rare fatal disease characterised by endothelial dysfunction and obliteration of small pulmonary arteries. The resulting increase in pulmonary arterial pressure and right ventricular afterload ultimately causes death by right heart failure. Current available treatments show little impact on patient mortality and there is thus an urgent need to better understand the causes of PAH pathology to derive new transformative therapies. Recently, a whole-genome sequencing study of >1000 idiopathic PAH patients identified pathogenic mutations in several new genes. In a small subset of patients, rare heterozygous variants in the SOX17 gene, predicted to be functionally deleterious, were significantly associated with PAH. SOX17 is an essential transcription factor involved in the development of the pulmonary vasculature and is highly expressed in endothelial cells. The aims of the studies underlying this thesis were to explore how SOX17 deficiency may compromise endothelial integrity and cause dysregulated vascular functions. To achieve this, I used SOX17-specific silencing RNA (siSOX17) to knock down SOX17 in pulmonary arterial endothelial cells (PAECs). siSOX17-transfected PAECs express only 10-20% of endogenous SOX17, without significant effects on the expression levels of other SOX family members including SOX2, SOX4, SOX7 and SOX18. SOX17-deficient PAECs exhibited decreased proliferation rates and transcriptional analysis suggested this to be a result of decreased cyclin A (CCNA1) and cyclin B (CCNB1) expression and increased expression of p15 (CDKN2B), a cyclin-dependent kinase inhibitor. Additionally, siSOX17-transfected PAECs showed reduced monolayer permeability and impaired cellular angiogenesis, namely through horseradish peroxidase (HRP) permeability assay as well as tube-network and bead-sprouting assays, respectively. In parallel, to recapitulate the discrete manner in which a patient-specific mutation in SOX17 may initiate PAH disease development, a heterozygous mutation found in patients was introduced into human wild-type (C2 SOX17+/+) induced pluripotent stem cells (iPSCs), resulting in the generation of the C2 SOX17+/R140P iPSC line via CRISPR-Cas9 and homologous recombination. The R140P mutation, replacing the amino acid arginine (R) to proline (P) at position 140, was chosen as it is predicted to substantially impair the DNA binding capacity of SOX17. To assess the impact on endothelial cell development, the gene-edited iPSCs were subsequently differentiated into iPSC-derived endothelial cells (iPSC-ECs). In mutant C2 SOX17+/R140P iPSC-ECs, the SOX17 mRNA expression level was substantially lower than in C2 SOX17+/+ iPSC-ECs, suggesting the mutation may impair a SOX17 autoregulatory positive feedback loop. Furthermore, transcriptional analysis of arteriovenous gene expression revealed a significant reduction in the arterial markers, NOTCH, HEY1 and EPHNB2 in mutant C2 SOX17+/R140P iPSC-ECs compared to C2 SOX17+/+ cells. Additionally, two SOX17 iPSC lines, SOX17147Δ8del and SOX17145Δ18del, containing an 8bp and 18bp deletion at amino acid position 147 and 145 respectively, were obtained via non-homologous end joining and studied in parallel to the C2 SOX17+/R140P iPSCs. Moreover, the effects of SOX17 mutations on protein biochemistry, namely SOX17 DNA binding capability and localisation, were also investigated in this study. WT SOX17 localised to the nucleus of both PAECs and C2 iPSC-ECs. Interestingly, C2 SOX17+/R140P iPSC-ECs exhibited nuclear SOX17 staining, while SOX17147Δ8del and SOX17145Δ18del iPSC-ECs demonstrated partial non-nuclear staining. Nuclear localisation site (NLS) prediction algorithms consistently identified a monopartite NLS within the SOX17 transcript at amino acid position 146-151. The deleted regions in both SOX17147Δ8del and SOX17145Δ18del overlapped with this predicted NLS, supporting the localisation data. To assess SOX17 DNA binding ability, a SOX17 luciferase reporter system was generated, though current data suggest further optimisations are required to increase specificity and remove confounding factors. Lastly, in vivo assessment of pulmonary haemodynamics in Sox17+/GFP mice showed Sox17 haploinsufficiency alone did not lead to PAH but may require pulmonary-artery specific deletion of Sox17 or additional stimulus such as hypoxia to precipitate PAH. Total RNA extraction for transcriptional analysis and immunohistochemistry for assessment of vascular remodelling are currently underway.

Chronic obstructive pulmonary disease (COPD) is a condition characterised by progressive lung function limitation. COPD is associated with airway inflammation and high numbers of inflammatory cells. Alveolar macrophages are the major phagocytic cell of the lung and control levels of pathogens through phagocytosis. Despite an increase in cell numbers in COPD subjects, there is evidence of bacterial colonisation possibly due to reduced phagocytosis. Reduced clearance of bacteria extends to fungal spores and apoptotic cells, suggesting defective phagocytosis of pathogens could contribute to colonisation, inflammation and disease progression. This thesis examines mechanisms underpinning defective bacterial phagocytosis. Monocyte-derived macrophages (MDM) from non-smokers, smokers and COPD subjects differentiated in GM-CSF were used to explore the role of the motor protein dynein in the context of defective phagocytosis in COPD. Defective bacterial phagocytosis of H. influenzae and S. pneumoniae was confirmed in COPD MDM, compared to non-smoker MDM and was associated with reduced trafficking of dynein. Inhibiting the movement of dynein in healthy cells also led to defective phagocytic uptake. To understand if these results were due to differences in cell signalling, leading to alterations in cytoskeletal rearrangements in COPD, the phosphorylation profile of MDM from COPD patients and controls in the absence or presence of H. influenzae was analysed. Analysis showed differential phosphorylation of several cytoskeletal associated proteins, including ezrin. Further analysis of ezrin suggested that reduced activation is linked to a reduction in phagocytosis in MDM, suggesting ezrin is important in this process. Changes to its expression or activity could contribute to defective phagocytosis, through its effect on microtubule instability, leading to reduced receptor recycling and early satiety. Cytoskeletal changes could be contributing to defective phagocytosis by COPD MDM. Investigations into defective phagocytosis aim to find an appropriate target for therapeutic modification, thereby restoring phagocytic capability, reducing bacterial load and diminishing inflammation.

The human lung is capable of endogenous repair, but the underlying mechanisms which control this remain unclear. Many of the processes involved in lung repair are thought to be similar to those required during alveolar development. As such, the targeting of factors involved in lung development provides a potential therapeutic route for the induction of endogenous repair mechanisms. Retinoic acid (RA), obtained wholly from dietary vitamin A (vit A), is an essential mediator of lung development and can promote regeneration in experimental animal models of alveolar insufficiency. We aimed to investigate the role of RA in mediating lung structural homeostasis and repair using a multi-disciplinary, epidemiological and lab-based approach. We identified a positive role for dietary vit A in the modulation of adult lung function in a large UK population-based study. Using genetic data from this study, we have also identified single nucleotide polymorphisms (SNPs) in RA pathway genes that modulate lung function, some of which interact with vit A intake. At a cellular level, we used an in vitro scratch assay model to show that treatment with exogenous RA directly promotes endothelial repair but does not affect epithelial repair, either directly or via paracrine signalling from the endothelium. To study the role of RA in 3D alveolar tissue, we developed the acid injury and repair (AIR) model, a novel ex vivo tool in which the response to injury and subsequent repair mechanisms can be determined. By examining established cellular markers of repair in the AIR model, we discovered that the number of progenitor cells increases in damaged regions of tissue whereas in the uninjured areas of the same PCLS, proliferation increases without a concomitant increase in progenitor cells. Manipulation of RA signalling using pharmacological inhibitors resulted in a significant reduction in proliferation following injury. RA reporter mice, which express GFP linked to the RA response element, were used to visualise active RA signalling. By combining this reporter line with the AIR model, we found GFP expression, indicative of active RA signalling, in postnatal day 7 PCLS and observed an increase in GFP signal following injury in adult PCLS. Overall, our findings suggest that RA signalling primarily affects the capillary endothelium during lung repair and that at a population level, a vit A rich diet could be used to modify lung function. Together this multidisciplinary approach provides important insight into the mechanisms underlying the role of RA in lung repair and regeneration and suggests that regulation of vitamin A levels through nutritional intake may provide a route to help maintain adult lung function.

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy persons, but it remains a major cause of pneumonia, bacteraemia and otitis media despite vaccines and effective antibiotics. There is an urgent need for novel therapeutic approaches, but such advances require a detailed knowledge of S. pneumoniae biology. To better understand pneumococcal biology and infections, we need sensitive in vivo imaging technologies. To this end, bioluminescence imaging can be used, for example, to evaluate anti-infectives, intraspecies interactions and pneumococcal virulence non-invasively. Pneumococcal strains containing click beetle luciferase (CBRluc) under the control of putative highly expressed pneumococcal promoters were constructed. The CBRluc constructs were integrated into known sites in the S. pneumoniae genome. The bioluminescent strains were compared to a lux-based system expressing bacterial luciferase using in vitro growth experiments and in vivo mouse model of pneumonia. The results revealed that CBRluc tagged bacteria showed robust activity of bioluminescence in exponential phase that is also maintained during stationary phase, whereas lux-expressing pneumococci emitted a light signal with high background that peaked during exponential phase and was significantly reduced in intensity during stationary phase. The presence of CBRluc did not affect the growth and virulence properties of the bioluminescent pneumococcal strains. In vivo bioluminescence activity obtained with CBRluc labelled bacteria was much higher than the lux containing strain under different promoters. This study also established that CBRluc reporter system can be used to study pneumococcal virulence in G. mellonella model. The findings demonstrate that the CBRluc reporter system is more efficient than lux, providing a potential platform for utilization in understanding of the mechanisms of pneumococcal pathogenesis in vivo.

Influenza viruses represent a major public health burden worldwide, resulting in an estimated 500,000 deaths per year, with potential for devastating pandemics. Considerable effort is expended in the surveillance of influenza, including major World Health Organization (WHO) initiatives such as the Global Influenza Surveillance and Response System (GISRS). To this end, whole-genome sequencning (WGS), and corresponding bioinformatics pipelines, have emerged as powerful tools. However, due to the inherent diversity of influenza genomes, circulation in several different host species, and noise in short-read data, several pitfalls can appear during bioinformatics processing and analysis. 2.1.2 Results Conventional mapping approaches can be insufficient when a sub-optimal reference strain is chosen. For short-read datasets simulated from human-origin influenza H1N1 HA sequences, read recovery after single-reference mapping was routinely as low as 90% for human-origin influenza sequences, and often lower than 10% for those from avian hosts. To this end, I developed software using de Bruijn 47Graphs (DBGs) for classification of influenza WGS datasets: VAPOR. In real data benchmarking using 257 WGS read sets with corresponding de novo assemblies, VAPOR provided classifications for all samples with a mean of >99.8% identity to assembled contigs. This resulted in an increase of the number of mapped reads by 6.8% on average, up to a maximum of 13.3%. Additionally, using simulations, I demonstrate that classification from reads may be applied to detection of reassorted strains. 2.1.3 Conclusions The approach used in this study has the potential to simplify bioinformatics pipelines for surveillance, providing a novel method for detection of influenza strains of human and non-human origin directly from reads, minimization of potential data loss and bias associated with conventional mapping, and facilitating alignments that would otherwise require slow de novo assembly. Whilst with expertise and time these pitfalls can largely be avoided, with pre-classification they are remedied in a single step. Furthermore, this algorithm could be adapted in future to surveillance of other RNA viruses. VAPOR is available at https://github.com/connor-lab/vapor. Lastly, VAPOR could be improved by future implementation in C++, and should employ more efficient methods for DBG representation.

Influenza is a common and widespread disease caused by influenza viruses. It causes approximately half a million deaths globally each year, and poses the constant risk of a novel pandemic strain emerging with a much higher mortality rate than seasonal influenza viruses. Reducing the burden of influenza depends on effective vaccines and antiviral drugs, and the rational design of these depends on a thorough understanding of the basic biology of influenza viruses. While influenza viruses have been intensely studied, several aspects of their biology have been neglected. Notably, most laboratory-adapted strains of influenza virus produce virions with a predominantly spherical morphology, but clinical isolates produce virions ranging from spheres with diameters of ~100 nm to filaments with lengths that can exceed 30,000 nm. Passage experiments suggest this pleiomorphy is adaptive, but it has been the focus of very little research and so its role is unknown. Furthermore, methods with which to examine filamentous virions are lacking, meaning this area of influenza biology is both understudied and requires extensive methods development. To solve these problems, I developed a range of tools with which to analyse filamentous influenza virions. These included an adaptable, semi-automated analysis pipeline using confocal microscopy to assess the concentration and lengths of filamentous virions in a sample; a set of validated procedures that can be used to handle filamentous virions without damaging them; and a robust method to enrich samples in filamentous or non-filamentous virions. Using these methods, I examined how the proteome of influenza virions varied with their morphology using mass spectrometry and found that all viral proteins except haemagglutinin and matrix protein were depleted in filamentous virions relative to their size. This suggested that filamentous virions may be less robustly infectious than non-filamentous virions, and may have different levels of glycoprotein activity. Using functional assays, I found that non-filamentous virions were indeed enriched in fully infectious particles compared to filamentous virions, and that the HA activity relative to NA activity of filamentous virions was much higher than that of non-filamentous virions. However, this difference in glycoprotein activity did not affect inhibition by mucus. These characterisations of the composition and function of filamentous influenza virions, and the tools used to perform them, provide a solid foundation to elucidate the role of, and potential pharmaceutical interventions exploiting, these complex particles in influenza virus infections.

Chronic lung disease is one of the main causes of morbidity and mortality worldwide. The recent discovery of the lung microbiome has transformed our understanding of the pathophysiology of respiratory infections and chronic lung disease. In the presented PhD thesis, the hypothesis that the composition of the whole microbial community rather than individual pathogens, is critical in the pathogenesis of chronic lung disease has been investigated. The airway microbiome was studied in a spectrum of chronic lung diseases: non-cystic fibrosis bronchiectasis, chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD) in adult survivors of extremely preterm birth and early pulmonary changes in people living with HIV (PLW-HIV) using culture independent approaches: next generation sequencing and quantitative polymerase chain reactions (qPCR). In all forms of the chronic lung diseases studied, a characteristic pattern of bacterial dysbiosis was identified. This was characterised by a significant decline in the bacterial community biodiversity and a shift in the bacterial community composition away from phylum Bacteroidetes; particularly genus Prevotella whose relative abundance was correlated with an important lung function parameter: FEV1% predicted. In PLW-HIV, some potential respiratory pathogens and gut bacteria were enriched in the airway microbiome which may place this population at higher risk to respiratory morbidities and pneumonia. Chronic lung disease is a sector employing extensive antibiotic prescription practices either to treat acute exacerbations, or as prophylaxis therapy. Substantial scientific evidence currently supports the clinical usefulness of macrolide prophylaxis therapy in managing chronic respiratory conditions. In this thesis, I investigated the effect of antibiotics on the homeostasis of the bacterial communities in the airways and how it contributed to the of antimicrobial resistance (AMR) among microbiota. The airway was found to harbour a rich source of AMR determinants and resistant microbiota. The AMR determinants were more related to the antibiotics used as rescue packs for prompt initiation of self-treatment of exacerbations. Antibiotic prophylaxis therapy was associated with lower total bacterial load and suppressed recognised pathogenic bacteria in the airways with minimal effect on the homeostasis of the respiratory microbiota. The airway bacterial community was resilient towards the disturbances caused by antibiotics use. No definite directional shift in the microbiome compositions associated with prophylactic antibiotics was identified at the group level.

Mycobacterium avium belongs to the group Nontuberculous Mycobacterium (NTM), which are acid fast, environmental bacteria belonging to the family Mycobacteriaceae. They are facultative intracellular bacilli causing opportunistic infections in individuals with pre-existing conditions such as respiratory and immune disorders. There has been a steady increase in the number of NTM infection reports in the past four decades and many of them show resistance to common anti-mycobacterial drugs. It is known that the bacteria manipulate the host immune response to their advantage; however, the intracellular trafficking and survival strategies are poorly understood. To address this, we have developed an image-based, single cell assay using a fluorescence dilution (FD) system to measure replication and persistence of M. avium in human THP-1 macrophage-like cells and assess new drug treatments. Accuracy of fluorescence signal as proxy of intracellular replication was confirmed against the gold standard technique of CFU plating. Moreover, the assay was validated using a panel of known anti-mycobacterial drugs, reproducing efficacy profiles similar to those reported in the literature. Based on our results, the fluorescence dilution system is an effective high throughput assay to test anti-mycobacterial drugs and host directed therapies. To dissect host responses to infection and identify potential targets for host-directed therapy, 10x single cell RNA sequencing (scRNA-seq) of M. avium infected THP-1 macrophages was performed, revealing for the first time the heterogeneity in the host response; i.e, several large cell clusters showing different degrees of an inflammatory response, as well as a small cluster of infected cells showing a strong type I interferon response, suggesting a dynamic interplay of the immune signals at the single cell level. Based on the differentially expressed genes identified in the scRNA-seq analysis, small molecule host targeted inhibitors were used in the FD assay, which led to the identification of importazole, a nuclear transport inhibitor, as a compound that inhibits intracellular replication. Further mechanistic studies showed that importazole has direct antibacterial activity against M. avium, causing a reversible increase in bacterial cell length and inhibition of replication, suggesting that the compound could inhibit bacterial cell division. Together, this work established and successfully employed a new image-based screening system to measure replication and persistence of M. avium in combination with scRNA-seq of infected macrophages, identifing the drug importazole as new inhibitor for M. avium replication, opening new avenues for the development of new therapies for M. avium infections.

Background: In patients with Chronic Obstructive Pulmonary Disease (COPD) daily physical activity (PA) levels are significantly lower than in healthy age-matched individuals. PA counselling has been employed to address the complex behaviour of PA and was shown via a systematic review and meta-analysis to be effective in improving daily PA levels (steps/day) both as a standalone intervention and alongside pulmonary rehabilitation (PR). However, in patients reporting low baseline levels of PA (≤4000 steps/day), PA counselling alongside PR did not induce clinically important improvements in daily PA levels. A plausible reason involves the concept of a "low functional reserve", indicating that these patients are less likely to become more physically active within their functional limits. Objectives: 1) to perform a systematic review and meta-analysis on PA counselling as a standalone intervention and alongside PR in COPD, 2) to determine the criterion validity and test-retest reliability of a commercially available pedometer used by COPD patients to self-monitor and report daily PA levels, 3) to evaluate daily PA levels, muscle function, anxiety and depression in patients with COPD living in the North East of England in comparison to healthy age-matched individuals from the same region in a cross sectional study design, and 4) to investigate the feasibility, acceptability and efficacy of combining PR, designed to improve exercise capacity, with comprehensive PA behavioural modification interventions, designed to translate PR-induced improvements in exercise capacity into improved daily PA, in COPD patients with low baseline PA levels. Methods: To accomplish the latter objective a prospective, single centre, two parallel-group, RCT, compared the efficacy of a PR programme combined with PA behavioural modification interventions (PR+PA: incorporating motivational interviewing, face-to-face twice weekly goal setting, step count monitoring and feedback) to a PR programme alone in 48 patients with COPD (FEV1: 49±19 % predicted) exhibiting low baseline exercise capacity (6MWT: 289±85m) and daily PA levels (3293±2000 steps/day). In both groups (PR+PA and PA alone) patients with profound anxiety and depression (≥8 HADS score) received sessions of Cognitive Behavioural Therapy (CBT) by a specialist respiratory nurse. Results: Compared to PR alone, PR+PA induced clinically important improvements in both PA levels (by 1016 steps/day; 95% CI 556 to 1474 steps/day, p = 0.001) and patients' PA experiences (by 7 points; 95% CI 4 to 11 points, p= 0.001) that were assessed by the European Medicines Agency qualified Clinical PROactive Physical Activity in COPD instrument (C-PPAC). Importantly, both groups reported clinically important improvements in the 6MWT (≥ 30 m) and CAT questionnaire (≥ -2 points), however improvements in upper and lower muscle strength were significantly greater in the PR+PA compared to PR group. These findings were supported by evidence of adequate PR completion rates (80%) and high patient acceptability of the PA behavioural modification interventions, with 75% of patients indicating that they "liked taking part in the intervention a lot", and 58% of patients claiming that the intervention "helped them a lot" regarding completing more PA outside of PR. Furthermore, patient adherence to the components of the behavioural modification interventions was high, including the weekly use of the pedometer (6.6±0.2 days) and interaction with the PA diary (93±17%) to self-monitor and report daily step counts. Conclusions: The findings suggest that in COPD patients with low baseline exercise capacity and daily PA levels, improvements in exercise capacity following completion of a standard PR programme may translate into clinically important improvements in daily PA levels only when tailored PA behavioural modification interventions are added to PR. In addition, PA behavioural modification interventions were proven to be feasible to incorporate into a standard PR programme and were well accepted by patients with COPD.

Introduction: A well designed randomised controlled trial is the best-known process to assess if a healthcare intervention works. The number of clinical trials registered and open for recruitment is increasing every year, through the global cumulative effort of many researchers and the large numbers of participants who volunteer their time. However, trials can be methodologically poor, ultimately leading to research waste whilst leaving important clinical questions unanswered. This thesis aimed to explore a series of novel methods as solutions in respiratory medicine. Methods: A number of studies were embedded within the CLEAR bronchiectasis trial. Studies investigated remote data collection in lung function using patient-led technology, the use of remote patient reported outcomes within the new standardised EMBARC framework for exacerbations and novel Studies Within A Trial (SWAT) to increase patient recruitment and retention. A systematic review was also completed for the use of mucoactives in acute respiratory failure, which together with the other methods explored in the CLEAR trial, informed the conditional design of a new trial in respiratory medicine. Results: Remote spirometry was completed by patients at home with moderate adherence and was not significantly different to supervised spirometry performed at clinic visits for a number of lung function tests (FEV1, FVC, FEF25-75 and PEF). The EMBARC definition was found to be equivalent to the Fuchs criteria for classifying exacerbations and it was feasible to assess key symptoms using remote patient reported outcomes. In addition, the symptoms contained within EMBARC corresponded to those that deteriorated most at the start of exacerbations. For the SWAT, modifications to the invitation letters had no significant effect on recruitment and giving patients enrolled in the trial a thank you card had no effect on retention, albeit both SWAT had small sample sizes. The systematic review found limited evidence for mucoactives in critically ill patients and justified the design of a large robust clinical trial. Conclusion: A comprehensive programme of important methodological research can be undertaken within a large clinical trial with aims that are distinct from those of the main trial. Remote spirometry and using the EMBARC definition with patient reported outcomes can be implemented in further bronchiectasis trials to increase pragmatism and quality. Further research is needed to identify methods to increase recruitment and retention. A large, randomised trial for common mucoactives in the critically ill is needed.

Bronchiectasis is a chronic airways disease characterised by permanent dilatation of bronchi, inflammation, daily sputum production and infective exacerbations. There is an unexplained female predominance in the prevalence of bronchiectasis in a range of small epidemiological studies. Assessment of quality of life in bronchiectasis uses several different health related quality of life questionnaires which are extensive and complex. Pregnancy Zone Protein (PZP) is an anti-proteinase initially found in the serum of pregnant women. It has been associated with several inflammatory conditions but it's exact role and significance is unclear. It has not previously been described in sputum. This thesis describes the largest cohort study of bronchiectasis patients with data collected via the EMBARC project. This includes patient demographics, clinical characteristics, microbiology and quality of life from 29 different countries. It will describe the differences between men and women with bronchiectasis including how they perceive their disease. A simple quality of life tool, the COPD Assessment Tool (CAT) will be validated for use in bronchiectasis. It will be examined for differences in the perceived quality of life between men and women. To investigate the hypothesis that differences in the inflammatory protein PZP, which is hormonally regulated, underlie some of the differences between men and women with bronchiectasis, PZP was measured in a cohort of patients with bronchiectasis and investigate the association between PZP and sex, severity of disease and the microbiome.

Acute respiratory distress syndrome is rapid pulmonary inflammation that can cause organ failure which is commonly caused by sepsis, severe pneumonia and trauma. ARDS has become one of the top research priorities since the SARS-CoV-2 outbreak in 2020. From injury to damaged tissue repair, cell proliferation is a mandatory mechanistic process which is triggered by neutrophils and tissue resident macrophages releasing various factors or cytokines to restore lung homeostasis. Recent research has highlighted some important evidence that the inflammasome may be a key player in lung injury progression. Importantly, inflammasomes have been shown to have roles in microbial clearance and neutrophil recruitment as part of the host defence against bacterial or viral infections. However, the contribution of inflammasome activation and the inflammasome-activated cytokines IL-1 beta and IL-18 in lung injury are still under deliberation. In particular, there is limited evidence of inflammasome involvement in the resolution of lung inflammation and repair. This study revealed that inflammasome proteins expressed in homeostatic and NLRP3-deficient lung tissue is not a key driver in the development of ARDS in this model. NLRP3 has a potential role in the resolution phase of lung injury because NLRP3 protein deficiency caused prolonged lung injury which was associated with increased total cell numbers in the lung on day 7 post-injury, i.e., the resolution phase in wild type mice. Caspase1 /11 (ICE) might be involved in protein leakage and recruitment or retention of cells and facilitates the initiation of the resolution stage by producing inflammatory cytokines to recruit immune cells to the site of injury. Thus, ICE might be a contributor in lung injury but NLRP3 mediates tissue repair. Further investigation of the underlying mechanisms during the resolution phase is needed for clarification of the role of NLRP3 in injury resolution.

Rationale: Cough is a common symptom within asthma that is both important and troublesome for patients and has been shown to be associated with a less well controlled disease. However, the role of cough within asthma is not well understood and there is little knowledge as to how cough is associated with typical markers of asthma disease control and severity. Methods: A series of studies were conducted to investigate how cough is associated with asthma. First, a systematic review to assess the extent to which cough has been studied within the asthma literature. Second, an observational study to investigate the associations of cough measurement tools with measures of asthma control, quality of life and associated inflammatory biomarkers. Third, a discrete choice experiment to gain an understanding of patient preferences for symptoms within asthma. Results: There are significant levels of cough burden within asthma with those with severe asthma being more affected by cough than those with mild/moderate asthma. Measures of cough-related quality of life are associated with measures of asthma control and can identify patients with inadequate asthma control. Cough measurement tools are suitable for repeated use within severe asthma. There appears to be a lack of significant levels of cough morbidity in patients where there is no evidence of raised Type 2 (T2) inflammation. Cough is an important symptom for patients with both severe and mild/moderate asthma and patients are willing to accept some additional asthma symptoms in order to reduce their cough burden. Conclusions: A significant level of cough morbidity can be identified in patients with asthma, even when not selecting for the symptom of cough. The use of cough measurement tools in asthma may provide additional insight into asthma control, quality of life and disease burden. However, as a significant level of cough burden was not observed in patients with low biomarkers of T2-inflammation, cough may not be suitable as a separate treatable trait in severe asthma. A logical first step for treating cough in severe asthma may be to achieve adequate suppression of T2 inflammation.

Rationale: Acute respiratory distress syndrome (ARDS) remains a common cause of significant mortality and morbidity for which there is currently no pharmacological treatment. Platelets play an important role in the pathophysiology of ARDS. There is in vivo, in vitro, observational and clinically relevant phase I evidence suggesting that aspirin may be of benefit in the treatment of ARDS. Objectives: The aim of the STAR trial (aSpirin as a Treatment for ARDS) was to test the hypothesis that enteral aspirin 75mg was both safe and effective in improving important surrogate outcomes in adult patients with ARDS. Methods: This randomised, double blind (patient and investigator), allocation concealed, placebo-controlled phase 2 trial was conducted in five intensive care units. Patients fulfilling the 2012- updated American- European Consensus Conference definition of ARDS (Berlin Definition) were randomly assigned in a 1:1 ratio to receive enteral aspirin 75mg or placebo for a maximum of 14 days using a computer-generated randomisation schedule with variable block size stratified by vasopressor requirement. The primary endpoint was oxygenation index at day 7. Secondary outcomes included respiratory compliance (Crs), PaO2/FiO2 ratio, change in sequential organ failure assessment (SOFA) score and safety parameters. Analyses were by intention to treat. Measurements and Main Results: The trial was stopped early after 49 of a planned 60 patients were recruited due to slow recruitment, with 24 patients allocated to aspirin and 25 patients allocated to placebo. There was no significant difference in oxygenation index at day 7 (unadjusted mean 54.4 [SD 26.8] in aspirin group, 42.4 [SD 25] in placebo group; mean difference 12.0, 95% CI -6.1 to 30.1, p= 0.19). Furthermore, aspirin did not have a significant impact on any of the secondary outcomes. However, the administration of aspirin in critically ill adult patients with ARDS was well tolerated with no difference in the number of adverse events (13 events in both groups; odds ratio 1.04, 95% CI 0.56 to 1.94, p=0.56). Conclusion: Aspirin was well tolerated but did not improve oxygenation index or other physiological outcomes in adult patients with ARDS.

Respiratory syncytial virus (RSV) is an enveloped virus with a negative sense, single-stranded RNA genome. RSV is responsible for upper respiratory tract infections such as rhinitis and croup mainly in babies and young children, but it can also infect the elderly and the immunocompromised. If RSV infection progresses into the lower respiratory tract then more severe diseases such as bronchiolitis and pneumonia can occur, which can lead to death. There is no licensed RSV vaccine and the only licensed antivirals are monoclonal antibodies, such as palivizumab, which are for prophylactic treatment only. During the RSV lifecycle, intra-cytoplasmic compartments named inclusion bodies (IBs) form. It is thought that RSV carries out transcription and replication within IBs. IBs contain viral proteins, viral RNA and some cellular host proteins. They have also been described to form through phase-phase separations and exist as membraneless structures. Here we employed a multi-modal correlative imaging approach to explore RSV cytoplasmic compartments during RSV infection, aiming to better understand RSV replication. Our use of RSV genome-specific fluorescent probes discovered a new RSV-induced cytoplasmic organelle: genome-rich bodies, GRBs. GRBs appear to be distinct from IBs, containing much of the viral genome within the cell as evidenced through confocal laser scanning microscopy. Using soft X-ray cryotomography we found that GRBs are granular, membranous cytoplasmic structures. Cryo-focussed ion beam milling followed by electron cryotomography revealed that GRBs are enclosed by an outer lipid bilayer and contain many internal membrane bound compartments, some of which have multiple membrane layers. Within the internal compartments are ring-like and tubular structures which we hypothesise are RSV nucleocapsids - viral genomes and anti-genomes coated by the RSV nucleoprotein. Morphologically identical structures were also seen within RSV filaments budding out of infected cells. The application of multiple imaging modalities has enabled the discovery of GRBs on different resolution scales, generating a detailed description of GRB structure and morphology. Further investigation to explore mechanistic aspects of these structures may inform the development of future RSV interventions.

Background: The global surveillance of human influenza viruses has resulted in the generation of a uniquely extensive collection of geographically and temporally comprehensive virus sequence data, which has provided an opportunity to explore the drivers behind the global spread of influenza viruses. However, due to the insufficient spatiotemporally representative virus sequence data from tropical and sub-tropical African countries, especially from sub-Saharan Africa, relatively little is known about the possible role these regions play in the global spread of influenza viruses. Using influenza A(H1N1)pdm09 virus and A(H3N2) virus sequence data, this study aimed to understand how seasonal influenza viruses are introduced and spread across geographically defined regions, whether local, national, continental or global, and their patterns of persistence across these regions. Methods: A laboratory method for whole-genome sequencing (WGS) of influenza A(H1N1)pdm09 and A(H3N2) viruses on Illumina next-generation sequencing (NGS) platform was established at Kilifi, coastal Kenya. This was then used to sequence samples collected between 2009 and 2018 from geographically defined regions: local community in Kilifi (n=66); countrywide in Kenya (n=383); and across Africa from 5 countries (n=100). The arising genomes were analyzed using phylogenetic and phylogeographical methods to investigate the patterns of spread, persistence and fade-out of seasonal influenza viruses at a local community in Kilifi, countrywide in Kenya, and across the African continent. Additionally, a global contemporaneous sequence dataset was analyzed in conjunction with the WGS data from this study in a Bayesian framework for inference of the situation of sub-Saharan Africa in the global network of spread of influenza viruses. Results: A total of 549 new influenza type A virus (IAV) whole genomes were generated during this study; 414 A(H1N1)pdm09 virus and 135 A(H3N2) virus genomes. Phylogeographical analyses revealed that local seasonal community epidemics of IAV were initiated by multiple independent introductions into the community, with each introduction commonly spreading to multiple locations within a relatively short period of time. Countrywide, in Kenya, circulation of IAV was predominantly characterized by virus migration from multiple locations to multiple destinations within the country and between locations in proximity; persistence of IAV countrywide might therefore be modulated by frequent virus introductions from outside the country and virus spread between locations in proximity. Continentwide, strains of IAV from Africa fell into strongly supported multinational lineages that suggested possible intra-continental spread of influenza viruses within Africa, which exhibited a significant northern to southern hemisphere migration. Globally, significant migration pathways from multiple geographical regions to multiple geographical destinations that also includes Africa were observed, which suggests that the seeding of epidemics of influenza viruses globally is driven by different geographical regions that also includes Africa. However, East or Southeast (E-SE) Asia acted as the major source of spread of influenza viruses globally, which is consistent with findings from other studies on the global circulation of influenza viruses. A greater global migration was observed for A(H3N2) virus compared to A(H1N1)pdm09 virus, consistent with greater global migration of A(H3N2) virus compared to (H1N1)pdm09 virus. Conclusions: The global migration dynamics of seasonal influenza viruses are well understood, and several models have been proposed to describe these patterns. However, analysis of virus sequence data from understudied regions, as exemplified in this study, suggests that these migration patterns are far more complex than those proposed by current models alone. For example, the findings from this study support the notion that influenza viruses persist as temporally structured migrating metapopulations in which new virus strains can emerge in any geographical region, including in Africa, with the location of the source population changing regularly. The epidemics across geographically defined regions (local community, countrywide, continentwide, and globally) are also interconnected at various scales of observation to different extents. Therefore, a more complete understanding of the global migration dynamics of influenza viruses requires deeper and wider sampling of viruses from understudied tropical and sub-tropical regions, notably, Africa, South and Central Asia, and South America. Understanding the circulation patterns of influenza viruses across geographically defined regions, together with their origins and patterns of persistence, is useful in selecting the most effective vaccine strains for the circulating seasonal influenza viruses. The rapid and widespread global mixing of viruses from all northern and southern hemisphere countries including in countries in Africa, Asia, Europe, North America, South America, and Oceania as reported in this study emphasize that global vaccine recommendations need well distributed, widespread global influenza A(H1N1)pdm09 virus and A(H3N2) virus sampling from as many localities as possible.

Chronic Obstructive Pulmonary Disease (COPD) is associated with comorbidities. Cardiovascular (CV) disease, cognitive impairment, dementia, and depression are common comorbidities in patients with COPD, leading to increased morbidity and mortality. However, there are considerable gaps in how these comorbidities are assessed, while the burden of others remains inadequately researched. This PhD applied several methodologies and used different databases to 1) explore whether circulating biomarkers from the blood (soluble Receptor for Advanced Glycation End-products - sRAGE) and urine (microalbuminuria) might have a role in COPD related to prognostication of CV risk (assessed by aortic stiffness and carotid intima-media thickness), 2) evaluate the prevalence and incidence of cognitive impairment and dementia, 3) examine the incident risk of depression, and 4) assess the risks of respiratory-related morbidities associated with antidepressant use in patients with COPD. Using data from a multisite UK study, sRAGE was not associated with aortic stiffness, carotid intima-media thickness, or CV disease in patients with COPD. However, there was a weak direct association between sRAGE and spirometric lung function measures. Data from the same cohort also showed that urinary albumin creatinine ratio (UACR) was not strongly associated with physiological CV risk measures. Nevertheless, patients with COPD and either diabetes, ischaemic heart disease, or cerebrovascular disease have increased UACR, compared to patients without these comorbidities. Microalbuminuria was also prevalent in all patients with COPD. Using a large primary care database, this PhD demonstrates that patients with COPD have increased prevalence of cognitive impairment compared to subjects without COPD, matched by age, gender and GP surgery. The incidence of cognitive impairment following COPD diagnosis was 23.1 per 1,000 person-years compared to 16.3 per 1,000 person-years in subjects without COPD. However, the prevalence and incidence of dementia were less frequently recorded in patients with COPD compared to individuals without COPD, indicating the possibility of underdiagnosis of dementia and highlighting the need for systematic assessment. The incidence of depression was also greater following COPD diagnosis, compared to subjects without COPD, which indicates the need to stay alert and target accordingly. Antidepressant use was associated with increased risks of pneumonia and COPD exacerbations relative to periods of unexposed to antidepressants in patients with COPD, raising the concern of potential side-effects and adverse events. This thesis addresses several aspects of COPD comorbidities and contributes new evidence for assessing, recognising, and managing comorbidities in patients with COPD.

Respiratory infections are of significant concern for individuals with chronic inflammatory lung diseases and there is an unmet need for novel antibiotics. Antimicrobial peptides possess antibiotic potential due to their antimicrobial and/or immunomodulatory properties. The project aim was to assess the therapeutic potential of several snake-derived AMPs and to evaluate the suitability of peptide delivery by nebulisation. In in vitro assays the peptides exhibited antimicrobial activity and/or anti-inflammatory effects with minimal cytotoxicity. We considered the impact of lung proteases on the peptides. Peptide incubation with CF sputum and protease inhibitors revealed the peptides were susceptible to proteolytic cleavage. Cleavage predictions highlighted neutrophil elastase (NE) as most likely responsible. All peptides were susceptible to cleavage by NE, and several peptides retained function following NE-incubation. Mass spectrometric analysis and cleavage predictions informed truncated derivative development, aiming to reduce production cost and potential immunogenicity. SnE1 and derivatives were tested as therapeutics for acute lung infection using a murine model of chronic lung disease. SnE1N treatment reduced lung bacterial load, whereas, SnE1 did not. SnE1 increased protein and IL6 concentration in the BAL fluid and reduced the number of immune cells, possibly due to cytotoxic effects, indicating increased inflammation; whereas, SnE1N did not. In nebulised delivery suitability testing, nebulisation did not alter peptide function. Sn1b and C2 were shown to be respirable in an adult spontaneously breathing model and all test peptides were found to be respirable in a mechanically ventilated breathing model. Assessment of nebulised droplet distributions suggested all test peptides would likely reach the lower airways. To conclude, AMPs remain an attractive candidate for antibiotic development. In this study, we found that SnE1N reduced the bacterial burden during acute lung infection in mice with chronic lung inflammation, warranting further investigation. Additionally, peptide delivery via nebulisation is likely feasible.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of mortality and morbidity worldwide. COPD is a complex and heterogeneous disease that exhibits a wide range of clinical phenotypes and it is characterised by numerous pulmonary and extra-pulmonary manifestations. Despite the rising incidence, prevalence and burden associated with COPD, the condition remains underdiagnosed and undertreated. There is a clear and urgent need for the development of diagnostic and disease management strategies that account for COPD heterogeneity. Tremendous efforts have been made in the field of biomedicine for the discovery of biomarkers that reflect disease activity; however, the clinical translation and implementation of these biomarkers have not matched those efforts. This study attempted to identify the most promising combination of biomarkers for the diagnosis and monitoring of COPD by employing functional genomics and next generation sequencing approaches on sputum. Many protein biomarkers that reflect the pathophysiology of COPD occurring with the onset and development of the disease have been identified. Additionally, a number of clinical phenotypes of COPD driven by variables such as body mass index, smoking status and treatment with oral steroids have been elucidated. Differences in the total microbial load, communities' richness and evenness appear to fluctuate at both genera and phyla level in relation to the onset, progression and disease stability. Lastly, differences in the proteins of microbial species provide insights into the pathology of COPD characteristic of every stage of the disease. The implementation of those biomarkers in clinical practice shows the most sustainable avenue for diagnosing and monitoring COPD. The impact associated with the translation of these findings could be linked to a decrease in the figures associated with COPD-driven mortality and morbidity, increased survival, improved quality of life and better patient outcomes.

Introduction: Pulmonary rehabilitation (PR) is a cost-effective management strategy in chronic obstructive pulmonary disease (COPD) patients which improves exercise performance and health-related quality of life. However, adherence to PR is common issue and this may be compounded by reduced muscle mass and/or malnutrition. The use of nutritional supplementation to overcome malnutrition and enhance outcomes for COPD patients during PR has been limited by the absence of rigorous evidence. Aim: To investigate relationships between malnutrition, nutritional supplementation, and PR in COPD. Specifically, to investigate the effect of protein-supplementation (Fortisip Compact Protein, FCP) during a COPD PR program on exercise capacity, peripheral muscle strength, anthropometrics measurements, anxiety and depression, health related quality of life, and physical activity. To assess whether any changes in exercise capacity, peripheral muscle strength, anthropometrics measurements, anxiety and depression, health related quality of life, and physical activity associated with nutritional supplementation are maintained at six weeks following PR. Methods: A systematic review was conducted to summarise the current evidence for using nutritional supplementation during PR in stable COPD to enhance PR outcomes. The systematic review facilitated the development of the research questions and main hypothesis. To identify the prevalence of malnutrition in our population, we conducted a study in a COPD population referred to PR, examining relationships between nutrition and disease severity. We then conducted a double-blind randomised controlled trial using FCP as an intervention and preOp (a carbohydrate supplement) as control in COPD patient who participated in a six-week PR programme. Participants were required to consume the intervention twice a day and attended PR sessions two hours long, twice each week for six weeks, with complete pre- and post-measures, including incremental shuttle walk test (ISWT) as the primary outcome. We conducted a follow-up study to identify if changes in exercise capacity and other outcomes were maintained six-week following completion of PR. Finally, participants' experience using both products was assessed in a survey. Results: It was impossible to draw definitive conclusions in the systematic review due to heterogeneity, but nutritional supplements may enhance the benefit of PR with a need for further well-designed and rigorous research to address this area. In our PR population, the prevalence of malnutrition was 17% and lower BMI was significantly associated with lower FEV1, FEV1% and FEV1/FVC. There were no statistically significant differences between intervention and control groups in exercise capacity measured by ISWT at the end of the PR, however, there was a clinically meaningful difference favouring the intervention group (intervention: 342 m ±149 vs. control: 305 m ±148, p >0.05). Individuals who reached that improvement had larger mid-thigh circumference (responder: 62 cm ±4.5 vs. non-responder: 55 cm ±6.2; p <0.05). Appetite did not change and the majority of participants were satisfied with product Conclusion: Malnutrition is common in stable COPD patients referred for PR and lower BMI is associated with lower lung function (FEV1). Using a high protein nutritional supplementation in individuals with COPD who were enrolled in PR resulted in a clinically meaningful difference favouring the intervention in exercise capacity measured by ISWT, and that improvement was maintained least six weeks later. A larger study would be necessary to demonstrate statistical significance. Individuals who reached that improvement had larger mid-thigh circumference. Nutritional supplements are acceptable to patients.

Introduction: Refractory chronic cough (CC) is a debilitating condition where the majority of patients complain of noxious somatic airway sensations (e.g. throat irritation) which precedes coughing. I propose these sensations drive the urge to cough (UTC) and cough. Furthermore, I hypothesise that low-dose, slow-release morphine sulphate (MST) works by modulating these sensations. I also hypothesise that patients respond differently to tussive agents dependent on the transient receptor potential (TRP) receptors triggered and their own demographics. Methods: We devised a novel cough challenge study in healthy volunteers (HV) to identify if VAS rated somatic airway sensations were associated with UTC and cough. This study was repeated using opioid responsive CC patients who withdrew from their MST therapy. The CC patients subsequently entered a placebo controlled randomised control trial assessing the effect of MST on these sensations and UTC. Morphine's antitussive efficacy was assessed simultaneously. I performed another randomised control trial involving four different tussive agents (each acting on a distinct combination of TRP receptors) in both CC and HV. The cough response to each agent was compared by group and other variables in order to determine influencing factors. Results: • Somatic sensations/UTC were rated higher per dose citric acid in CC patients versus HV. Somatic sensations were perceived more potently than UTC at low citric acid doses and predicted the UTC and cough in the CC cohort. • MST reduced somatic sensations in the CC cohort. This effect was negated at the point of coughing twice (C2). MST reduced both 24-hour cough frequency (-72.4%, p < 0.0001) and patient reported outcomes versus placebo. • Single breath jet nebulised hyper/hypo-osmolar saline was ineffective at provoking cough and so ultrasonic nebulisation was used for these agents. • Gender, age, lung volumes, methods of nebulisation and subject cohort all influenced the cough response to each challenge differently. Conclusions: There is a clear association between somatic sensations, UTC and coughing in CC, with strong evidence of sensory sensitisation in this condition. In clinical responders, MST is a highly effective antitussive, with its mechanism of action likely to be a modulatory effect on noxious sensations. Opioid receptors in the periaqueductal gray, a nucleus that inhibits processing of noxious signals, are a postulated site of action. The results of the multi challenge study suggests that cough is a heterogeneous disorder, where various factors impact upon the cough response. Taking this into consideration, it is unlikely that CC stems from a purely central or peripheral sensitisation problem. This has implications for therapeutics in chronic cough; some patients may need centrally acting medications, others will need peripherally acting medications, and some will need a combination of the two.

Chronic Obstructive Pulmonary Disease (COPD) is a common, yet preventable and treatable disease. COPD is characterised by emphysema, chronic bronchitis, and by a constant airflow limitation. Even with treatment, in 2016 COPD was the cause of more than 3 million deaths. It is the third disease in terms of mortality, and it has been associated with a significant health and economic burdens through hospital admissions and absenteeism from work. Cigarette smoking is one of the main risk factors to develop the disease. Exacerbation periods are associated with the worsening of symptoms and is linked with inflammatory responses and microbial infections, with a decrease of lung function and quality of life. However, greater understanding is needed into COPD to provide insight into the underlying disease and to provide biomarkers for diagnosis and treatment. In this study the use of microbiomic and metabolomic technologies were explored to assess patients with COPD, and to correlate the disease with its severity status, exacerbation periods, antibiotics and corticosteroids treatments, smoking status and different time points of collection. Firstly, flow infusion electrospray ionization mass spectrometry (FIE-MS) was tested if it could reveal biologically meaningful data using biopsies of lung squamous cell carcinoma (SSC) samples paired with histologically normal adjacent tissues. Lung SSC represented a well-characterised tissue and there is an increased risk of lung cancer in COPD patient. A total of 8 paired biopsies samples (tumour tissue and matched histologically normal tissue) were collected from patients with lung SCC and assessed using FIE-MS. These analyses showed the established metabolomic changes in SCC but revealed some novel aspects, particularly in vitamin metabolism. Having validated the metabolomic approach, the sputum metabolomes of 17 patients with COPD collected in four different time points, over the period of 18 months were assessed. The saliva of 22 patients was also examined and collected at four different time points over 18 months, using metabolomics approaches as FIE-MS. Sputum metabolomics suggested mitochondrial dysfunction associated with disease severity. Putrescine was shown to be associated with oxidative stress during exacerbation periods and found fatty acids changes associated with antibiotics treatment was seen. The saliva metabolome showed that oxidative stress has an important facet of disease severity and that ammonia recycling might be linked to amino acid metabolism due to muscle wastage in severe and very severe patients. Alterations in energy metabolism pathways and mitochondrial function were also observed. The microbiome of 14 patients using paired saliva and sputum samples was also investigated. Samples were collected in four different time points over 18 months and assessed by 16S rRNA amplicon sequencing. The results demonstrated that Firmicutes was the most abundant phylum in COPD oral cavity and lower respiratory tract. It was also found that the oral cavity microbial population may linked to the lower respiratory tract. Antibiotics and corticosteroids affected the microbiome of patients, proving an environment for other commensal pathogens to grow. The findings also showed that Firmicutes and Proteobacteria were associated with exacerbation periods in sputum and saliva samples. In summary, metabolomic aspects were identified in saliva and sputum that characterise changes in the energy metabolism, mitochondrial function and oxidative stress which could have systemic effects, especially on skeletal muscle. The microbiome showed that oral cavity microbial population may have an impacted in the lower respiratory tract.

Asthma affects ~5,300,000 people in the UK. It is a significance public health burden costing the NHS an estimated one billion pounds per year. Approximately 50% of asthma patients suffer from hyperpnoea-induced bronchoconstriction (HIB), defined as a transient narrowing of the airway following hyperpnoea. HIB affects ~10% of the general population but is very prevalent in athletes, affecting approximately 35% of athletes and is severely under diagnosed and undertreated. Both asthma and HIB can be treated through pharmacological therapies, but these are neither curative nor preventative and chronic use can cause significant side-effects. The purpose of this thesis was to further understand the prevalence of HIB in athletes, and assess the effect of the dietary prebiotic, Bimuno-Galactooligosaccharide (B-GOS), supplementation on asthma and HIB. To establish the effect of B-GOS we first sort to evaluate the efficacy of a novel method to measure airway inflammation. Consequently, this thesis investigated: (i) the prevalence of HIB in university field hockey athletes and assessed the effect of sex and diagnostic criteria on prevalence rates. Additionally, the thesis assessed the relationship between symptoms of dyspnoea and HIB in situ; (ii) the efficacy of the RTube device to collect exhaled breath condensate (EBC) with measurable cytokine concentrations; (iii) the effect of B-GOS supplementation on HIB severity, asthma control, and markers of airway inflammation in asthma participants with HIB. The prevalence of HIB in university field hockey players was found to be 19% with a higher prevalence in males (30%) than females (5%). Diagnostic criteria influenced prevalence rates by 19% suggesting that less stringent criteria overestimate HIB prevalence. Symptoms of dyspnoea namely the "unpleasantness or discomfort of breathing" and the intensity of sensory dimensions (IPDS) were higher in HIB+ than HIB- athletes, correlated with HIB severity, and were highly sensitive and specific in predicting HIB (A1: Sensitivity = 100%; Specificity = 63%; IPDS: Sensitivity = 89%; Specificity = 66%). This is the first time respiratory symptoms and HIB have been assessed in situ, and one of the few reports to find an associate between symptoms and HIB. This thesis found that IL-13, TSLP, IL-5, and TNF-ɑ could not be detected in EBC samples collected with the RTube device in participants with HIB and/or asthma, or healthy controls. Device material and protein adsorption was suspected in the poor recovery of biomarkers. Attempts to reduce biomarker adsorption by coating the RTube in 1% bovine serum albumin and 0.01% Tween20, alongside an 8-fold sample concentration did not results in the detection of biomarkers. As such, future work should use alternative methods of measuring airway inflammation to assess the effect of B-GOS supplementation on airway inflammation in participants with asthma and HIB. It was found that 21 days supplementation with 3.65g/d of B-GOS did not affect HIB severity, asthma control and systemic markers of airway inflammation. These findings contrasted previous work (Williams et al, 2016) and sheds doubt on the efficacy of B-GOS to attenuate HIB. GOS dosage, however, was noted as a potential influencing factor, as such, future work should look to confirm the efficacy of B-GOS using a B-GOS dosage providing ≥5.28g/d of GOS in a larger sample size and taking direct measures of airway inflammation.

Dyspnoea is a complex and unpleasant sensation that can have a severe impact on a person's quality of life. Evidence for there being distinguishable types of breathlessness arising from distinct neural networks has been produced, and experimental models have also been produced to reproduce these sensations in healthy individuals. Current knowledge of the cerebral mechanisms has been gleaned predominantly through the use of brain imaging studies, however alternative methods also present opportunities to further elucidate the mechanisms behind intractable dyspnoea in ways that brain imaging cannot. Aim: The ultimate purpose of this thesis was to investigate the cerebral mechanisms behind breathlessness using neurological populations. Using deep brain stimulation (DBS), it is a primary aim of this thesis to further investigate the role of different brain structures in dyspnoea perception, specifically 'air hunger' which is a particularly unpleasant form of breathlessness often experienced by those with Chronic Obstructive Pulmonary Disease (COPD). An additional aim was to investigate whether neurological damage in the form of Parkinson's disease, and glioma of the insular cortex alters the perception of breathlessness. Methods: (i) Experimental air hunger was induced in participants with deep brain stimulation of the motor thalamus to assess whether perception of dyspnoea is changed when stimulation is turned ON compared to when it is turned OFF. Experimental air hunger was also induced in one patient with low grade glioma to explore the impact of this damage on the insular cortex. (ii) A validated tool for measuring breathlessness called the Dyspnoea 12 questionnaire was utilised in patients who were breathless at rest and whom had DBS of the motor thalamus or the STN. This questionnaire was completed with their stimulation turned ON and turned OFF. This same questionnaire was also used to assess dyspnoea in individuals in the community with Parkinson's disease to determine the prevalence of dyspnoea in this group (iii) Local field potentials (LFPs) where recorded from the motor thalamus whilst air hunger was induced. These LFPs are signals which correlate with neural activity. Results: (i) Participants with DBS of the motor thalamus rated air hunger significantly lower with stimulation ON in comparison to OFF by (median change 12mmVAS; range +19 to -50mmVAS). (ii) The participant with low grade glioma of the insular cortex did not rate an increase in breathlessness when air hunger was experimentally induced (increase in PCO2 of 11mmHg with VE fixed at 4.4 L/min). (iii) Participants with breathlessness at rest and DBS of the STN reported an overall increase in breathlessness when stimulation was turned on (an increase of 10% fullscale in the D12 total score). Conversely, DBS of the motor thalamus in participants with breathlessness at rest due to pre-existing COPD experienced a reduction in breathlessness with stimulation ON (-20.2% full scale for D12 total score) (iv)The prevalence of dyspnoea in individuals with Parkinson's disease was 63.6%. (v) an LFP signal was detected when air hunger was induced that was not present in other breathing conditions. Conclusions: The motor thalamus and the STN have opposite roles in modulation of breathlessness, the mechanism of which is yet to be determined. The prevalence of breathlessness among patients with Parkinson's disease is considerable and warrants more attention. Glioma of the insular cortex confirms the importance of this region in breathlessness perception. Study of patients with neurological interventions (Deep brain stimulation) and with neurological damage (low grade glioma or Parkinson's disease) will lead to a better understanding of the cerebral network for dyspnoea perception and ultimately to identify new treatment options for intractable dyspnoea.

Allergic conditions affect increasing numbers of the population, are associated with considerable morbidity and in their most severe forms can be life-threatening. A prominent feature in allergic conditions is the activation of mast cells and basophils by allergen resulting in the release of inflammatory mediators. A unique product of basophils is basogranulin, a protein stored and released from the secretory granules on cell activation. Our aim has been to develop new assays for assessing basophil activation based on basogranulin measurements, and applying these assays for measuring basophil sensitivity in samples from allergic patients. In addition, we have investigated basogranulin in saliva and BAL fluid as a marker for basophil activation in vivo. Basophils stimulated with fmlp, anti-IgE and grass pollen in vitro and the basogranulin released into supernatants was quantified by dot blotting. In addition, flow cytometric assays were developed for measuring alterations of intracellular and surface basogranulin expression following basophil activation in vitro with fmlp, anti-IgE anti-FcɛRI or specific allergens. There was an apparent depletion in intracellular basogranulin stores in activated basophils when compared with that in non-stimulated basophils. The depletion of intracellular basogranulin was inversely associated with increased expression of CD63. Surface basogranulin expression was barely detected in nonstimulated basophils but was increased following stimulation. Membrane expression of basogranulin was correlated with that of CD63 in nonstimulated basophils (0.829, P< 0.005, n=10), and in basophils stimulated with anti-IgE (r=0.877, P< 0.001, n=51), anti-FcɛRI (r=0.680, P< 0.0001, n=20), or fmlp (r=0.914, P< ii 0.0001, n=9). When basophils were stimulated with extracts of various allergens including D. pteronyssinus, D. farinae, crab, shrimp and oyster increased basogranulin expression wasobserved in cases for where this was not detected for CD63. Alterations in intracellular and surface basogranulin expression were also visualised by confocal microscopy, and by this technique considerable heterogeneity in marker expression was observed between individual cells. Basogranulin could be detected in saliva samples of peanut allergic children, suggesting a potential role in assessing the contribution of basophils in clinical disease. In BAL fluid from patients with atopic asthma basogranulin levels were higher than that from healthy subjects, but not from those with severe asthma. Basogranulin measurement within basophils, on the cell membrane and in biological fluids represent novel approaches for assessing basophil activation and for establishing the contribution of basophils in clinical disease. They show promise as new means for the diagnosis of allergic sensitivity and confirmation of allergic reactions mediated by basophils.

During the pathogenesis of chronic obstructive pulmonary disease (COPD) and in subsequent exacerbations, neutrophilic inflammation predominates and contributes to disease progression. Levels of neutrophil-derived microvesicles (NMVs; small extracellular vesicles formed from membrane blebbing in resting and activated cells) have previously been shown to be elevated in COPD patient sputum and in the circulation during inflammation. Evidence of the pro-inflammatory activity of NMVs in several cell types, along with an ability to increase epithelial monolayer permeability, led to the hypothesis that NMVs are released upon neutrophil activation in the circulation and the inflamed airway environment and are subsequently internalised by lung epithelial cells, inducing their pro-inflammatory activation and dysfunction. The overall aim of my PhD was to investigate the role of NMVs in lung inflammation. This was firstly done by characterising circulating MVs in COPD patients as both potential disease biomarkers and as mediators of inflammation using stored plasma samples from two COPD patient cohorts. Subsequently, it was crucial to isolate human neutrophils from the peripheral blood of healthy volunteers so as to generate, isolate and characterise a pure population of NMVs from these cells and to investigate the functional effects of these vesicles using the bronchial epithelial cell line BEAS2B. Finally, to further understand the biological relevance of NMV effects in the lung, C57BL/6 mice were utilised in a series of experiments to explore lung inflammation and NMV fate. Key findings: 1. Plasma MV matrix metalloproteinase-9 (MMP-9) content is significantly increased in COPD patients compared with age-matched controls, however, numbers of circulating leukocytederived MVs are not differential between these groups. 2. NMVs from fMLP-stimulated healthy participant neutrophils contain active MMP-9 and can degrade extracellular matrix proteins, but activated and quiescent neutrophils do not produce differential NMV numbers. 3. NMVs are rapidly taken up by bronchial epithelial cells, likely via an endocytic mechanism, with functional effects of NMVs on epithelial activation, barrier integrity and proliferation. 4. In vivo in mice, airway administration of NMVs does not induce or augment lung inflammation, instead, mass uptake and clearance of these NMVs by alveolar macrophages occurs. In this thesis I have shown that MV-associated protein content is altered in COPD, and investigation of these differences will be important going forward in defining the role of NMVs in chronic lung diseases. Degradation of extracellular matrix shown here and epithelial cell activation and permeability induced by NMVs when applied directly to these cells define key pathological events in COPD and indicate a role of these vesicles in epithelial dysfunction. In contrast, NMV uptake by alveolar macrophages was identified as a mechanism of NMV clearance in healthy lung tissue. Together, these findings provide novel insight into neutrophil interactions and activity in the lungs. This work contributed to our understanding of the processes occurring in inflammation and identified several valuable avenues for future investigation to understand more about complex conditions like COPD.

Cigarette smoking is a prime cause of morbidity and mortality worldwide. It is a major risk factor for the development of cardiovascular diseases, various cancers, respiratory disorders, and skeletal muscle dysfunction. The adverse effects of cigarette smoking beyond the respiratory system has been an area of rising interest, particularly its impact on skeletal muscles. Considering the capital role of skeletal muscles in movement, maintenance of posture, metabolism, and vital functions such as respiration, a proper understanding of the manifestations and mechanisms of smoking-induced skeletal muscle dysfunction and development of strategies to curb the ensuing morbidities and mortality is necessary. In humans and animal models the manifestations of smoking-induced skeletal muscle dysfunction include: atrophy, weakness, decreased mitochondrial function and bioenergetics, reduced force and fatigue resistance, decreased protein synthesis, increased proteolysis, capillary regression, reduced vasodilation, decreased perfusion, increased inflammation and oxidative stress. Smoking cessation is the most efficient and cheapest way to avert these deleterious alterations in skeletal muscle structure and function as a result of smoking. In fact, long term smoking cessation has been shown to restore whole body and skeletal muscle mass, increase muscle mitochondrial function, and improve muscle plasticity in humans and animal models. However, prior to our study, evidence of short-term (1 to 2 weeks) benefits of smoking cessation were lacking. Our study in mice reveals that smoking cessation for as short as 1 to 2 weeks leads to immediate benefits on the diaphragm and limb muscles demonstrated by the reversal of muscle mass loss, recovery of whole-body fat and especially lean mass, and improvement of mitochondrial function. We also show that the constantly active diaphragm muscle is most affected by cigarette smoking and recovers rapidly after smoking cessation. Considering the fact that the devastating effects of smoking on skeletal muscles occur slowly and may only manifest later in life, these results are important as they could motivate smokers to stop smoking as soon as possible to avoid long term muscle wasting and weakness. The maintenance and improvement of skeletal muscle mass and plasticity is highly recommended, especially for people suffering from muscle wasting such as patients with COPD, cancer, dystrophic muscle diseases, chronic smokers and the elderly in whom smoking and vitamin D deficiency is also prevalent. Skeletal muscle hypertrophy is a normal response of skeletal muscles to resistance training and overload which lead to increased mass and fiber cross-sectional area. It is usually accompanied by beneficial adaptations, such as muscle mitochondrial biogenesis, increased protein anabolism, reduced proteolysis, enhanced vasodilatory capacity and capillary proliferation that are associated with an increased force generating capacity and fatigue resistance of the muscle. However, cigarette smoke may impair the hypertrophic response to overload. Vitamin D plays a vital role in skeletal muscle plasticity and regeneration and vitamin D deficiency, which is highly prevalent worldwide, leads to skeletal muscle dysfunction with similar manifestations to those orchestrated by cigarette smoke such as fiber atrophy, loss of muscle mass and muscle mitochondrial dysfunction. Our second study was therefore designed to understand the impact of smoking alone, vitamin D deficiency alone or a combination of these factors on the skeletal muscle hypertrophic response in mice. Our results showed that smoking or vitamin D deficiency alone did not diminish the hypertrophic response to overload in the plantaris muscles, but this hypertrophic response was attenuated in the presence of both conditions, as indicated by the attenuated fiber hypertrophy in vitamin D-deficient smoking mice. Therefore, a combination of these risk factors impairs the skeletal muscle hypertrophic response. These data are relevant for consideration in the design and implementation of strategies to improve muscle mass, especially in smokers and vitamin D deficient individuals. Our studies report the short-term benefits of smoking cessation that include an improved mitochondrial function and limb muscle mass. This is particularly relevant as an incentive to encourage smoking cessation in smokers and individuals suffering from muscle deterioration yet struggling with smoking cessation. We also show the synergistic deleterious effect of smoking combined with vitamin D deficiency on the skeletal muscle hypertrophic response to overload. This is relevant as it may help in understanding the differences in skeletal muscle response to exercise training and could improve the interventions to restore or improve muscle mass.

Background Echinacea purpurea has long been used for the treatment and prevention of respiratory tract infections. The medicinal plant has originally been discovered by the Native American population, who squeezed the sap from fresh plant or chewed dried roots. Today, commercially available preparations vary greatly in terms of Echinacea concentration, manufacturing methods, plant parts used, et cetera. This heterogeneity results in tremendously fluctuating bio-activities between products. Some of these products finally failed to show efficacy in large clinical studies raising principal questions about the value of the medicinal plant. Objectives The aim of this doctoral work was to explore in vitro and in vivo pharmacological effects, as well as clinical efficacy in prevention and acute treatment of respiratory tract infections for a single, phytochemically characterized and standardized Echinacea purpurea extract (Echinaforce®, EF). Methodology In vitro antiviral and anti-inflammatory (immune-modulatory) activities of EF were researched studying the most relevant respiratory pathogens including influenza, respiratory syncytial virus (RSV) or coronavirus (CoV) and using cytokine assays for interleukins IL-6 or IL-8 in airway epithelial cells. Test tube experiments were sought to be confirmed in organotypic tissues and upon peroral administration of EF ex vivo, whereas bio-availability of alkylamides in the extract was investigated to estimate systemic relevance. Two clinical studies aimed to investigate efficacy of EF for the treatment and the prevention of respiratory tract infections and subanalyses served to confirm above proposed pharmacological actions in vivo. Finally, EF's therapeutic potential beyond its traditional use for colds and flu was researched by looking at the prevention of bacterial superinfections including bronchitis or pneumonia. Results EF was demonstrated to inhibit a wide range of respiratory agents, showing a primary specificity to enveloped viruses (e.g. influenza, RSV and CoV) in vitro and in a clinical prevention study. The extract modulated overexpression of inflammatory cytokines in epithelial cells and in the presence of respiratory pathogens. It is thereby expected not only to impact occurrence but also the symptomatic development of viral infections. Alkylamides were found to play a role in the systemic immune-modulation as bio-availability was demonstrated after peroral ingestion of EF. Next, EF was administered for the treatment of clinically diagnosed influenza (flu) to find non-inferiority to the gold-standard therapy Oseltamivir in this indication. Comparable recovery rates were observed for virologically confirmed influenza infections, demonstrating clinical efficacy in the treatment of viral respiratory infections. Another large clinical study investigated EF for the prevention of respiratory tract infections. In the placebo group a total of 188 cold episodes were identified, which lasted for 850 days in comparison to 149 episodes and 672 sick days with EF extract (p < 0.05). Enveloped viruses (influenza, RSV and CoV) were found in 24 patients with EF and in 47 with placebo (p < 0.05). In vitro EF reduced the expression of bacteria-binding receptors (e.g. of intracellular adhesion molecule, ICAM-1) on airway epithelial cells after infection with respiratory viruses. Thereby, EF prevented the attraction of pathogenic bacteria potentially inducing bacterial superinfections of initial viral infections. Conclusion The evidence generated by this PhD work substantiates the medicinal value of Echinacea purpurea for the treatment and prevention of respiratory tract infections. By focussing on a single, chemically standardized extract (Echinaforce®) problems with heterogeneity between Echinacea products could be overcome to reach more consistent conclusions. EF exhibits antiviral and anti-inflammatory effects to not only prevent occurrence but also the symptomatic development of infections. Alkylamides are bio-available and capable to systemically modulate the immune response. Bacteria-binding receptors on the epithelium are controlled with EF to finally prevent respiratory complications including bronchitis or pneumonia.

The bacterial Type II Secretion System (T2SS) is a transmembrane multiprotein assembly that ejects folded protein substrates from the periplasm into the extracellular milieu. These substrates are essential for pathogenesis in humans, animals, and plants. In the intracellular pathogen Legionella pneumophila, the T2SS has been shown to play an important role in evading recognition by the host during infection. Although much is known about the structures of the compositional proteins of the system, recognition and recruitment of substrates for secretion by the system is a poorly understood process. Previous studies have demonstrated binding between the inner membrane protein XcpPGspC and T2SS substrates in Pseudomonas aeruginosa thus implicating its involvement in this process. In this thesis, the periplasmic linker region of the L. pneumophila homologue of XcpPGspC (LspCGspC) and the T2SS substrate Novel Type II secreted protein A (NttA) (13 kDa) were recombinantly expressed and puri ed in Escherichia coli. Using NMR spectroscopy titration experiments, a weak fast-exchange interaction between these two proteins was observed, showing similar a nities to that previously demonstrated in the study in P. aeruginosa. Solution structures of the two proteins were then solved by NMR spectroscopy. Then, through the computational docking of these two proteins and guided by NMR titration data, a recognition complex was characterised revealing residues essential in the binding process. This is the rst discovered structure of a recognition complex between the T2SS and its substrates in L. pneumophila. This study suggests a novel pharmacophore model that may be used in antibiotic drug design of inhibitors to the formation of the recognition complex.

Background: Exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are common and account for approximately 12% of UK hospital admissions. A significant proportion will be complicated by respiratory acidaemia which has a high mortality. Non-Invasive ventilation (NIV) confers a 2-3 fold reduction in mortality in this setting. Despite this, practice is suboptimal; the intervention is underused, infrastructure is lacking, and complex decisions are made by a wide range of clinicians. It is feasible that pessimism contributes. Aims: To derive and separately validate a simple, bedside, clinical tool to predict in-hospital mortality in exacerbations of COPD complicated by respiratory acidaemia requiring assisted ventilation. Methods: The study was split into two parts with similar methods. The derivation study was a single trust (2 sites: one urban and one rural) retrospective study. In patients meeting selection criteria, data were collected, and multivariable regression analysis identified independent predictors of in-hospital death. A simple predictive model was created. The validation study captured a more limited dataset in prospectively recruited patients across 10 trusts. The predictive model's performance was assessed. Results: 489 patients were identified in the derivation study and 733 in the validation. Independent predictors of outcome were confirmed, and a final, simple bedside model entitled the NIVO score produced. Using atrial fibrillation, chest X-ray consolidation, eMRCD score, Glasgow coma scale, timing of acidaemia relative to admission time and pH in a simple scoring system stratified risk was obtained with an area under the receiver operated curve of 0.79 in the validation cohort. Discussion: Using only simple, readily available indices good prediction of in-hospital mortality is feasible. The NIVO score outperformed pre-identified comparator scores in both its derivation and validation studies. Potential practical applications include but are not limited to guiding level of care, setting treatment limitations and objectifying discussion with patients or family members.

Human Rhinovirus (hRV) is a major cause of upper respiratory tract infections (URTIs) and is linked to lower respiratory tract infections (LRTIs) and airway diseases exacerbations. Cough, airway inflammation and hypersensation are common symptoms of hRV infections however, the mechanisms involved remain elusive. A promising hypothesis is neuromodulation, whereby stimulation of transient receptor potential channels (TRPV4) in airway epithelial cells release ATP, subsequently activating the purinoreceptor P2X3 on vagal afferent nerve fibres. Here it is hypothesised that hRV infection will alter expression of channels and receptors known to be involved in cough and airway inflammation, causing lung tissue to become hyperresponsive to cough and bronchoconstrictive agents. A co-culture model of human airways using a lung epithelial cell line (A549 or Beas-2b) and astrocytes (1321N1 transfected with P2X3) to mimic neurones showed that activation of TRPV4 on lung epithelial cells led to ATP release which could evoke a measurable calcium influx into astrocytes. Additionally, A549 and Beas-2b cells responded differently to RV16 infection, with A549 cells exhibiting a reduced cytotoxicity, increased viability and proliferation compared to Beas-2b cells. Furthermore, RV16 infected ex vivo tissue showed limited impact on viability and cytotoxicity, reduced TEER measurements, increased bronchoconstriction and upregulated ICAM-1 protein expression. Finally, comparison of microfluidic devices to standard culture conditions demonstrated A549 cells could be cultured for 72 hours with similar rates of cell proliferation and viability, whilst PCLS could be cultured for 6 days with minimal impact on tissue viability or morphology. Overall, findings suggest that the TRPV4-ATP-P2X3 neuromodulation theory may play a role in the mechanism of cough and supports observations indicating hRV causes URTIs more frequently than LRTIs. Furthermore, hRV infection of ex vivo tissue appears promising as a model that can replicate in vivo and in vitro observations. Whilst the microfluidic devices used to replicate elements of the aforementioned models provide a promising start to better recapitulating the in vivo environment.

Acute Respiratory Distress Syndrome (ARDS) is an inflammatory disorder of the lungs, for which there is no effective pharmacotherapy. 11β hydroxysteroid dehydrogenase type 1 (HSD-1) acts as a reductase to convert cortisone into cortisol, and is normally expressed by alveolar macrophages (AMs). A previous study has shown evidence of impaired alveolar HSD-1 reductase activity and relative alveolar cortisol deficiency in ARDS patients. Mesenchymal stem cells (MSCs) administration has been effective at attenuating inflammation in pre-clinical models of lung injury. This thesis reports a series of investigations into the role of AM dysfunction in ARDS pathogenesis, and whether use of transgenic MSCs (tMSCs) expressing HSD-1 offers a potential therapy for ARDS by enhancing AM efferocytosis. It also investigates the impact of expansion on the ability of MSCs to attenuate lung injury. Expansion of MSCs for 1 month had no effect on the ability of MSCs to promote alveolar epithelial wound healing, however the ability of MSCs to stimulate IL-10 release from macrophages was impaired. Lentiviral transfection was used to create HSD-1 tMSCs, which expressed functional HSD-1. Development of sepsis-related ARDS is associated with decreased AM HSD-1 reductase activity and efferocytosis, which contributes to the increased alveolar neutrophil apoptosis, alveolar inflammation and mortality observed. Dysregulated AM function contributes to ARDS pathogenesis, therefore upregulation of AM efferocytosis may offer a therapeutic strategy for ARDS patients. ARDS broncho-alveolar lavage fluid (BALF) treatment of healthy AMs in vitro can decrease rac1 gene expression and impair efferocytosis, thereby replicating the AM functional defect observed in ARDS patients. Following ARDS BALF treatment, AMs increase expression of pro-efferocytosis receptors CD206 and MerTK, as an unsuccessful compensatory mechanism to restore normal efferocytosis. HSD-1 tMSC co-culture was unsuccessful at restoring AM efferocytosis both ex vivo and in an in vitro model of ARDS, indicating that HSD-1 independent mechanisms contribute to impaired AM efferocytosis in ARDS. HSD-1 tMSC therapy reduced cellular inflammation in a model of murine peritonitis, but also caused bacterial overgrowth due to immunosuppression, indicating that HSD-1 tMSC therapy is unlikely to have utility in patients with sepsis-related ARDS. However, further studies using multiple low doses of HSD-1 tMSCs in HSD-1 knockout mice in conjunction with antibiotic therapy are required to fully assess the therapeutic potential of HSD-1 tMSCs. Rho-associated protein kinase (ROCK) inhibitor treatment partially restored AM efferocytosis in an in vitro model of ARDS, indicating that targeting of the ROCK pathway may offer therapeutic potential in ARDS patients.

Pulmonary arterial hypertension (PAH) is a rare disease and often heterogeneous at clinical presentation. There has been significant interest in the use of clinical parameters to assess risk, and two risk stratification tools exist. I aimed to assess whether additional non-invasive investigations could improve upon current approaches. In addition, I sought to evaluate specific phenotypes in pulmonary hypertension, including patients with idiopathic PAH (IPAH) who have mild lung disease. Data were obtained from clinical databases at a large PAH referral centre. When assessing clinical phenotypes, I identified that the presence of minor lung disease in patients with IPAH was a strongly negative prognostic marker, and these patients did not demonstrate an improvement in exercise capacity in response to PAH targeted therapy. For risk stratification I aimed to identify thresholds for low (<5%), intermediate (5-10%) and high (>10%) risk of one-year mortality, as in other widely-used approaches. Risk could be stratified using three non-invasive assessments: cardiac MRI, incremental shuttle walking test (ISWT) and emPHasis-10 quality of life score. In contrast to current risk stratification approaches, thresholds for cardiac MRI were able to identify a large proportion of patients (63%) at low-risk of one-year mortality. In addition, cardiac MRI was able to add discriminative value to currently used risk stratification scores. EmPHasis-10 was an independent predictor of mortality, and in a risk stratification approach was able to identify patients with distinct levels of one-year mortality. For the ISWT, a 10% improvement in exercise capacity was an independent prognostic marker of survival, and thresholds derived at baseline accurately stratified risk at follow-up. This thesis demonstrates that non-invasive assessments can be used either in isolation or in conjunction with other prognostic parameters in patients with PAH. The thresholds proposed could be considered for incorporation into widely-used risk stratification scores.

The question of whether occupational asbestos exposure is an under- recognized cause of idiopathic pulmonary fibrosis arises because it is clinically and epidemiologically plausible, and consistent with fibre studies, case-control, and toxicological data. This thesis examines the question by means of a literature review and a novel hospital based case-control study, the idiopathic pulmonary fibrosis job exposures study (IPFJES). In a literature review and meta-analysis of studies reporting on occupational exposures in idiopathic pulmonary fibrosis (IPF) I found significant associ- ations with metal, wood, and stone dust, but not asbestos. However, there was considerable heterogeneity and confidence in the meta-analysis result is tempered by a high risk of bias arising from selection, lack of blinding, exposure misclassification, incomplete exposure data, and selective report- ing of exposures. In a mortality analysis I found that the UK incidence of IPF continues to rise and appears to be correlated with mesothelioma mor- tality. I did not find clear evidence of an association between IPF, pleural mesothelioma, and asbestosis at a regional level. In a critical review of methods for assessing occupational asbestos exposure I found support for the use of a job exposure matrix based on proportional mortality rates for mesothelioma and validated by quantification of asbestos fibre lung burden. I also found support for using a structured interview tool to provide a quantitative estimate of previous exposure which was validated using historic and simulated exposure data. In a review of MUC5b and IPF I found evidence supporting a common MUC5b driven pulmonary fibrosis endotype and a candidate mechanism for 3occupational asbestos exposure contributing to this; alveolar macrophage NLRP3 inflammasome activation resulting in increased IL-1 β driven airway MUC5b expression. Occupational asbestos exposure alone was not associated with IPF in IPF- JES. It was associated with dyspnoea independent of smoking and case status. It was associated with IPF in participants who also had smoking exposure and the strength of this association was greatest for participants with the minor allele of the MUC5b promoter variant rs3505950 and when a stricter case definition (definite UIP rather than definite UIP or possible UIP) was used. These studies suggest that occupational asbestos exposure in smokers, cou- pled with genetic susceptibility factors, may be an important cause of IPF.

Asthma exacerbations are frequently caused by respiratory viral infection, most commonly rhinoviruses, in up to 80% of cases. Virus-induced asthma exacerbations have been associated with increased type 2 airway inflammation and impaired interferon induction, however the mechanisms driving virus-induced asthma exacerbations are not well understood. Suppressor of cytokine signalling 1 (SOCS1) is an inhibitor of both interferon signalling and induction and is induced by the cytokines IL-4 and IL-13. This thesis sought to investigate if SOCS1 expression is increased in asthmatic bronchial epithelial cells and if SOCS1 is associated with impaired interferon responses or worse symptoms upon rhinovirus infection. In bronchial biopsies from atopic asthmatics, SOCS1 expression was associated with worse symptoms and greater falls in lung function upon rhinovirus infection. However, in cultured bronchial epithelial cells, there was no difference in SOCS1 mRNA expression between atopic asthmatics or healthy controls. In bronchial biopsies and cultured bronchial epithelial cells, skin prick test positivity was associated with increased SOCS1 expression and induction, respectively. This thesis has also shown that IL-4 and rhinovirus interact to increase SOCS1 expression; IL-4 induces SOCS1 via activation of the adjacent promoter, while rhinovirus functions through a distinct and uncharacterised mechanism, which may involve distal regulatory elements. The knockout of SOCS1 in a fibroblast cell line enhanced rhinovirus-induced interferon induction. However, this thesis was unable to demonstrate a role for nuclear SOCS1 in inhibiting rhinovirus-induced interferon or determine if IL-4 or rhinovirus increases the nuclear localisation of SOCS1. Collectively, these findings support the hypothesis that increased SOCS1 is associated with worse clinical outcome following rhinovirus infection. These findings support a role for increased type 2 inflammation in enhancing SOCS1 expression, which may be of interest in asthma exacerbations. The identification of the precise mechanism by which SOCS1 is induced and undergoes nuclear localisation will require further study.