Background Airways diseases are increasingly recognised to be poorly defined phenomena with overlapping pathophysiology and symptoms. They are a significant and growing cause of morbidity, with increasing numbers of people affected globally and no improvement in key outcomes in the UK for the last decade despite ever increasing expenditure. The classification of airway diseases has changed little in the last 50 years, and may no longer be fit for purpose due to the growing appreciation of the complexity and heterogeneity of airways disease and the advent of molecular-based diagnostic techniques to target specific treatment. Aim To investigate whether strategies based on the measurement of selected phenotypic and biological characteristics of airways disease can help to improve the understanding of their pathogenesis and targeting of treatment. Methods Three characteristics of airways disease, namely (1) exhaled nitric oxide, (2) chronic productive cough of unknown cause and (3) the airway microbiota were described/measured in selected cohorts of patients in three clinical studies. Measurement of each of these characteristics was used to answer focused clinical questions regarding the pathogenesis and treatment of aspects of airways disease. Results (1) The baseline measurement of FENO in steroid naïve subjects with symptoms suggestive of asthma had a low diagnostic value for asthma but was an excellent predictor of inhaled steroid treatment response. (2) A cohort of subjects with chronic productive cough of unknown cause was described. These subjects tended to have radiological evidence of airway dilatation and chronic inflammatory changes but not significant bronchiectasis. Their cough responded well to treatment with azithromycin, with ongoing neutrophilic airway inflammation a particularly strong predictor of treatment response. (3) There were no significant differences in the abundance or community structure of the bacterial communities in the airways between subjects with mild (BTS 2) or severe (BTS 4) asthma or between severe (BTS 4) asthma patients taking inhaled fluticasone or budesonide. However a number of differences in relative abundance of certain species (including enrichment of Haemophilus parainfluenzae in the fluticasone group) were noted on comparison of the groups. Conclusions This thesis provides support for a new approach to the classification and treatment of airways disease. The recognition of pathologically important processes (treatable traits) which can be used to predict response to targeted treatment has the potential to revolutionise the management of airways disease and result in improved patient outcomes.

The urokinase plasminogen activator receptor (uPAR, PLAUR) is a cell surface receptor actively involved in the regulation of cell homeostasis. Expression is elevated in the bronchial epithelium in vivo and also in serum and sputum in asthma and elevated expression often indicates poor prognosis in a number of human diseases. The relative contribution of uPAR to asthma disease mechanisms is not fully understood and the functional roles of uPAR isoforms remains to be resolved. The key aims of this thesis were to i) investigate how the uPAR pathway may influence bronchial epithelial barrier properties; ii) investigate the gene expression patterns in the bronchial epithelium in asthma; iii) identify functions of different forms of uPAR in human bronchial epithelial cells (HBEC) and to iv) investigate the association between genetic polymorphisms spanning the PLAUR gene with clinical features and the presentation of asthma in moderate to severe asthma. Using two cell based approaches we identified an inverse relationship between soluble-cleaved uPAR expression and epithelial barrier properties. Importantly, we demonstrated that blocking uPAR-integrin interactions provides a potential therapeutic opportunity to improve epithelial barrier function. Using whole transcriptome analysis genes differentially expressed between cultured asthma and control subjects were identified which were related to cell growth, repair and immune regulation. Furthermore, uPAR expression was elevated in epithelial cells in asthma subjects compared to healthy controls, suggesting expression is inherently altered in the bronchial epithelium in asthma. Transcriptomics was used to provide novel insight into the specific and overlapping functions of uPAR isoforms and to determine the effects of elevated uPAR expression on HBEC function. Finally, the contribution of PLAUR genetic variants to clinical and immunological traits within asthma were investigated and found that PLAUR single nucleotide polymorphisms (SNPs) did not show an association with the traits measured in a severe asthma population. Overall this work has provided new insight into the function of uPAR as a regulator of the bronchial epithelium in asthma.

Lung cancer is the most commonly diagnosed cancer and the most common cause of cancer related death worldwide. Population-based lung cancer screening programmes have been initiated in the USA and could soon be implemented in other countries. The overarching purpose of this thesis was to explore the emotional impact of lung cancer screening. The research was conducted as part of a clinical trial that was investigating the effectiveness of a blood autoantibody test, EarlyCDT®-Lung, in identifying individuals at the risk of lung cancer. A systematic review was conducted that aimed to identify factors associated with the emotional impact of screening for lung cancer. Participants with indeterminate test results, current smokers and females were more likely to experience negative non-specific and specific emotional outcomes. In addition to highlighting several key factors associated with higher levels of emotional distress following screening, factors that warranted further research were also identified. Such factors included age, education level, marital status, ethnic origin, and perceived risk of developing lung cancer. Finally, important methodological and theoretical limitations in the literature were identified. One key methodological limitation was that no studies measured positive emotional outcomes. A longitudinal study was conducted exploring the impact of lung cancer screening on positive affect, negative affect, lung cancer worry and distress specific to screening for lung cancer. Participants from each of the EarlyCDT®-positive, EarlyCDT®-negative, and control groups completed questionnaires containing emotional outcome measures at pre-randomisation and then at one, three, six and 12 months post randomisation. Scores for each outcome measure were described by groups over time and multilevel regression modelling was used to compare scores over time within and between groups. Results were reassuring as screening was found to have no clinically important impact on positive affect, negative affect, frequency of lung cancer worry or impact of lung cancer worry on mood and ability to perform daily activities. Although screening specific distress in the EarlyCDT®-positive group was significantly higher than that of the EarlyCDT®-negative group, it did reduce over time. Statistically significant and clinically important increases in the proportion of participants reporting anxiety about the results of future tests/treatments were identified. As a result of this finding, a further study was carried out to identify factors that could influence an individual’s level of anxiety about the results of future tests/treatment. Participants more at risk of reporting anxiety about the results of future tests/treatment were younger participants, non-white participants, current smokers and participants who did not own or have a mortgage on their home. Psychological variables associated with increased anxiety were: higher general anxiety scores, higher depression scores, higher negative affect scores, participants who reported that they were upset when they thought about their risk of lung cancer, participants who were worried about getting lung cancer, and those who reported the highest impact of lung cancer worry on mood and ability to perform daily activities. The final chapter of this thesis presents the results of a randomised controlled trial embedded within the emotional outcomes study (described above), which evaluated the effect of timing of monetary incentives (£5 voucher sent with questionnaire vs. £5 voucher sent on receipt of questionnaire) on the following outcomes: study participation rates, questionnaire response rates over time, the number of reminders sent and the completeness of returned questionnaires over time. Previous research had found that monetary incentives were useful in increasing response rates in clinical trials. Results from this trial extended the evidence base by showing that the timing of monetary incentives makes no difference to the above outcomes. In each chapter the findings of this thesis are discussed in terms of their contribution to knowledge. Recommendations for future research and clinical practice are also made within each chapter.

Introduction Airway remodelling describes the histopathological changes in tissue architecture observed in obstructive lung diseases such as asthma and may have a negative impact on lung function. These changes do not appear to be treated by current asthma treatments. Changes observed during airway remodelling include increased thickness of airway smooth muscle (ASM) layer and enhanced extracellular matrix (ECM) deposition. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes, which facilitate tissue remodelling via ECM protein degradation. Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of patients with asthma (but not in healthy people). MMPs expression is highly regulated in lungs and is increased in disease states. My project aimed to assess MMP-1, -2 and -9 expression and activity in asthma airways. Furthermore, the underlying mechanism of MMP-1 activation and subsequently its role in airway remodelling and worsening asthma severity was investigated in the context of asthma exacerbation, which is thought to be an exaggerated lower airway inflammatory response to an environmental exposure such as respiratory virus infection. Methods Patients with stable asthma and healthy controls underwent spirometry, methacholine airway (PC20 ) challenge, exhaled nitric oxide (FeNO) test, bronchoscopy/bronchial washings and primary airway smooth muscle (ASM) cell cultures. A second asthma group (mild to moderate severity) and controls had symptom scores, spirometry and bronchoalveolar lavage (BAL) before and after rhinovirus inoculation. ECM was prepared from decellularised primary ASM cultures. MMP-1 protein levels and activity were assessed in bronchial fluid samples by enzyme-linked immunosorbent assay (ELISA), western blotting and fluorescent activity assay. ASM cell growth was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reduction assay and cell counts. Bronchial fluid gelatinase (MMP-2 and -9) expression and activity was assessed by gelatin zymography. Results MMP-1 and MMP-9 expression was enhanced in both stable asthma and during asthma exacerbations, whilst MMP-2 expression was only increased during asthma exacerbations. MMP-1 can be activated by tryptase, which is an inflammatory product of mast cell degranulation. Activated (degranulated) mast cells enhanced proliferation of both control and asthma ASM cells via the production of a pro-proliferative ECM in vitro and the proliferative effect was dependent on MMP-1. In patients with asthma, mast cells numbers within ASM bundles were associated with ASM growth. MMP-1 protein levels were related to bronchial reactivity and MMP-1 activity increased during asthma exacerbations, where its levels were related to exacerbation severity. Conclusion This study suggests that MMP-1 plays an important role in asthma pathophysiology and that ASM/mast cell interactions contribute to asthma severity by transiently increasing MMP-1 activation, ASM growth and airway responsiveness. Moreover, there is increased expression of MMP-2 and -9 during asthma exacerbations compared with stable asthma. As both MMP-2 and -9 act as mediators of inflammation (Okada, S. et al., 1997) (Elkington, P.T.G., 2006) and tissue remodelling (Oshita, Y. et al., 2003), an increase in gelatinolytic activity linked to MMP-2 and MMP-9 is also likely to play a significant role in the pathophysiology of asthma exacerbations.

Genome-wide association studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3’end processing of small nuclear RNAs. An interrogation of bioinformatic datasets showed that in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighboring GSTCD expression. In contrast to the previous reports in other species, a minor alteration of small nuclear RNA processing was observed following INTS12 depletion. RNA sequencing analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, chromatin immunoprecipitation and sequencing experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, INTS12 regulome was defined which includes genes belonging to the protein synthesis pathways. INTS12 has functions beyond the canonical snRNA processing and evidence is presented showing that it regulates translation by directly controlling the expression of genes belonging to protein synthesis pathways. This thesis provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the understanding of biology behind the genetic association for lung function at the 4q24.

Background Evidence from the UK suggests the incidence of idiopathic pulmonary fibrosis is increasing, but there is a lack of data from elsewhere in the World. The cause of the disease remains unknown. New anti-fibrotic therapies may increase the use of surgical lung biopsy for accurate diagnosis, although the risks of this (and other surgery) are not clear. Methods Collated international mortality statistics and a systematic review of the literature were used to assess the incidence and mortality of idiopathic pulmonary fibrosis worldwide. Primary care data from the United Kingdom were used to assess the association between recent major surgery and a new diagnosis of idiopathic pulmonary fibrosis. Secondary care data from the United States and United Kingdom were used to assess the risk of surgical lung biopsy for the diagnosis of interstitial lung disease, and the risk of other major surgery in those with idiopathic pulmonary fibrosis. Results Mortality from idiopathic pulmonary fibrosis in increasing steadily worldwide. Incidence varies worldwide but is in the range of 3-9 per 100,000 in the West. No association was identified between recent major surgery and a new diagnosis of idiopathic pulmonary fibrosis. Surgical lung biopsy for the diagnosis of interstitial lung disease has an in-hospital mortality of under 2% for elective procedures, but this is higher for non-elective surgery, and in those who are older with co-morbidities. In those with idiopathic pulmonary fibrosis undergoing major surgery, in-hospital mortality was higher than the general population. Conclusion Idiopathic pulmonary fibrosis seems to be increasingly common worldwide. Surgery has risks, particularly in unwell older patients, and less invasive diagnostic methods are needed.

Rationale Chronic obstructive pulmonary disease (COPD) is a progressive lung condition with extrapulmonary manifestations- cardiovascular diseases (CVD), impaired physical function, activity and increased frailty. Integrating measures of function into community assessments is hindered by the space and time required. The association of function, activity and CVD has not been extensively investigated in COPD. Objectives Explore the potential utility of Time Up and Go (TUG) as a measure of physical function in COPD Assess association of non-invasive measures of haemodynamics to physical function and self-reported activity Explore ambulatory haemodynamics in COPD and controls Methods Subjects with COPD (n=119) and controls (n=58) were recruited. Ethical and governance approvals were obtained. A medical history including falls, spirometry, peripheral and central haemodynamics, self-reported physical activity questionnaires and functional assessments (TUG and six-minute walk distance (6MWD)) were obtained from all subjects. Ambulatory 24-hour haemodynamics including aortic pulse wave velocity (aPWV) and blood pressure were measured in patients (n=20) and controls (n=19). Results TUG mean(SD) was increased in patients 11.9(3.7)s compared to controls 9.5(1.8)s, p < 0.001. In patients, fallers had longer TUG than non-fallers (p=0.02) and a cut-off time of 12s had the highest sensitivity and specificity to detect fallers and non-fallers. Aortic stiffness was not associated to physical function or physical activity, p > 0.05. In the pilot study, significant nocturnal dip in aPWV was seen in controls, p < 0.01, but not in patients, p=0.07. Conclusion TUG could be a useful measure of function and possibly be incorporated into COPD assessment, particularly where time and space are limited. Finally, ambulatory haemodynamic machine, the Mobil-O-Graph, is feasible and offers opportunity to assess 24-hour haemodynamics profile including aPWV as opposed to a one-off measurement.

A thesis submitted in fulflment of the requirements for the degree of Doctor of Philosophy in the School of Computer Science and Applied Mathematics. November 2015 / The understanding of uid ow at microscale geometrics is an increasingly important eld in applied science and mechanics, especially in bioinspiration and biomimetics. These elds seek to imitate processes and systems in biology to design improved e cient engineering devices. In this thesis, inspired by the e ciency of the insect tracheal system in transporting respiratory gases at microscale, mathematical models that both mimic and explain the gas exchange process are developed. Models for the simultaneous movement of respiratory gases across the insect spiracle, gas transfer from one respiratory chamber to the next, end di usion and tissue absorption at the tracheole tips, and tracheal uid transport are presented. Expressions for tracheal partial pressures of the respiratory gases, rate of change of gas concentrations, rate of tracheal volume change, spiracle behaviour on net gas ow, cellular respiration and tissue absorption, and global gas movement within the insect are presented as well. Two versions of bioinspired pumping mechanism that is neither peristaltic nor belongs to impedance mismatch class of pumping mechanism are then presented. A paradigm for se- lectively pumping and controlling gases at the microscale in a complex network of channels is presented. The study is inspired by the internal ow distributions of respiratory gases produced by rhythmic wall contractions in dung beetle tracheal networks. These networks have been shown to e ciently manage uid ow compared to current produced micro uidic devices. The insect-like pumping models presented are expected to function e ciently in the microscale ow regime in a simple or complex network of channels. Results show the ability to induce a unidi- rectional net ow by using an inelastic channel with at least two moving contractions. These results might help in explaining some of the physiological systems in insects and may help in fabricating novel e cient micro uidic devices. In this study, both theoretical and the Di erential Transform Method are used to solve the exible trachea with gas exchange problem as well as the 2D viscous ow transport with or without prescribed moving wall contractions problem. Both Lubrication theory and quasi- steady approximations at low Reynolds number are used in the derivation of theoretical analysis. ii Moreover, an analytical investigation into the compressible gas ow with slight rarefactions through the insect trachea and tracheoles is undertaken, and a complete set of asymptotic analytical solutions is presented. Then, estimation of the Reynolds and Mach numbers at the channel terminal ends where the tracheoles directly deliver the respiratory gases to the cells is obtained by comparing the magnitude of the di erent forces in the compressible gas ow. The 2D Navier-Stokes equations with a slip boundary condition are used to investigate the compressibility and rare ed e ects in the respiratory channels.

No description available.

The chronic infection of the cystic fibrosis (CF) lung with Pseudomonas aeruginosa strongly correlates with critical outcomes. Pseudomonas alkyl-quinolone signal (PQS) is a diffusible cell-density dependent signal controlling the production of virulence determinants. The PQS amount in the CF lung was proportionate to P. aeruginosa colonisation and PQS molecules have been demonstrated to inhibit pro-inflammatory signalling. However, how PQS influence the recognition of P. aeruginosa by the human lung is unknown. The contribution of PQS to the interaction of P. aeruginosa with human bronchial epithelial cells (HBECs) was characterised using a PQS-deficient mutant ΔpqsA in comparison with its isogenic wild type (WT). Although ΔpqsA appeared attenuated, the pathogenesis of WT and ΔpqsA upon infection of HBEC did not differ in bacterial growth, actin and junctional protein degradation, and pro-inflammatory activation. Despite PQS being highly secreted by a CF isolate LESB58, preliminary data showed that LESB58 was less cytotoxic than the laboratory WT. Our results suggest that PQS does not alter P. aeruginosa pathogenicity on HBECs. Idiopathic pulmonary fibrosis (IPF) is characterised with heterogeneous pathological patterns caused by scarring leading to irreversible destruction of lung architecture. Emerging evidence suggests that dysregulated immunological events could cause the failure of tissue-healing. Systemic immune responses of patients with IPF and age- and sex-matched healthy donors were determined by quantifying cytokines produced by peripheral blood mononuclear cells (PBMCs) upon an array of stimuli. The results showed that PBMCs in patients with IPF were less likely to produce IL-17A, IL-10 and IL-13 than healthy controls (OR 0.14-0.3, 95% CI 0.003-0.03). Patients with lower levels of cytokines had a four to six-fold increased risk of death (HR 4.31-6.13, 95% CI 0.0052-0.0176). This study contributes to a better understanding of the role of PQS in P. aeruginosa pathogenesis and identified cytokine production as a novel biomarker in IPF.