Search

Environmental and pathogenic Gram-negative bacteria are exposed to lipid peroxidation emerging from oxidative stress conditions. However, the mechanism to survive this stress is essentially unknown. The opportunistic pathogen Burkholderia cenocepacia encodes three genes that are upregulated under oxidative stress conditions such as sub-lethal concentration of antibiotic and superoxide ion stress. One of these genes, bcnA, encodes a periplasmic protein from the lipocalin family, which is usually associated with a gene encoding a cytochrome b561 inner-transmembrane protein that we named lcoA; this synteny is conserved in the majority of the β-proteobacteria. Compared to the wild type, mutants lacking these proteins and psrA, a gene encoding a cytoplasmic aldehyde reductase that reduces toxic aldehydes, are more susceptible to bactericidal antibiotics and produce more lipid peroxidation by-products. Fluorescent microscopy analysis using a probe that interacts with lipid peroxyl radicals shows an accumulation of these molecules at the bacterial cell poles and septum. Additionally, using a dye that interacts with anionic phospholipids, we found that peroxidation is associated with a redistribution of anionic phospholipids in the membrane. From this, we conclude that BcnA, LcoA, and PsrA are components of an evolutionarily conserved, previously obscure peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals.

CF (Cystic Fibrosis) is the most prevalent genetic disease among Caucasians. Here, a sodiumbicarbonate channel, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), is impaired, dehydrating all epithelia. The principal cause of morbimortality of CF is CF lung disease (CFLD) where CF airways have impaired mucociliary clearance and undergo constant remodelling and chronic inflammation, ultimately leading to irreversible loss of lung function. Interestingly, within CF patients carrying the same CFTR mutation resides a high variability in terms of CFLD severity and response to treatments. These variabilities are thought to be due to differentially expressed non-CFTR genes. To locate these genes, Genome-Wide Association Studies (GWAS) collected and analysed data from large patient cohorts, identifying four loci (5p15, 3q29, 11p12-13, Xq22-23) associated with more severe clinical manifestations of CFLD. These loci could sit close by genes potentially impacting CFLD penetrance, and patient responsiveness to therapeutics. We aim to understand how genes outlined by GWAS influence CFLD. To this end, we first optimized a model system which relies on human induced pluripotent stem cells (hiPSC)-derived Airways Epithelial Cells (AECs). We first explored two differentiation protocols that generate lung AECs as organoids and Air-Liquid-Interface (ALI) cultures and outlined their respective advantages and limitations. Then, using the most robust differentiation protocol, we explored the expression of GWAS-outlined genes and observed that E26 Transformation-Specific Homologous Factor (EHF) was expressed at comparable levels to primary Human Bronchial Epithelial Cells. Thus, we decided to study EHF in our AECs in-vitro model. First, we knocked out EHF using CRISPR-Cas9 from two non-CF hiPSC lines and selected, within the pool of edited cells, homozygous and heterozygous knock-out (KO) clones alongside wild-type clones, then used as internal controls. The KO was confirmed by finding decreased or absent levels of EHF mRNA and protein in all clones. Second, the resulting hiPSCs lined were differentiated into AECs differentiation. The absence of EHF led to a decreased expression of MUC5B in organoids and alterations in the transcriptomic signature of Basal Cell (BCs) in ALI cultures. Finally, we assessed physiological functions (trans-epithelial electrical resistance (TEER), cilia motility, and cell migration) and functions known to be faulty in CFLD (CFTR function, survival to infection, and response to hypoxia). EHF-/- clones had significantly increased TEER values and forskolin-induced swelling while reducing the HIF1a-induced response to hypoxia, thereby modifying the pH of EHF-/- epithelia, compared to EHF+/+ clones. Taken together, our data outlined several mechanisms by which EHF could be linked to CFLD. In conclusion, we hypothesise that any polymorphisms increasing the expression of EHF could lead to more severe CFLD. Our study sheds light on how a single gene can impact the severity of disease through multiple mechanisms and draws perspectives onto broader targets for personalized therapies against CF.

The development of pH sensing tools for in vitro cell cultures is imperative to maximize the pathophysiological knowledge the scientific community can extract from disease models. Surface-enhanced Raman scattering (SERS) has emerged as a method well suited to pH measurements in cell cultures because SERS optical sensors are readily miniaturised and biocompatible. SERS probes use the protonation state of a reporter molecule adsorbed to a plasmonic surface to measure pH and can be delivered to the intracellular or extracellular space of cells for sensing applications. This thesis aimed to improve the understanding of the pH sensitivity of 4-mercaptobenzoic acid (MBA) SERS probes; to create extracellular SERS substrates for integration into advanced cell culture platforms; and to develop user-friendly SERS probes and assays to complement state of the art cell culture models. Through the course of addressing these aims, three SERS substrates were explored and evaluated for in vitro pH sensing applications: gold nanoparticles (AuNPs), gold nanofibres (AuNF), and gold nanoparticle decorated microspheres (SERS-MS). In Chapter 2, the pH sensitivity of SERS nanoprobes functionalised with MBA (MBA-AuNPs) was explored using lab-based and computational methods. The pH sensitive carboxylate peak in the MBA-AuNPs SERS spectrum is typically used for pH sensing and its sensitivity is widely attributed to the protonation state of MBA molecules at the nanoparticle surface. This thesis demonstrated the intensity of the carboxylate peak in MBA-AuNPs pH calibration curves increases beyond what is expected for a response solely originating from the protonation state of MBA molecules. Chapter 2 demonstrated that, for MBA-AuNPs, the carboxylate SERS peak experiences a pH-dependent increase in SERS enhancement. The potential of MBA-AuNPs as intracellular pH probes were then explore in hepatocellular carcinoma cell line HepG2/C3A. Transmission electron microscopy and SERS pH measurements showed the majority of intracellular MBA-AuNPs were in the lysosome; a pH environment too acidic for SERS sensing. This chapter yielded new insight into the pH sensitivity of MBA SERS probes and examined the limitations of their use to measure intracellular pH in a commonly used hepatocellular carcinoma cell line. In Chapter 3, a SERS-active nanofibre mesh (AuNF) was developed to collect spatially localized extracellular pH measurements in the cellular microenvironment of hepatocellular carcinoma cell line HepG2/C3A cells grown in 2D. AuNF were fabricated by coating electrospun polyurethane nanofibres with gold and functionalised with MBA to make MBA-AuNF. HepG2/C3A were cultured on the surface of MBA-AuNF to facilitate pH measurements in close proximity to the cellular membrane. This method could be used to detect extracellular pH changes following the exposure of HepG2/C3A to staurosporine, an apoptosis-inducing drug. This work demonstrated the integration of a novel pH sensor into 2D cell culture for non-invasive extracellular pH measurements. Finally, Chapter 4 and Chapter 5, developed the application of SERS microsensors (SERS-MS) for the study of airway-surface liquid (ASL) pH in 2D and 3D airway models grown from patient-derived basal epithelial cells. The ASL pH of people with cystic fibrosis is a matter of scientific debate and SERS pH sensors for in vitro epithelial models have the potential to provide insight into airway pH regulation. SERS-MS were fabricated by adhering MBA functionalised AuNPs to polymer microparticles. Using SERS-MS, ASL pH was measured in CF and non-CF 2D epithelium models grown from donor-derived basal epithelial cells. This work demonstrated a 'use friendly' SERS pH assay where pH measurements were collected minutes after administering the probes. SERS-MS were also integrated into 3D airway organoids for pH sensing in the lumen and extracellular environment of organoids. This work demonstrated organoids could be cultured in the presence of SERS-MS for several weeks and Raman spectroscopy used to measure pH in the lumen and extracellular matrix surrounding organoids.

Cystic Fibrosis (CF) is the most prevalent, life-limiting, multi-system, genetic disorder, affecting over 100,000 individuals globally. The condition is caused by mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, which corrupts the instructions for making the corresponding CFTR protein. In the lungs, the defective protein leads to a reduced depth to, and acidification of, the airway surface liquid, a sticky and viscous outer mucous layer, and impaired mucociliary clearance. This in turn leads to airway obstruction and chronic bacterial infection. Recurring, sudden, clinical deteriorations in respiratory symptoms - termed acute pulmonary exacerbations (APEs) - cause cumulative damage to the lungs, and are the most significant driver of mortality and morbidity in CF. However, despite this, currently little is known about their pathophysiology, nor what triggers them. The ability to accurately predict impending APEs would permit earlier treatment, reduce inflammatory lung damage, and directly benefit life expectancy. The average time to treatment varies from a few days to around a month, and so there is a significant opportunity to reduce the delay from current levels. Achieving this outcome was the driving force behind my research. First, I developed an unsupervised machine learning model (the Alignment Model) that, for the first time, was able to generate a characteristic profile of the changes in physiology and symptoms during an APE, and to define an accurate start point for exacerbations. Of particular interest was the existence of a partial interim recovery approximately 10 days after the start of the APE. By extending the model I was also able to identify three distinct classes of APE - one closely resembled the global profile, another showed declines in symptoms before FEV1, and finally one that showed signs of repeated exacerbation and a steeper decline. Second, I used the inferred exacerbation start dates from the Alignment Model to categorise the full set of study days into stable vs unstable (APE episodes). Using this training data, I developed a supervised ML model (the Predictive Classifier) that was able to predict the onset of APEs with 83.6% reliability, and on average 9.5 days earlier than current clinical practice. For this reseach, I leveraged the SmartCareCF study home monitoring data-set, and importantly, I was also able to quantify that including physiological measures in the data collection process resulted in a 51% improvement over self-reported symptom scores alone. Given the already time-consuming treatment regimen for people with CF, this is a key justification of the value of providing these additional measurements. I observed that the performance of the Predictive Classifier was negatively affected by increasing amounts of missing input data. In order to use the predictive algorithm in a clinical setting, it is critical to understand the extent to which any missing input data might have affected the predictions, and, therefore, whether any given prediction can be trusted (is safe) or not. Consequently, in my next area of research, I generated a synthetic data-set that represented the sensitivity of performance of the Predictive Classifier to the amount and pattern of missing data points. I then used this to train a separate ML model (the Safety Classifier) that was able to determine whether the APE predictions were safe or not. It achieved a PR-AUC of 89.7% and an ROC-AUC of 91.2%. Additionally, I was able to use the Safety Classifier iteratively to determine the minimum amount of data that was required to guarantee a safe APE prediction, which could be used in future studies to optimise the data collection requirement. Finally, I was able to apply my research to a new ongoing adult CF home-monitoring study (Project Breathe). The results for both the Alignment Model and the Predictive Classifier were consistent with my earlier findings. However there were three significant environmental factors that should be noted: i) The study period coincided with the broader rollout of triple modulator therapy, which I was able to show reduces the frequency of APE's by 75%; ii) Also there was an extended period of covid-19 enforced isolation, which reduced cross-infection risk; iii) The data collection compliance was significantly lower - for the five most important measures, it was less than half that of SmartCareCF. Despite the overall data-set being nearly five times the size, there were relatively fewer usable APE events - both in absolute count (55) as well as in overall proportion (1%) - and so this reduces my confidence somewhat in the generalisability of the results. Additionally, the Safety Classifier showed that only 10% of the overall Project Breathe study days would be determined as safe to make an APE prediction which was approximately five times lower than for SmartCareCF. A material increase in data collection would be required to be able to use the algorithms in a clinical setting. Overall I was able to meet my research objectives, demonstrating there is a signal in the home measurement data that can be used to identify APEs, building an ML model that could reliably and accurately predict the onset of APEs over a week earlier than current clinical practice, and developing a framework to ensure these APE predictions are safe in the context of missing input data. I am excited to be taking the results of my research into a clinical trial later this year.

Cystic fibrosis (CF) is a hereditary disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The lung disease is, however, also associated with dysregulation of the epithelial sodium channel (ENaC) which leads to dehydrated airways predisposing the individual to chronic cycles of infection and inflammation. ENaC is activated by trypsin-like channel activating proteases (TLPs), inhibition of which increases airway surface liquid volume and improves mucociliary function of primary human airway epithelial cells (hAECs) grown at air-liquid interface (ALI).

Cystic fibrosis (CF) is an autosomal recessive disease affecting over 100,000 patients globally. Current treatments are limited due to several types of mutations affecting the CFTR gene and are costly to healthcare providers. Therefore, new treatment options are required. Studies at Queens University Belfast have shown that Cathepsin S (CatS), a cysteine protease, is elevated in 100% of CF patients. Further studies have shown that CatS is involved in structural damage of the lung tissue, antiproteases degradation, mucus production, and antimicrobial peptides degradation. Moreover, CatS knockout has been shown to improve bacterial clearance and to reduce inflammation. To effectively treat CF patients, we are aiming to synthesize an inhaled CatS inhibitor. Inhaled drugs are more efficient in treating respiratory diseases due to the delivery of higher concentrations locally than systemically administered compounds. Therefore, there are certain essential physical and chemical properties that are required to achieve an effective inhaled drug such as, high potency and selectivity, low systemic exposure, and long duration of action. In chapter 1, we attempted to synthesize cyanopyrimidine CatS inhibitors based on the work of Irie and colleagues. They were able to produce a few potent CatS inhibitors by switching the selectivity of a CatK inhibitor. In addition, the selectivity of those inhibitors against CatK, and L can be massively improved. Therefore, a series of SAR (Structure-activity relationship) optimization was carried out to improve the potency and the selectivity of the scaffold however, the results did not meet our potency criteria. The work of chapter 2 was based on previous (unpublished data) in our lab, which resulted in developing very potent CatS inhibitors with excellent potency and cellular activity, but poor solubility. Therefore, optimization of the inhibitors to improve the Solubility was needed. The optimization was achieved via a series of SAR studies of the P3 and P2 moieties of the inhibitors. Although our findings revealed high potency and selectivity, further optimization is required to improve the cellular activity of the inhibitors.

Pulmonary exacerbations (PEx) are clinically impactful events that accelerate Cystic Fibrosis (CF) lung disease progression. Current PEx treatment approaches have suboptimal outcomes and the frequency of PEx experienced by people with CF (pwCF) is variable and poorly predicted by demographic or clinical factors. There is an unmet need to improve the prediction, treatment, and management of PEx, however this has been hampered by a lack of understanding of the pathophysiology of PEx. Characterisation of the underlying host immune and microbiological mechanisms underlying PEx could improve therapeutic management of PEx and inform a more individualised approach to treatment. In this thesis we provide novel insights into the pathophysiological mechanisms underlying infrequent and frequent exacerbations which we anticipate will be highly informative for improving the personalisation of treatment of PEx through targeting the underlying disease processes.

Background: Musculoskeletal problems in CF are reported in the literature, including an inflammatory arthritis. However, defining and delineating the diagnoses underlying these has been limited. We used a mixed methods approach to understand the scale of the problem and to identify a subset presenting with an inflammatory arthritis phenotype not attributable to other rheumatic disease. Methods 1: Collaborative design of a questionnaire which was then used to gain an overview of the scale and impact of musculoskeletal symptoms. Results: 49% of participants reported musculoskeletal symptoms impacting their activities of daily living in the last year. 44% reported back pain in the last week. The knee was the most commonly painful peripheral joint, with a quarter of participants reporting knee pain within the last week and 50% within the last year. Early morning stiffness and joint swelling were markedly less common, suggesting that the majority of musculoskeletal pain in CF is not due to an inflammatory arthritis. Methods 2: Assessment of people reporting joint swelling and early morning stiffness using ultrasound and blood tests alongside history and clinical examination to identify patterns of disease. Results: Cystic fibrosis associated arthritis (CFA) with clinical and ultrasound evidence of inflammatory arthritis was seen in 25 participants. 8 were found to have other rheumatological diagnoses. Methods 3: A literature review of the use of disease modifying drugs for any indication in people with CF pre-lung transplant alongside departmental data. Interpretation of this was supported by data from disease modifying drugs used in other lung pathologies to consider the safety issues associated with treating inflammatory arthritis in CF. Results: Disease modifying treatment does not appear to be associated with worsening of CF lung disease. Conclusion: Musculoskeletal problems are common in CF. A minority of these are inflammatory arthritis, some of which is CFA and some other rheumatological disease. Those who have non-CFA inflammatory arthritis should be treated according to national guidelines for that disease. Pre-treatment screening should be tailored but follow both national and local protocols. In the context of CFA, no treatment guideline exists, but where there is definitive evidence of synovitis treatment with disease modifying medications would seem appropriate and safe.

People with bronchiectasis and cystic fibrosis (CF) frequently suffer from recurrent infective pulmonary exacerbations (PEx) which lead to a progressive loss of lung function, reduced quality of life and increased morbidity and mortality. Determining how bacterial load and community composition changes with time may improve our understanding of the natural history of these chronic airway diseases. The aims of this thesis were to (i) to determine the psychometric properties of HRQol questionnaires in bronchiectasis, (ii) to measure the longitudinal change in bacterial load and community composition in patients with bronchiectasis when clinically stable and following antibiotic treatment of a PEx, (iii) to determine the antimicrobial susceptibility of P. aeruginosa and H. influenzae isolates cultured from sputum samples collected from patients with bronchiectasis and (iv) to determine changes in total bacterial and P. aeruginosa load following antibiotic treatment of a PEx in people with CF who received either microbiome directed orempirical antibiotic treatment. Results showed that all HRQol questionnaires had good internal consistency and short-term test-retest reliability in at least some domains. Responsiveness was reasonable with the meta-analysis showing a difference between the treatment and placebo effect. In people with bronchiectasis, no statistical difference was apparent in total bacterial, P. aeruginosa and H. influenzae load and microbiome composition during periods of clinical stability and following antibiotic treatment of a PEx. However, P. aeruginosa load was associated with lower lung function and H. influenzae load was associated with increased systemic inflammation .P. aeruginosa isolates were susceptible to tobramycin, ceftazidime and ciprofloxacin, while H. influenzae isolates were susceptible to ampicillin, ciprofloxacin and co-amoxiclav but resistant to azithromycin. In the CF study, no difference was observed in total bacterial and P. aeruginosa load between patients who received microbiome directed and patients who received empirical antibiotic treatment. Resilience in bacterial load and microbiome composition was observed in the lungs of patients with chronic airway diseases with no change observed during periods of clinical stability and following antibiotic treatment of a PEx.

British Lung Foundation research estimates that lung disease affects almost one fifth of the UK population and is one of the UK's three biggest killers alongside cardiovascular disease and non-respiratory cancers. Unfortunately, as chronic respiratory diseases progress, the efficacy of supportive treatments tends to decline. Mesenchymal stem cell (MSC) therapies offer a novel approach due to the immunomodulatory and reparative/regenerative properties of these cells. So, while the bulk of current respiratory disease treatments are aimed at managing symptoms and delaying disease progression, MSC therapies have the potential to promote pulmonary repair/regeneration and restore lung function. Therefore, the Scnn1b-transgenic (Tg) overexpression mouse model of chronic inflammatory lung disease was used to assess the therapeutic potential of MSCs and their secretome in a whole-body approach. MSC-based therapy significantly reduced inflammation and lung damage, with a concomitant improvement in pulmonary function in Scnn1b-Tg mice. Subsequently, administration of the MSC secretome, in a complementary cell-free approach, was investigated to see if similar effects could be achieved. Scnn1b-Tg mice were subcutaneously injected or intranasally instilled with control media or MSC-conditioned media (MSC-CM) for two weeks, after which they were sacrificed at postnatal day 14 (prophylactic) or 6 weeks of age (therapeutic). Analysis of the bronchoalveolar lavage fluid revealed significantly reduced neutrophilia in Scnn1b-Tg mice systemically treated with MSC-CM. Histological and pulmonary function analyses revealed that prophylactic, but not therapeutic, MSC-CM administration significantly reduced lung damage in Scnn1b-Tg mice. Overall, these findings highlight the potential of MSCs and their secretome to limit lung damage and/or promote pulmonary repair/regeneration in the Scnn1b-Tg mouse model of chronic lung disease. Given that MSC-CM significantly restored peroxisome proliferator-activated receptor γ (PPARγ) nuclear localisation and enhanced the phosphorylated levels of adenosine monophosphate-activated protein kinase (AMPK) in the distal conducting airway epithelia of 2-week-old Scnn1b-Tg mice, PPARγ induction and AMPK activation studies were conducted to investigate the underlying mechanisms of action. The results suggest that while the MSC secretome appears to exert its immunomodulatory effects, at least in part, through PPARγ induction, it primarily affects its changes through AMPK activation.

In cystic fibrosis (CF), airways dehydration and impaired mucociliary clearance (MCC) is due to a lack of cystic fibrosis transmembrane conductance regulator (CFTR) chloride secretion and overactive epithelial sodium channels (ENaC), which leads to hyper-absorption of Na+ and water from the airway lumen. The resulting thickened secretions and airway plugging give rise to devastating cycles of chronic infection and inflammation, leading to a progressive decline in pulmonary function strategies to improve MCC in the airways include the inhibition of overactive ENaC. As proteases released from host epithelial and inflammatory cells and bacteria in CF airways, principally trypsin-like proteases (TLPs), play a role in activating ENaC, inhibition of these channel activating proteases (CAPs) represents a novel therapeutic strategy to counteract airway surface dehydration. A library of serine trypsin-like protease (TLP) inhibitors, previously synthesized by Ferguson (2015), was initially screened in an aim to identify a potential ENaC inhibitor. Compounds which had the highest inhibitory effect against TLP activity across various models were then investigated for their ability to inhibit ENaC. Electrophysiological assessment of the four compounds however, found that none were able to effectively prevent the proteolytic activation of ENaC in differentiated primary CF HBE cells. Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in the regulation ENaC. The impact of pharmacological agents stimulating the AMPK signalling pathway in modifying ENaC and inflammation was previously reported with the AMPK activator, metformin. As these experiments were carried out using an unphysiologically high concentration of metformin (5 mM), an alternative AMPK activator, salicylate at a physiological dose of 3 mM was tested which, if able to inhibit ENaC, could potentially have clinical utility in PWCF. Salicylate was shown to activate AMPK, as evident by western blotting. However, electrophysiology showed that salicylate was unable to inhibit ENaC. Inflammation in the lungs of PWCF is difficult to treat, even with established regimens that often include azithromycin (AZ). Given the emergence of elexacaftor/tezacaftor/ivacaftor (ETI), which is expected to restore CFTR activity in 90% of the CF population. The aim of this part of the study was to examine the effect of AZ and ETI, singly and in combination, to assess potential anti-inflammatory effects and investigate the effects on ion channel activity. A significant reduction in cytokines was obtained by ETI in stimulated CF HBE cells. AZ had no effect on inflammation nor on ion channel activities and it had neither synergistic or antagonistic effect when used with ETI. The incorporation of ETI to the standard of CF care provides new opportunities to reduce overall treatment burden and assess drug combination efficacy in ETI-treated patients. Potential rationalisation of regimens should be explored through clinical trials and if successful could reduce drug burden and improve medication adherence.

Mycobacterium abscessus (M. abscessus) is a rapid growing non-tuberculous mycobacteria (NTM) that is increasingly recognised as a major pathogen owing to its inherent resistance to a wide range of the currently available antibiotic therapies. In particular, it causes problems in patients with Cystic Fibrosis (CF) with evidence of marked inflammation, frequent infective exacerbations and an accelerated decline in lung function. Current treatment outcomes are extremely poor. There is therefore an urgent need to understand how M. abscessus resists killing by existing antibiotics and to find new treatments. I have attempted to address these issues in this thesis, particularly relating to the development of novel treatment regimens and antimicrobial agents. I first analysed the treatment outcomes for the CF patients infected with M. abscessus at the Royal Papworth Hospital to examine whether specific antibiotic combinations were associated with better treatment outcomes and could therefore form the basis for a novel treatment regimen. I showed that, despite expert care, the rates of achieving a sustained culture conversion following often prolonged treatment was around 30%, matching the results available from the limited published data. I could find no strong evidence that particular antibiotic combinations were better, further motivating my search for new treatment strategies. I then designed a novel regimen of existing antibiotics (based on synchronous targeting of cell wall, protein synthesis and cellular energetics) to use in CF patients with either treatment-naïve or refractory disease. I demonstrated good efficacy in vitro against a range of clinical isolates of M. abscessus. Subsequent GWAS analysis revealed several potential resistance pathways, including a variety of metabolic pathways and membrane transport systems, which could be used to stratify patients in a future clinical trial. I also developed and evaluated a novel highly effective formulation of acidified nitrite as a new nebulised antimicrobial therapy for M. abscessus which has been shown to have in vivo activity and is currently being evaluated in a clinical trial. Finally, I have developed a method using ultra-performance liquid chromatography to detect intracellular permeability of compounds to support fragment-based antibiotic discovery. I screened a library of 960 fragments to assess for permeation into M. abscessus. Machine learning was then used to generate a predictive algorithm for bacterial permeation that can be used in future fragment-based campaigns.

Cystic fibrosis (CF) is a life-shortening, autosomal recessive disease caused by loss-of-function mutations in the CFTR gene. The primary cause of morbidity and mortality in CF is respiratory failure following progressive loss of lung function and characterised by recurrent infections and an intense, chronic, neutrophil-dominated inflammatory airway response that is highly excessive relative to the weight of the actual infectious burden. As such, the inflammatory response has been one of the central focuses of CF research for the past 30 years, with an array of studies elucidating the dysregulation of several key immune processes in CF. Macrophages are the most plastic of all immune cells and their immense functional diversity allows them to play a part in orchestrating all major stages of the inflammatory response, from initiation, to resolution and repair. Macrophages, often overlooked in CF research despite their integral role in other chronic inflammatory pathologies, have increasingly become recognised as potential orchestrators of the excessive inflammatory response in CF. The discovery of functional CFTR expression in several immune cells, monocytes and macrophages in particular, has also opened the possibility that some immune cells, much like epithelial cells, are intrinsically dysregulated in CF and pre-programmed to contribute to an excessive inflammatory response. The aim of this project was to investigate the role of macrophages in driving progressive lung disease in CF and gain a better understanding of the mechanisms underlying dysregulation of CF macrophage immune function, with a focus also on the extent of the CFTR-dependent nature of such dysfunction. Another aim was to track longitudinally the effects of novel CFTR modulator therapies, which work to correct the basic underlying defect in CF, on patient inflammation and myeloid function. Whole-blood flow cytometry was used to show that patient circulating monocytes exist in a significantly higher state of activation than non-CF controls and that this correlates with predicted lung function (FVCpp). This activation phenotype was reversed with CFTR modulator (Elexacaftor/Tezacaftor/Ivacaftor) treatment suggesting that such treatment modulates levels of inflammation in patient circulation. CF monocyte-derived macrophages (MDMs) were found to exhibit a hyperinflammatory response to activation with P. aeruginosa LPS and produce elevated levels of several important proinflammatory cytokines by qRT-PCR and ELISA. Given that such cells were cultured for 9 days in the same conditions as non-CF controls, such a phenotype would point towards intrinsic immune dysregulation in the cells. In order to gain a more high-resolution, genome-wide view of transcriptional changes in CF MDMs responding to inflammatory challenge, a highly unbiased approach of global transcriptome profiling was carried out using the cap analysis of gene expression (CAGE) method, which provides additional layers of information regarding promoter usage, enhancer activity and other important regulatory elements that drive gene expression. CAGE sequencing revealed substantial differences in gene expression patterns of CF LPS-treated MDMs compared to non-CF controls, particularly involving genes involved in the immune response, and elucidated a significantly blunted type I IFN signalling response in CF macrophages at 24 hours post-LPS. Such results provide scope for investigating the impact of defective type I IFN signalling in macrophages on bacterial inflammation in CF and also on antiviral immunity, with acute respiratory viral infections representing important, and common, precipitants of pulmonary exacerbation in CF. These findings provide novel insights into macrophages dysfunction in CF and further underline the potential of these cells as a target for novel, and thus far elusive, anti-inflammatory interventional strategies to effectively slow CF lung function decline in combination with novel CFTR modulators and the standard treatment regimen.

Cystic fibrosis (CF) is the most common, inherited, life-limiting, multi-system disorder among the Caucasian population. Disease arises from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene. In the lung, defective protein leads to the accumulation of thick viscid mucus, depletion and acidification of the airway surface liquid and impairment of the normal muco-ciliary clearance mechanisms, providing a permissive environment for chronic infection and progressive airway damage. Pseudomonas aeruginosa is a versatile, Gram-negative opportunistic human pathogen with a predilection for establishing chronic infection in the CF lung. Its extraordinary capacity to cause infections is due to its vast repertoire of secreted and cell-associated virulence determinants, which are subject to a complex regulatory network of intracellular and intercellular signals. Acute pulmonary exacerbations (APE), are the main cause of morbidity and mortality in CF. Despite their clinical significance, the mechanisms that trigger these events are poorly understood. In this dissertation, I investigate whether home monitoring for changes in patient physiology and symptoms was feasible and could permit advanced detection of an APE. I additionally concentrated on whether temporal fluctuations in the behaviour and structure of established P. aeruginosa populations within the CF lung may trigger APEs, and whether such changes could function as a bacterial biomarker(s) and be correlated with home monitored data to facilitate APE diagnosis and prompt initiation of treatment. First, I conducted a single-centre, pilot study (TeleCF) of 15 adults with CF in order to determine whether daily home monitoring of a single sputum bacterial biomarker (P. aeruginosa exotoxin A [PEA]) along with several clinical parameters might provide advanced warning of an APE. Home monitoring was well tolerated and provided high resolution data on physiological and biomarker changes preceding, during and following antibiotic therapy for an APE. On its own, PEA did not prove an effective biomarker for early detection of APEs in adults with CF, but the study did provide proof of concept for the application of home-monitoring and sputum profiling for bacterial biomarkers to inform further work. Next, I collected longitudinally sampled sets of 95 isolates per sputum sample from 9 adults with CF before, during and after antibiotic treatment for an APE. Isolate populations were analysed for a series of phenotypic traits associated with P. aeruginosa virulence to determine whether changes in phenotype composition were related to exacerbation onset. I also investigated for differences in phenotype composition between isolate populations of the P. aeruginosa Liverpool epidemic strain (LES), Manchester epidemic strain (MES) and local non-epidemic strains. I found strong evidence for the uncoupling of the traditional quorum sensing (QS) regulatory hierarchy in CF isolates, with the rhl subsystem playing a more dominant role in virulence expression in certain strain types. Importantly, no link was found between APEs and the emergence of a particular sub-population of morphotypic or phenotypic variants. Finally, I conducted a multicentre UK-based study (SMARTCARE) of 147 adults with CF to assess the acceptability and feasibility of daily monitoring of symptoms and physiology using novel sensor technology and mobile phones. Linked-anonymised data were analysed using machine learning (ML) methods to define the profile of APEs and predict their onset. Survey patient feedback confirmed that home monitoring was easy to do and helped patients track their health over time. Unsupervised machine learning analysis uncovered the typical signal profile of an APE and revealed three distinct classes of APE. We developed an ML predictive classifier that can detect an impending APE on average 11 days earlier than current clinical practice. This work has contributed greater insights into the day-to-day variation in symptoms and physiology prior to, during and following periods of APE in adults with CF. It has confirmed the important role for home monitoring in CF care delivery and highlighted the power of machine learning methods when applied to high frequency data to advance our understanding of APEs in adults with CF.

The airways of persons with cystic fibrosis (CF) are prone to lifelong colonisation with dense microbial ecosystems comprised of a diverse combination of bacteria and fungi. The role of interspecies interactions in modulating changes in gene expression and the metabolism of individual members of a polymicrobial community, compared with growth as an axenic population, is becoming increasingly apparent within the field of medical microbiology. In part, these poorly understood interactions explain why, regardless of intense research efforts, therapeutic treatments designed to clear such chronic airway infections often fail within the clinical setting. The paucity of existing in vitro and in vivo models used to study CF-associated infections greatly hinders research into the behaviours of polymicrobial communities. Species that coexist within the same in situ environment often outcompete one another when grown as a mixed population using traditional culture methods. The work presented in this dissertation redresses these problems. I describe the development and characterisation of a novel in vitro continuous-flow culture model. This experimental system permits the stable co-cultivation of distinctly different microbial species commonly-associated with CF airway infections; species that ordinarily readily outcompete one another in existing models. I demonstrate that the metabolic state and mutation rates of species cultured within the model remain stable for extended periods of time. Furthermore, I show that antimicrobial compounds display decreased efficacy against their target organism when grown as part of a polymicrobial community. I also show that different combinations of microbial species display different biofilm-forming potential. This work provides the basis of future research efforts aimed at the in vitro recapitulation of an entire polymicrobial community directly derived from persons with CF. Ultimately, this will help to bridge the bench-to-bedside gap for the development of more efficacious airway infection management regimens.

Cystic fibrosis (CF) is a life-limiting, inherited disorder caused by a faulty gene, which affects many parts of the body, most predominantly the lungs and gut. Currently no cure exists for CF and treatment regimens are intensive and time consuming. CF results in substantial psychosocial sequelae. The salience of disclosure of an individual's CF diagnosis has increased as the average life expectancy has extended. The integrative review aimed to synthesis evidence exploring the experiences of individuals who disclose CF to others. Five electronic databases were searched and eight studies met the inclusion criteria. Findings indicated that disclosure of CF is a complex process, with the potential to elicit positive support and development, whilst, conversely, being associated with high anxiety and possible negative effects. As such, individuals with CF undergo a multifaceted decision-making process before disclosing. Findings were discussed in relation to relevant literature and theories, clinical implications and future research. CF is predominantly diagnosed during infancy. However, a small proportion of individuals, who present clinically and socially differently, are diagnosed in adulthood. Therefore, the empirical study aimed to explore the experiences of individuals who received a diagnosis of CF as adults, including psychological impact and support around diagnosis. Semi-structured interviews were conducted with six participants and analysed using Interpretative Phenomenological Analysis. Four super-ordinate themes, comprised of 12 sub-themes, emerged from the data. The super-ordinate themes were: 'Inexplicable and failing'; 'The blow of diagnosis'; 'Processing journey' and 'Managing others'. Themes were discussed in relation to relevant literature and theory, with recommendations made for clinical practice and future research.

Survival in cystic fibrosis (CF) is increasing exponentially and we are now seeing an ageing population. At the Manchester Adult Cystic Fibrosis Centre (MACFC), 20% of the total patient group are aged 40 years and above. Survival is likely to increase further in this exciting era of cystic fibrosis transmembrane conductance regulator protein (CFTR) modulation. Although there has been a paradigm shift in CF management and disease perspectives in recent years, there remains a paucity of research in this important and fascinating older CF patient population, particularly over the last decade. The CF population aged 40 years and above at MACFC is heterogeneous. There was a high proportion of patients with class I-III CFTR mutations and associated severe phenotypic disease. However, the prevalence of residual function CFTR mutations increased with age and were associated with lower sweat chloride values and later age of CF diagnosis. Cardiovascular risk exists in this older CF cohort, with 34.9% of patients showing elevated QRisk®3 scores of 10% or greater. The prevalence of cardiovascular disease may increase with an ageing CF population. The recognition and management of cardiovascular risk is essential to reduce further comorbidity in this already complex and challenging disease. At 3.6%, the prevalence of chronic kidney disease (CKD) was not as high in this older adult CF cohort as perhaps initially predicted. However, the measurement of estimated glomerular filtration rate (GFR) can be inaccurate in CF patients. There may be some justification for the wider use of measured GFR methods, such as iohexol, to improve the reliability of renal monitoring and in the assessment of GFR decline in select phenotypic groups of older CF patients. Cardiac magnetic resonance imaging (CMR) is feasible and well tolerated in this older adult CF group with a range of pulmonary disease severity. The use of CMR may provide an opportunity to analyse cardiac morphology and function in greater detail in CF than can be done with echocardiography alone, and cardiac imaging may become increasingly relevant in an ageing CF population.

Cystic fibrosis (CF) is the most common fatal genetic disease in the Caucasian population. It is a multisystem disease, however respiratory disease is almost universal and is characterised by colonisation of respiratory pathogens, repeated infection resulting in inflammation and structural change, with death occurring secondary to progressive respiratory failure. One such respiratory pathogen commonly found in CF patients is the fungus Aspergillus fumigatus, which is the most common disease causing fungus in CF. Aspergillus can cause a spectrum of disease, and novel immunological classification of Aspergillus disease phenotype was suggested by Baxter and colleagues (Baxter et al. 2013a) to comprise; Class 1, no disease; Class 2, allergic bronchopulmonary aspergillosis (ABPA); Class 3, Aspergillus sensitised; and Class 4, Aspergillus bronchitis. This thesis provides the first long-term follow-up of the original patient cohort to investigate what influence Aspergillus disease phenotype may have on the clinical outcomes of adult CF patients, revealing no difference in mortality, transplant rates, decline in forced expiratory volume in 1 second (FEV1), body mass index (BMI), or intravenous (IV) antibiotic therapy required over time when comparing Aspergillus Class 1-4. Indeed, baseline FEV1 remains the sole predictor of survival in this patient cohort. Further to this, this study re-classifies the same patient cohort 10 years after Baxter's original work to ascertain if patients change disease class over time. It appears that using the corrected diagnostic algorithm set forth by Baxter and colleagues reveals a large shift towards the Class 4 phenotype. It is unclear however, if this represents a true migration of disease, or is reflective of sputum galactomannan levels which were significantly raised in comparison to the previous study, and likely reflects an older population and more expansive lifetime exposure to Aspergillus. Given that Aspergillus fumigatus is a ubiquitous environmental fungus, work was also undertaken to investigate the environmental fungal burden within the Manchester Adult Cystic Fibrosis Centre, and in patient's homes, to determine if environmental Aspergillus could influence clinical disease. A seasonal rise in Aspergillus fumigatus was noted in the ward environment over the testing period, which also coincided with building works being undertaken, in-keeping with published literature. Examination of protective environmental factors in the form of ventilation systems and air filtration were therefore undertaken to elucidate if a minimum ventilation and filtration requirement could be suggested for a specialist CF centre, with >10 air changes per hour (ACH) appearing to be reliable in effectively reducing fungal burden, however high-efficiency particle air (HEPA) and Q40 filters did not appear to effectively reduce environmental fungal burden. Patient homes grew relatively low counts of Aspergillus and other fungi. There appears to be no relationship between Aspergillus levels, either in the CF centre or in patient homes, and Aspergillus disease phenotype.

Chronic lung conditions such as cystic fibrosis and bronchiectasis are characterised by cycles of inflammation and infection. Long term antibiotics are commonly prescribed in these conditions to decrease exacerbation frequency and improve quality of life. Azithromycin is often given orally for this purpose with its benefit thought to be due to a dual antibacterial and anti-inflammatory effect. In this thesis, the aim was to develop an inhaled liposomal loaded azithromycin formulation that could be delivered via dry powder inhaler for use in chronic lung infection. By delivering the antibiotic directly to the site of action in the lungs, higher local concentrations could be achieved whilst overcoming issues with oral therapy such as systemic side effects and the exposure of bacteria at non-target sites. Furthermore, the encapsulation of azithromycin within liposomes should offer advantages in both sputum and bacterial biofilm penetration. Results from this study showed the formulation could be manufactured reproducibly, with good stability retained following one-year storage. The liposomal loaded azithromycin formulation was as effective as free drug against both Gram positive and Gram negative clinical respiratory isolates, as well as equally effective at clearing Staphylococcus aureus infection in an in vivo mouse model of lung infection. The formulation showed no toxicity in vitro, at relevant concentrations, against human and mouse cell lines with no increase in release of inflammatory mediators. Interestingly, the liposomal formulation itself, without azithromycin, resulted in a decrease in pro-inflammatory cytokine CXCL1 from a lipopolysaccharide stimulated murine alveolar macrophage cell line in vitro. Finally, the adjustment of protectant sugar to a mixture of trehalose and dextran offered further improvements in glass transition temperature and water content. The results of this study support the potential of this formulation as an alternative to oral azithromycin in the treatment of chronic lung infection.

Section A This is a literature review of patient empowerment in children and young people (CYP) with cystic fibrosis (CF), using a meta-synthesis of 17 qualitative studies. Thematic synthesis identified analytic themes: ‘relational support’, ‘information and understanding’, ‘feeling heard and respected’, ‘mastery and competence’, appeared to facilitate empowerment; ‘prejudice and assumptions’ was identified as a potential barrier. ‘Navigating being different’ appeared to both influence and be influenced by empowerment. Findings provide an initial understanding of factors influencing empowerment in CYP with CF. Potential clinical and research implications are considered. Section B This is an empirical paper presenting a grounded theory study that aims to develop a preliminary theory of empowerment in CYP with CF. Seven young people with CF, five parents and four professionals were interviewed. The emerging model suggests that ‘thriving alongside CF’ may be supported by interactions between ‘having a team’ and ‘taking charge and having a voice’, leading to ‘being able to just be a child/getting on with life’. ‘Concealing self’ may get in the way of ‘thriving alongside CF’. These processes occur within wider medical and developmental contexts. Study limitations, clinical and research implications are discussed.

Cystic Fibrosis-Related Diabetes (CFRD) is the most frequently observed extra-pulmonary co-morbidity associated with Cystic Fibrosis (CF). It is an ever-increasing burden on the CF population and is estimated to be present in up to 50% of people with CF over the age of 30. People with CFRD have a six-fold greater mortality rate compared to people with CF without diabetes, however the exact mechanism for CFRD development currently remains unclear. This thesis will aim to elucidate the role of CFTR in the development and function of pancreatic islets. A novel in vivo model of CFTR inhibition model was used in this thesis and validated against commonly used transgenic models. Short-term CFTR inhibition in C57BL6 mice was shown to cause a significant reduction in the size of pancreatic islets alongside a significant reduction in insulin secretion whilst sub-chronic CFTR inhibition lead to the development of insulin resistance consistent with other models of CFRD. Expression of islet connectivity markers Connexin 36 and E-cadherin were consistently reduced across multiple CF mouse models which may contribute to altered islet morphology and dysfunction observed in CFRD. An in vitro model was used to investigate the role of exocrine secretions on endocrine function, and whilst a number of differences in inflammatory makers were observed in the presence of different CFTR mutations, these were found to have little effect on the insulin secretory ability of beta-cells. This thesis supports a role for CFTR in the regulation of islet size and function, suggesting that therapeutic approaches using CFTR modulators may be beneficial for the restoration of islet architecture and glucose homeostasis in people with CFRD.

Cystic Fibrosis (CF) lung disease is characterised by dysfunctional innate immunity. Even before microbial colonisation is established, there is an accumulation of neutrophils within the airways. Neutrophil extracellular traps (NETs) are an evolutionarily conserved antimicrobial defence mechanism of neutrophils. However, NETs can also cause inflammation and damage when in excess and as such are implicated in the pathogenesis of CF lung disease. Within the airways, NETs are a source of proinflammatory proteins including myeloperoxidase, neutrophil elastase and calprotectin. Treatment of CF patients with DNase reduces airway inflammation and sputum viscosity, which may be due to the drug increasing clearance and/or decreasing the production of NETs. Our group has previously found that NETs are proinflammatory to monocyte-derived macrophages (MDM) in co-culture, and that this was more exaggerated in CF MDM, so it is likely that an interplay between the innate immune cells exacerbate airway damage. Several defects have been reported in CF MDM previously, which may exacerbate airway infection and inflammation. One such reported defect is failure of the macrophage phagolysosome to acidify, which impairs bacterial killing and may lead to more bacteria surviving in the airway as a driver of inflammation. Throughout this PhD, sputum samples from both CF and healthy control (HC) participants were collected and lung function measured to investigate whether NETs are associated with airways inflammation and increased severity of lung disease. Using ELISA quantification of NETs (by an in-house ELISA which measures histone-bound calprotectin) and proinflammatory cytokines, CF participants were found to produce higher levels of sputum NETs than HC. Those CF participants taking DNase had significantly decreased levels of sputum NETs relative to those not on the drug. Furthermore, positive associations were found between NETs and proinflammatory proteins, whilst negative correlations were demonstrated between these proteins and lung function. Furthermore, when forced expiratory volume in one second (FEV1) was predicted by multi-variate linear regression, the level of sputum NETs was a significant independent indicator of FEV1. A murine model of lipopolysaccharide (LPS)-induced acute lung injury was used to characterise how CFTR alters NET formation and associated inflammation following a sterile inflammatory challenge and whether DNase affected this. It was demonstrated that CFTR-/- mice have an exaggerated inflammatory response to LPS-induced acute lung injury relative to wild type (WT) littermates in terms of airway proinflammatory cytokine concentrations and histopathological scoring of acute lung inflammation. This was despite no acute increase in airway neutrophil or macrophage numbers, suggesting intrinsic defects exist within these innate immune cells due to absence of CFTR. The receptor for advanced glycation end-products (RAGE) had higher gene expression in CFTR-/- vs WT littermates, which leads us to speculate that up-regulation of NF-κB signalling could be responsible for increased inflammation in CF mice. Importantly, airway NETs were not significantly different between genotypes and DNase had no effect on inflammation, suggesting the CF murine model is imprecise in mimicking human disease. This is perhaps unsurprising given that people with CF have non-resolving airways inflammation, rather than an acute injury. Final experiments investigated the role of CFTR in regulating macrophage phagolysosomal pH. Using MDM from both HC and CF donors, we developed a novel technique compatible with real-time analysis of phagocytosis, which showed that surface-enhanced Raman spectroscopy-based nanosensors exhibit superiority over conventional fluorescence spectroscopy in measuring macrophage phagolysosomal pH in terms of sensitivity, ratiometric quantification, and both spatial and temporal resolution. Human MDM phagolysosomal acidification was found to be CFTR-independent and may not be critical in the pathophysiology of CF lung disease, although further experiments using alveolar macrophages would strengthen these conclusions. To summarise, NETs are associated with inflammation and disease severity in human CF lung disease. Our mouse work suggested that intrinsic defects of innate immune cells exist in CFTR-/- mice, manifest by an exaggerated response to sterile inflammation, possibly via RAGE-NFκB signalling pathways. We also demonstrated that macrophage phagolysosomal acidification was not impaired in human CF MDM. Further research investigating the underlying mechanisms causing innate immune cell dysfunction will help identify therapeutic targets for the treatment of inflammation in CF lung disease.

Cystic fibrosis (CF) is an autosomal recessive disease, caused by mutations in the gene which codes for the CF transmembrane conductance regulator (CFTR). Mutations in CFTR cause multi-organ dysfunction, including in the lungs, pancreas, gastro-intestinal system and the reproductive system. However, lung disease is the major cause of morbidity and mortality in people with CF PWCF. CF lung disease is characterised by hyper-susceptibility to infection coupled with mucus obstruction, exaggerated inflammatory responses and resulting pulmonary damage and decline. Dysregulation of ENaC, secondary to the loss of functional CFTR in CF, leads to increased Na+ absorption which contributes to airways dehydration, thickened mucus, impaired mucociliary clearance and is thought to be a major contributor to the pathogenesis of lung disease. Previous work has shown that QUB-TL1, an active site directed inhibitor of trypsin and trypsin-like enzymes, reduces ENaC-mediated sodium absorption from the surface of airway epithelial cells in CF, which was associated with improved mucociliary clearance due to a concomitant increase in airways hydration. The initial aim of this project, therefore, was to synthesize and evaluate three novel inhibitors of trypsin-like channel activating proteases to determine their effect on inflammatory signaling via PAR-2 and also on ENaC activity in CF airway epithelial cells (AECs). Further, two highly selective and potent furin inhibitors, BOS-981 and BOS-318 were characterised, with the aim of determining whether selective inhibition of furin has the potential to reduce ENaC-mediated sodium transport and improve airways hydration. The effect of BOS-981 and BOS-318 on inflammatory signalling and TGF-β secretion in CF AECs was also investigated.

Introduction: A number of studies have shown an increased prevalence of gastro-oesophageal reflux in adult and paediatric cystic fibrosis (CF) populations. To date it remains unproven if the increased amounts of reflux shown can affect CF lung disease. The most commonly proposed mechanism by which this may occur is reflux aspiration. The study's aim was to assess if a relationship exists between reflux and measures of lung disease severity. Methods: A prospective observational study was conducted in stable adult CF patients, measuring reflux with combined oesophageal pH-impedance (n=41). This allowed the following analyses: (I) The reflux measures were described for the entire cohort; (II) The influences of various factors on the amount of reflux were examined, including oesophageal motor function, prescribed medications and co-morbidities; (III) The effect of reflux on respiratory endpoints was assessed; (IV) Mass spectrometry was used to assess the effect of reflux on relative protein abundance within sputum. Results: Reflux was increased in 54% of this cohort using total reflux events. Reflux characteristics assumed to be high-risk for reflux aspiration (proximal and/or supine events) were raised in 41%. Dysfunction of the oesophago-gastric junction correlated with acid exposure. Methylxanthines but no other medications correlated with the number of reflux episodes. No correlation was shown between reflux measures and any respiratory endpoint tested. No differences in relative protein abundance within sputum were demonstrated between those with the highest and lowest measures of reflux. Conclusions: Although there was a high prevalence of increased reflux using pH-impedance, no effect of reflux was demonstrated on the tested respiratory endpoints or relative protein abundance within sputum. This may reflect that it is currently not possible to directly measure the amount of reflux aspiration, which is a major limitation.

Pseudomonas aeruginosa is an opportunistic pathogen that can invade and colonise the lungs of people with cystic fibrosis (CF), cause septic shock through bacteraemia infections, and lead to serious infection of burn injuries. It is one of the most critical multi-drug resistant bacteria, and is associated with high morbidity and mortality. A total of 4,094 P. aeruginosa isolates were sampled from nine patients with CF over a six-month time period. These isolates were collected from sputum samples during stable, acute, and recovery timepoints from periods of sudden and rapid lung function decline, called acute pulmonary exacerbations (APEs). These isolates were previously analysed for the presence and absence of ten virulence-related phenotypes. The P. aeruginosa isolates were whole-genome sequenced to investigate the inter- and intra-patient genotypic diversity, associations with phenotypic diversity, and adaptation within the CF lung. Each of the nine patients with CF were colonised with a distinct clone of P. aeruginosa. Six patients were infected with well-characterised, highly-transmissible strains of either the Liverpool Epidemic Strain (LES) or the Manchester Epidemic Strain (MES). The remaining three patients were infected with novel sequence types (STs); ST3307 or ST3308. Putative transmission was identified between the two patients infected with ST3307. Two large deletions in genetic regions commonly associated with progression from acute to chronic infection were identified in ST3307. The acquisition of the LES by one of the patients was very recent, estimated to have occurred within the two years prior to the study. This recent acquisition provides an insight into the immediate adaptation of P. aeruginosa to the CF lung, with adaptation observed in genetic regions associated with progression from acute to chronic P. aeruginosa infection. The timepoints for each APE within the individual patients were not associated with variation in the diversity of the populations of isolates. This was confirmed by random distribution of phylogenetic clusters with respect to each APE timepoint for most patients, suggesting that APEs, and the treatment of APEs, do not substantially affect the diversity of the P. aeruginosa population within the patient lung. Genome-wide association studies (GWAS) were carried out on the CF isolates, to investigate any associations with the ten previously-tested virulence-related phenotypes. Population structure could be effectively controlled for in this highly structured dataset, using linear mixed models. Multiple GWAS approaches were required to capture the different classes of genetic variation, resulting in the identification of biologically relevant associations for complex phenotypes, most notably a premature stop-codon in the global transcriptional regulator rhlR, as well as several novel, potentially significant associations. An additional 352 P. aeruginosa isolates from patients with bacteraemia were also whole-genome sequenced. These isolates were sourced from both a local collection and from a UK-wide surveillance collection, and broadly match the defined population structure of P. aeruginosa. Three STs were overrepresented in this dataset, which are associated with virulence and multi-drug resistance; ST175, ST253 and ST395. One of these overrepresented STs, ST175, was distributed across the UK, shows significant geographical clustering and temporal signal, and is predicted to have been introduced into the UK between the late 1980s and the early 1990s. Antimicrobial resistance profiles showed that current therapeutic options are still viable for most P. aeruginosa bacteraemia infections, and that colistin is still effective against the most multi-drug resistant isolates.

The Mycobacterium abscessus} species complex (MABSC) consists of three subspecies, M.a. abscessus}, M.a. massiliense} and M.a. bolletii}. All three of these subspecies are capable of causing opportunistic pulmonary and skin and soft tissue infections in immunocompromised individuals. Infections caused by the MABSC are particularly serious in people with underlying lung conditions such as Cystic Fibrosis (CF) as these organisms are highly antibiotic resistant and the currently available treatment is toxic. This has resulted in treatment of MABSC infections failing in up to 50% of cases. Given the increasing prevalence globally of infections caused by the MABSC, particularly in individuals with CF, there is increased urgency to improve the treatment available for MABSC infections. The overall aim of this project was to use genomic analyses to increase our understanding of how the MABSC has evolved to become an opportunistic pathogen, which in turn could potentially uncover promising targets for the development of novel antibiotics. Genomic analysis of the MABSC has already shown that lineages of the MABSC are capable of indirect person to person transmission and the extent to which transmission contributed to the increasing prevalence of MABSC in people with CF became evident when the MABSC global population structure was determined. This showed that 70% of the MABSC isolates from people with CF were attributed to lineages made up of densely clustered isolates, where acquisition via indirect person-to-person transmission, as opposed to from the environment, was more likely. This thesis used population genomic approaches to i) investigate the genetic factors that drove the emergence of the most prevalent MABSC lineages, ii) look for evidence of convergence after the clonal expansion of these lineages to understand how the MABSC was continuing to adapt and spread amongst people with CF and iii) to examine the within host evolution of these pathogens to uncover how these environmental organisms were adapting to the CF lung. This thesis also used whole genome sequencing to explore the largest known outbreak of MABSC infections, a post-surgical wound infection epidemic in Brazil. Through this research the emergence of the most prevalent MABSC lineages were found to be driven by increased opportunity, probably due to the increased number of people with CF surviving longer, as opposed to the acquisition of a common genetic determinant. Not enough signal was detected after the clonal expansion of the most prevalent MABSC lineages to come to strong conclusions about genetic factors driving their continuing expansion, but strong evidence of convergent evolution was detected between MABSC isolates evolving over time within the host. The MABSC was shown to be potentially using a similar central regulatory network in response to environmental cues from the phagosome to that of M. tuberculosis}. The investigation into the epidemic of post-surgical wound infections in Brazil showed that a single M.a. massiliense} lineage was introduced into Brazil just prior to the initial outbreak and that this lineage subsequently spread, through several waves of transmission, to multiple cities in geographically distant areas of Brazil. This highlighted how the MABSC was capable of long distance transmission and emphasised the potential of the MABSC as a nosocomial pathogen capable of causing large scale outbreaks.

The work described in this thesis is focused on the application of fragment-based approaches for two essential mycobacterial target proteins, fumarate hydratase (fumarase) from Mycobacterium tuberculosis (Mtb) and tRNA (m1G37) methyltransferase (TrmD) from Mycobacterium abscessus (Mab). With Mtb fumarase a high-throughput screening (HTS) hit was used to design a small library of fragments in a deconstruction-reconstruction approach. These fragments were screened using a range of both biochemical and biophysical methods. The resultant fragments showed evidence of weak protein binding. As an alternative strategy, derivatives of the HTS hit were synthesised and screened by a biochemical assay, which identified nanomolar inhibitors of this enzyme. In addition, X-ray crystallography was also carried out with a range of these compounds. Selected compounds were subsequently screened by collaborators at the NIH against Mtb. With the enzyme TrmD from Mab, the fragment hits identified were used as the basis of a fragment-merging approach to develop potent inhibitors, guided by structural biology. In the implementation of this approach, synthesis and biophysical techniques were extensively utilised, including both differential scanning fluorimetry and isothermal titration calorimetry. This approach led to the development of novel inhibitors with low nanomolar affinity. Select compounds were screened by collaborators against both Mab and Mtb in vitro. In light of encouraging activity against Mtb, the TrmD homolog in Mtb was expressed and screened against select compounds to demonstrate the broader applicability of the lead series.

Eukaryotic organisms rely on the coordinated beating of motile cilia for a multitude of fundamental reasons. In smaller organisms, such as Paramecium and the single cell alga Chlamydomonas reinhardtii, it is a matter of propulsion, to swim towards a higher concentration of nutrients or away from damaging environments. Larger organisms use instead the coordinated motion of cilia to push fluid along an epithelium: examples common to mammals are the circulation of cerebrospinal fluid in the brain, the transport of ovules in the fallopian tubes, and breaking the left/right symmetry in the embryo. Another notable example, and one that is central to this thesis, is mucociliary clearance in human airways: A carpet of motile cilia helps keeping the cell surface free from pathogens and foreign particles by constantly evacuating from lungs, bronchi, and trachea a barrier of mucus. The question of how motile cilia interact with one another to beat in a coordinated fashion is an open and pressing one, with immediate implications for the medical community. In order for the fluid propulsion to be effective, the motion of cilia needs to be phase-locked across significant distances, in the form of travelling waves (``metachronal waves''). It is still not known how this long-range coordination emerges from local rules, as there is no central node regulating the coordination among cilia. In the first part of this thesis I will focus on studying the coordination in carpets of cilia with a top-down approach, by proposing, implementing, and applying a new method of analysing microscope videos of ciliated epithelia. Chapter 1 provides the reader with an introduction on motile cilia and flagella, treating their structure and motion and reporting the different open questions currently tackled by the scientific community, with particular interest in the coordination mechanisms of cilia and the mucociliary clearance apparatus. Chapter 2 introduces Differential Dynamic Microscopy (DDM), a powerful and versatile image analysis tool that bridges the gap between spectroscopy and microscopy by allowing to perform scattering experiments on a microscope. The most interesting aspects of DDM for this work are that it can be applied to microscope videos where it is not possible to resolve individual objects in the field of view, and it requires no user input. These two characteristics make DDM a perfect candidate for analysing several hundred microscope videos of weakly scattering filaments such as cilia. In Chapter 3 I will present how it is possible to employ DDM to extract a wealth of often-overlooked information from videos of ciliated epithelia: DDM can successfully probe the ciliary beat frequency (CBF) in a sample, measure the direction of beating of the cilia, and detect metachronal waves and read their direction and wavelength. In vitro ciliated epithelia however often do not show perfect coordination or alignment among cilia. For the analysis of these samples, where the metachronal coordination might not be evident, we developed a new approach, called multiscale DDM (multiDDM), to measure a coordination length scale, a characteristic length of the system over which the coordination between cilia is lost. The new technique of multiDDM is employed in Chapter 4 to study how the coordination among cilia changes as a response to changes in the rheology of the mucous layer. In particular, we show that cilia beating under a thick, gel-like mucus layer show a larger coordination length scale, as if the mucus acted as an elastic raft effectively coupling cilia over long distances. This is corroborated by the coordination length scale being larger in samples from patients affected by Cystic Fibrosis than in healthy samples, and much shorter when the mucus layer is washed and cilia therefore beat in a near-Newtonian fluid. We then show how it is possible to employ multiDDM to measure the effectiveness of drugs in recovering, in CF samples, a coordination length scale typical of a healthy phenotype. In the second part I will focus instead on the single cilium scale, showing how we can attempt to link the beating pattern of cilia to numerical simulations studying synchronisation in a model system. In particular in Chapter 5 I will describe our approach to quantitatively describe the beating pattern of single cilia obtained from human airway cells of either healthy individuals or patients affected by Primary Ciliary Dyskinesia. Our description of the beating pattern, and the selection of a few meaningful, summary parameters, are then shown to be accurate enough to discriminate between different mutations within Primary Ciliary Dyskinesia. In Chapter 6 instead I report the results obtained by coarse-graining the ciliary beat pattern into a model system consisting of two ``rotors''. The rotors are simulated colloidal particles driven along closed trajectories while leaving their phase free. In my study, the trajectories followed by the rotors are analytical fits of experimental trajectories of the centre of drag of real cilia. The rotors, that are coupled only via hydrodynamics interactions, are seen to phase-lock, and the shape of the trajectory they are driven along is seen to influence the steady state of the system.

Recent years have seen the emergence of Mycobacterium abscessus, a highly drug-resistant non-tuberculous mycobacterium, which causes life-threatening infections in people with chronic lung conditions like cystic fibrosis. This opportunistic pathogen is refractory to treatment with standard anti-tuberculosis drugs and most currently available antibiotics, often resulting in accelerated lung function decline. This project aims to use a structure-guided fragment-based drug discovery approach to develop effective drugs to treat M. abscessus infections. During the early stage of the project, three bacterial targets were identified, based on analysis of the structural proteome of M. abscessus and prior knowledge of M. tuberculosis drug targets, followed by gene knockout studies to determine target essentiality for bacterial survival. The three targets from M. abscessus were then cloned, expressed and purified and suitable crystallization conditions were identified leading to the determination of high resolution structures. Further, a large number of starting fragments that hit the three target proteins were determined, using a combination of biophysical screening methods and by defining crystal structures of the complexes. For target 3, PPAT (Phosphopantethiene adenylyl transferase), a chemical linking of two fragments followed by iterative fragment elaboration was carried out to obtain two compounds with low micromolar affinities in vitro. However, these compounds afforded only low inhibitory activity on M. abscessus whole cell. All starting fragments of target 2, PurC (SAICAR synthase), occupied the ATP indole pocket. Efforts were then made to identify further fragment hits by screening diverse libraries. Sub-structure searches of these initial fragment hits and virtual screening helped to identify potential analogues amenable to further medicinal chemistry intervention. While fragment hits of target 1, TrmD (tRNA-(N1G37) methyl transferase), were prioritized, whereby two chemical series were developed using fragment growing and merging approaches. Iterative fragment elaboration cycle, aided by crystallography, biophysical and biochemical assays led to the development of several potential lead candidates having low nano-molar range of in vitro affinities. Two such compounds afforded moderate inhibition of M. abscessus and stronger inhibition of M. tuberculosis and S. aureus cultures. Further chemical modifications of these compounds as well as others are now being done, to optimize cellular and in vivo activities, to be ultimately presented as early stage clinical candidates.

Burkholderia cenocepacia, an opportunistic Gram-negative bacterium that causes serious respiratory infections in patients with cystic fibrosis, produces the extracellular bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures a range of antibiotics outside bacterial cells, providing a global extracellular mechanism of antimicrobial resistance. In this thesis, I report that watersoluble and liposoluble forms of vitamin E inhibit antibiotic binding by BcnA. In vitro, both vitamin E forms bind strongly to BcnA and contribute to reduce the MICs of antibiotics. Expression of BcnA was required for the adjuvant effect of vitamin E. In vivo, vitamin E and norfloxacin treatment significantly increased Galleria mellonella larva survival upon infection in a BcnAdependent manner. Together, my findings suggest that vitamin E can be used to increase killing by bactericidal antibiotics through interference with lipocalin binding. The transcription of bcnA gene increases upon antibiotic stress, which stimulates the oxidative stress and membrane lipid peroxidation. BcnA lipocalin, and associated proteins BcoA cytochrome and BarA reductase are involved in membrane peroxidation stress response to antibiotics and other forms of oxidative stress. I report that bcnA, bcoA and barA deletion mutants display enhanced membrane lipid peroxidation and fail to survive under conditions that stimulate peroxidative stress (e.g. Tellurite treatment, cold stress and salt stress). Peroxidation also affects the functionality of the bacterial outer membrane. Absence of BcnA compromises the permeability of the outer membrane, resulting in the release of extracellular DNA leading to a depletion aggregation phenotype and cell death. Together, my findings uncover a novel peroxidation quenching mechanism based on B. cenocepacia BcnA, BcoA and BarA, which protects the bacterial cell envelope against lipid peroxidation stimulated by antibiotic or metal stress, in the presence of oxygen.

The polymeric mucins, MUC5B and MUC5AC provide the structural and functional backbone of the respiratory mucus barrier, conferring both viscoelastic and antimicrobial properties onto these complex secretions. Within the mucus barrier, polymeric mucins are believed to be highly organised, arranged in a network-like formation. Structural details regarding how mucins are packaged, secreted and expand to form a functional barrier remain elusive. In addition, despite recent investigations into mucin secondary structure, it is unclear as to how the heavy glycosylation found within mucin domains can influence polymer conformation. Through the use of electron microscopy techniques in combination with biophysical analyses we performed an in depth structural analysis on purified MUC5B and MUC5AC, we then utilised these same techniques to investigate mucin packaging and expansion in response to changes in calcium concentration and pH levels. Finally through structural investigations into the native organisation of the salivary mucus barrier we aimed to investigate the hierarchical mucin topology present within mucosal systems, thus enabling us to draw conclusions about how this organisation may be compromised in disease states such as Cystic Fibrosis (CF). Through transmission electron microscopy (TEM) combined with single particle analysis, we provide evidence to suggest that MUC5B contains bead-like structures, which repeat along the polymer axis and suggest that MUC5B may consist of repeating motifs derived from distinct glycosylation patterns, rather than the disorganised random coil conformation previously assumed. Moreover, through TEM and rate zonal centrifugation, we demonstrate that respiratory mucins form highly entangled linear polymers, the structures of which are sensitive to calcium concentration and fluctuations in pH. In the presence of Ca2+ at pH 5.0, MUC5B adopted a compact conformation which was lost upon removal of calcium, via EGTA, and through increasing the pH to 7.4. We further suggest that the expansion of mucins following secretion occurs via two-step process, with an initial rapid expansion followed by a slower protease-independent maturation process, which is driven by distinct changes to the mucin interactome. Through isolation of mucins derived from CF sputum we have assessed the structural and biophysical changes that occur to the mucin network during CF progression, we suggest that the CF mucin network is severely degraded, with mucin glycopeptides predominating. Furthermore, we suggest that poly (acetyl, arginyl) glucosamine (PAAG) may represent an attractive treatment for CF lung disease, here we highlight its ability to directly bind MUC5B and induce its linearisation, resulting in a less viscous mucin network. Here we provide novel insights into the structural organisation of MUC5B and MUC5AC on both intra- and intermolecular levels, and shed light onto the processes required for mucin packaging and expansion, which have allowed us to understand the mechanisms behind aberrant mucus transport in CF.

Background: Lung clearance index (LCI), obtained by multiple breath washout testing (MBW), is a sensitive measure of lung disease in infants. It has been identified as a particularly suitable endpoint for clinical trials in cystic fibrosis (CF), but has potential applications in many other conditions. However, MBW in infants presents a number of technical challenges. Conventional MBW is based on simultaneous measurement of flow and gas. These two signals are then aligned and combined to derive expired gas volumes and measures of ventilation inhomogeneity: this process becomes increasingly vulnerable to errors in gas signal alignment at rapid respiratory rates. At present, no existing system for infant MBW meets all the criteria set out in international guidelines, and there is no simple method of assessing lung function outside research laboratories in this population. This thesis describes an alternative method of performing MBW in infants. In this method, expired gas is collected and analysed to derive functional residual capacity (FRC) and LCI. There is no need to simultaneously measure flow, and therefore no need for the complicated step of integrating flow and gas signals. Dead space is also significantly reduced by removing the flowmeter. Methods: In the first phase of testing, an existing lung model was modified to generate realistic infant breathing parameters with high accuracy. The prototype system was modified to improve accuracy and subsequently tested at FRC of 100-250mls with respiratory rates of 20-60min-1. In the second phase, testing proceeded to an in vivo pilot study of the novel method in children with cystic fibrosis and healthy controls. Practical applicability of the system was determined by the number of successful duplicate tests, and within-subject repeatability. Comparison was made with LCI measurements obtained using a respiratory mass spectrometer, currently considered the gold standard for infant LCI. Results: In a total of 103 tests performed in the lung model, overall mean error (standard deviation) of FRC measurement was -1.0(3.3)%, with 90% of tests falling within +/-5%. 13 patients were excluded from the clinical study due to being unsedated or inadequately sedated and therefore failing to tolerate the test. A total of 25 patients (7 children with CF, 18 healthy control children) were deemed to be adequately sedated at the start of the test, of these 20 patients (7 with CF) successfully underwent duplicate testing (80% success rate). Mean FRC for healthy controls was 19.5ml/kg, and mean LCI 6.45. For children with CF, mean FRC was 21.8ml/kg and mean LCI 6.98. Mean within-subject coefficient of variation for FRC was 7.18% and for LCI 5.94%. Of 4 infants assessed with both the novel method and the respiratory mass spectrometer, there was good correlation in FRC measurement (mean difference -8.1%). Comparison of LCI with the mass spectrometer was affected by technical difficulties with the test; in those patients who underwent technically adequate tests with both methods, mean difference in LCI between the two methods was 1.65%. Discussion: FRC measurement using the novel method has superior accuracy in vitro than previously described systems. Data from the pilot study suggest that this is a feasible and reproducible method of performing LCI in infants and young children, as long as they are adequately sedated. Results in both children with CF and controls fall within the expected range, and well within accuracy limits set by international guidelines. However, the system and testing protocol could be further improved to reduce the number of technically inadequate tests having to be excluded. This could provide a more accessible alternative to previously described systems for infant MBW.

Background: Adults with cystic fibrosis (CF) experience, on average, two to three pulmonary exacerbations per year. Exacerbations are associated with a decline in lung function and increased mortality, result in high symptom and treatment burden and present self-management challenges for patients. Little is known about the emotional impact of exacerbations and how it relates to self-management. There are no patient-reported outcomes measures (PROMs) currently available to assess symptom distress and self-management during a pulmonary exacerbation in CF, limiting the ability to evaluate interventions in support of exacerbation-related self-management. Aim and objectives: The aim was to develop and conduct preliminary validity testing of a PROM for measuring a concept of relevance for symptom management during pulmonary exacerbation in adult CF patients, guided by the Food and Drug Administration (FDA) framework. Objectives were to: i) critically review PROMs related to symptom management in CF and appraise their suitability of use during exacerbations (Phase 1); ii) identify and describe current gaps in knowledge regarding CF patients experience of a pulmonary exacerbation (Phase 2); iii) explore patients’ experience (Phase 3); iv) develop a conceptual framework for symptom management in exacerbations (Phase 4); and v) develop a draft item list and assess content validity and clarity (Phase 5). Methods: A multistage study was undertaken, comprising qualitative methods (Phase 2), and two convergent mixed-method studies (Phases 3 and 5) Results: Phase 1: Five PROMs were included in the review; none measured the concept of symptom distress or self-management during exacerbation. Phase 2: The thematic synthesis of qualitative studies identified a broad spectrum of physical and emotional symptoms experienced by patients but data relating to experiences during exacerbation were limited. Phase 3: The mixed-method study (n=18) revealed that pulmonary exacerbations are characterized by an increase in emotional distress, which was linked to the meaning associated with the exacerbation. Experiencing a pulmonary exacerbation meant being ‘thrust out of normality’ and indicated a period of threat and life domination by CF. Symptom and treatment burden consumed energy and placed restrictions on physical activity and daily life roles. Phase 4: Based on a review of relevant theoretical frameworks, a conceptual model describing illness-related emotional distress was developed. Phase 5: A list of 27 items were extracted from patient interviews (Phase 3) which were underpinned by the conceptual framework (Phase 3 & 4). Clinical experts (n=12) and patients (n=8) rated the concept ‘emotional distress’ as relevant and comprehensively covered by the item list, and agreed that the draft PROM layout and wording were easily understood. Following this consultation, three further items were added (giving a total of 30 items) and yielded a draft PROM which is ready for refinement and psychometric testing in future quantitative studies. Conclusion: During exacerbation, illness-related emotional distress increases and is multidimensional, acting as a driver for self-management. The preliminary item list with confirmed face validity provides a basis for guiding and evaluating interventions.

The bronchiectatic lung is a diseased state in which the airways are chronically damaged and dilated. This state is found in the clinical entities of cystic fibrosis and non-cystic fibrosis bronchiectasis. These are two highly relevant chronic suppurative lung diseases in which an understanding of the microbiology of these patients is considered key to appropriate management. This has traditionally been via the use of traditional culture techniques. However, with the development of molecular methodologies, the previously perceived wisdom is being challenged. In both cystic fibrosis and non-cystic fibrosis bronchiectasis, Pseudomonas aeruginosa is considered the most significant pathogen. In CF there has been considerable concern surrounding the risk of transmission of Pseudomonas aeruginosa between patients on the basis of a significant quantity of research into this matter. In contrast, there has been very little research performed into the equivalent risk in non-cystic fibrosis bronchiectasis. In this thesis we describe an extensive single-centre epidemiological review of Pseudomonas aeruginosa spanning both these diseases. Via this we have shown evidence of cross-infection within a non-cystic fibrosis bronchiectasis cohort. This epidemiological review has included multiple genotyping methods including multilocus sequence typing and whole genome sequencing, As an extension of the epidemiological review, we have performed an in silico prediction of hypermutator status from the whole genome sequencing data to provide greater understanding of the likelihood of cross-infection, and have also demonstrated a culture-independent adaption of multilocus sequence typing for potential screening for cross-infection. In addition to Pseudomonas aeruginosa, we have also looked at the wider bacterial community in the lungs of patients with these two conditions via culture-independent techniques. We have shown that whilst Pseudomonas aeruginosa is often an important component, these are clearly complex communities. We have primarily investigated the cohort with non-cystic fibrosis bronchiectasis, but we have demonstrated associations between clinically-relevant markers and complexity of the bacterial communities within the lungs of both these cohorts of patients. Whilst we have used the gold-standard technique of 16S rRNA sequencing, we have also shown the validity of a simple and potentially more feasible profiling technique for standard clinical care. In summary, through the application of culture-dependent and independent molecular techniques, this research has shed light on the epidemiology of Pseudomonas aeruginosa within our respiratory cohorts, and the complexity and clinical relevance of the wider microbial communities within these patients. Such studies are essential if we are to advance our understanding of the bronchiectatic lung and optimise strategies for patient management.

Aspergillus fumigatus is increasingly recognised as a cystic fibrosis (CF) pathogen with reported infection rates up to 50% and associated with increased hospitalisations and lung deterioration. Research investigating immune responses of CF phagocytes against A. fumigatus has been scarce and the role of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in anti-Aspergillus immune responses is not known. The studies in this thesis aimed to explore innate immune responses by CF phagocytes against A. fumigatus. Peripheral blood mononuclear cells (PBMC), monocytes (MNC) and polymorphonuclear cells (PMN) were isolated from blood samples of CF patients with at least one copy of the F508del mutation. CF phagocytes efficiently cleared A. fumigatus similar to healthy controls. Microtubuleassociated proteins 1A/1B light chain 3B (LC3) expression was found to be attenuated in CF PMN and MNC, indicating a disbalance in the autophagy or LC3-associated phagocytosis pathway. Regarding inflammatory responses, it was found that upon phagocytosis a resting A. fumigatus conidia, CF phagocytes produce up to 4-fold more reactive oxygen species (ROS) compared to controls. The excessive ROS was then shown not to be necessary for adequate killing, suggesting the increased ROS to be redundant in the antifungal response. Patient metrics obtained from the clinic showed that the excessive ROS production correlated to exacerbations and lung function. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that CF PMN only express 20-25% of the 4 haemoglobin subunits compared to healthy controls. Combining the LC-MS data suggests that CF PMN are under hypoxic stress. In conclusion, the effective clearance of A. fumigatus by CF phagocytes comes at the cost of an excessive respiratory burst which correlates to disease severity. Our data indicate that CF PMN are under hypoxic stress while circulating in the blood stream, which is likely to contribute to the hyperinflammatory phenotype observed upon interaction with A. fumigatus.

The Multiple Breath Washout (MBW) test is increasingly being recognised as a sensitive method of detecting early small airways lung disease. Indices of MBW include lung clearance index (LCI), Scond and Sacin. Factors that affect MBW variability have not been fully established. This thesis presents five studies which examine MBW repeatability in children with and without cystic fibrosis (CF) or asthma. MBW was performed using 0.2% sulphur hexafluoride and the modified Innocor (Innovision). Testing was performed at the Clinical Research Facility of the Royal Hospital for Sick Children in Edinburgh. (1) MBW and spirometry were performed in children with and without CF (n=20 in each group), initially while sitting and then 30 minutes after assuming a supine posture. LCI was found to significantly rise on lying supine in healthy children (p < 0.01) and children with CF (p=0.03). (2) Thirty two children with CF performed MBW and spirometry on four study visits, results were correlated with findings from high resolution chest computed tomography scans taken on the first visit. LCI showed the strongest correlation with extent and severity of bronchiectasis (r=0.66, p < 0.01 and r=0.69, p < 0.01 respectively). Variability of LCI was similar to FEV1 over the 4 visits. (3) MBW and spirometry of 66 healthy children were compared to 63 children with stable asthma; lung function of asthmatic children was related to symptoms and medication use. LCI was higher in the asthmatic group (6.7 vs 6.3, p < 0.01); within the asthmatic group LCI was significantly higher if asthma was less well controlled (p=0.02). (4) Children with and without asthma (n=21 in each group) performed MBW and spirometry before and after exercise and again after salbutamol, symptom data was collected from asthmatic children. Baseline LCI was abnormal in the asthmatic group who had severe exercise induced bronchospasm during testing. (5) Asthmatic children admitted to hospital due to exacerbation performed MBW and spirometry. Mean (SD) LCI was abnormally high at 8.5 (1.7) in the nine patients recruited and returned to normal 6.7 (0.6) in three patients who attended follow up. I have presented evidence that LCI is repeatable and sensitive to early disease in CF and asthma. I have described for the first time the effects of exercise and exacerbation on MBW indices in asthmatic children. MBW is potentially a very useful tool in paediatrics; standardisation of testing and equipment may enable clinical use.

Introduction: Puberty is a crucial period for rapid changes in bone mineral, size, geometry, and microarchitecture. The mechanostat theory postulates that increased mechanical loading will affect bone phenotype and strength during development and in later life. Individuals with cystic fibrosis (CF) have an increased risk of developing osteoporosis and fragility fractures in young adulthood, which may be caused by poor growth. The aim was to investigate whether sex and disease status modified the relationship between: 1) puberty and bone, and 2) muscle and bone. This would contribute to the understanding of how sex (males vs. females) and disease group (CF vs.controls) alters the relationship between bone and muscle in children and adolescents as they transition through puberty and who, on a population level, differ in the prevalence of osteoporosis and risk of fracture in later life. Methods and Analyses: This observational study used novel imaging and muscle assessment techniques to measure bone and muscle parameters in White Caucasian children and adolescents, aged 8 to 16 years, living in the UK, with children with CF (n=65) and controls (n=151). Anthropometry and pubertal status were assessed. Dual energy X-ray absorptiometry, peripheral quantitative computed tomography (pQCT), high-resolution pQCT, and jumping mechanography were used to measure bone and muscle outcomes. ANCOVA with Scheffé post hoc and multiple linear regression tests were performed. Data were adjusted according to the research aims and included covariates; sex, disease group, pubertal stage, age, quadratic age, height, weight, maximum force (Fmax), and maximum power (Pmax). Data are presented as beta-coefficient (%) and p-value, with the significance level set to p < 0.05. Results: In height adjusted analyses, among healthy participants, females had smaller bones and lower bone density compared to males. With pubertal maturation, females had lower apparent gains in the distal and proximal total area (Tt.Ar and CSA), distal cortical porosity (Ct.Po) and proximal bone strength (SSI) but higher apparent gains in distal and proximal cortical bone density(Ct.BMD, Ct.TMD, vBMD). Females had consistently lower distal total area (total CSA) and density (total vBMD), distal trabecular density(BV/TV) and number(Tb.N), and proximal cortical area(CSA) compared to males, across all stages of puberty. With increasing muscle force (Fmax), females had higher apparent gains in total body less head bone mineral (TBLH BMC) and bone area(BA), distal total and trabecular density (total and trab vBMD) compared to males. In contrast, with increasing muscle power (Pmax), females had higher apparent gains in distal total and cortical densities (D100, Ct.BMD and Ct.TMD), and distal trabecular thickness (Tb.Th), and proximal cortical density (cortical vBMD) but lower apparent gains in distal cortical porosity (Ct.Po) and trabecular number (Tb.N) compared to males. In height adjusted analyses, participants with CF had smaller bones and lower bone density compared to controls. With increasing pubertal maturation, participants with CF had lower apparent gains in total body less head bone mineral and bone area, and in distal trabecular density, cortical porosity, and trabecular thickness compared to controls. Participants with CF had consistently lower distal total and cortical area, distal total and trabecular densities and proximal bone strength compared to controls, across all stages of puberty. With increasing muscle force, participants with CF had lower apparent gains in total body less head bone mineral and bone area, distal total density, trabecular density, and trabecular number. In contrast, with increasing muscle power (Pmax), participants with CF had higher apparent gains in distal trabecular density (BV/TV) and trabecular number (Tb.N) compared to controls. Conclusion: These findings suggests that sex and disease status do modify the relationships between puberty and bone, and between muscle function and bone. Skeletal adaptation to muscle differs between sexes and in populations with chronic disease, which may explain sex and disease group differences in risks of osteoporosis and fracture. Bone adaptation to muscle in children with CF is altered, which may lead to narrow, under-mineralised bones, with lower bone strength in later life. Understanding better impairments in muscle functions may provide targets for intervention to improve skeletal health in later life.

Within the Cystic Fibrosis (CF) literature it is acknowledged that parents play a significant role in supporting children with treatment procedures. Furthermore, a number of parenting variables have been associated with treatment adherence within the paediatric CF population. Interventions that target parenting practices may therefore have the potential to improve CF treatment adherence. Paper one presents a systematic literature review of parenting interventions targeting treatment adherence in children and adolescents with CF. The majority of studies focussed on dietary adherence and overall findings from these studies suggested that combined behavioural and nutritional counselling parenting interventions led to improvements in calorie intake and positive parenting practices. Interventions specifically targeting exercise adherence and interventions targeting multiple aspects of the CF treatment regimen were also shown to improve treatment adherence. The review highlighted that interventions targeting some of the more laborious treatments (i.e. chest physiotherapy) were lacking, as were interventions specifically tailored to the needs of adolescents and their parents. Over recent years, CF life expectancy has increased substantially due to medical advances. As a result, more children are living into adulthood, therefore needing to adhere to an increasingly complex treatment regime in order to manage increasing symptoms. Adolescence is a particularly challenging time for treatment adherence as children increase their independence and parents begin to allow the child to manage their own disease management. The study described in Paper 2 aimed to explore the acceptability and feasibility of the Self-Directed Teen Triple P parenting intervention within the adolescent CF population. It also explored whether parent-reported treatment adherence, positive parenting practices, parent wellbeing, and child emotional and behavioural functioning were increased as a result of this intervention. Whilst data from two cases indicated increasing trends in treatment adherence and positive parenting practices following the onset of the parenting intervention, uptake and retention to the intervention was poor. Interviews with parents and CF nurses indicated low acceptability and feasibility of the intervention in its current form and a number of adaptations were reported. The study concludes that researchers need to include parents within the design of tailored parenting interventions within this population in order to increase acceptability. Following this, larger scale studies are required to increase the reliability and rigor of research findings in this area. Paper 3 is a critical reflection and considers both Paper 1 and Paper 2. Within this paper the approaches used, the challenges encountered, and future research are considered.

The purpose of this thesis was to extend our understanding of the assessment and interpretation of aerobic exercise function of paediatric patients with cystic fibrosis (CF). The first investigation sought to establish (1) the validity of traditional criteria to verify maximal oxygen (V ̇O2max) during a maximal cardiopulmonary exercise test (CPET); and (2) the utility of supramaximal verification (Smax) to confirm V ̇O2max. Traditional criteria significantly underreported V ̇O2max, whilst Smax was shown to provide a valid measurement in this patient group. The reproducibility of this CPET protocol, over the short- (48 h) and medium- (4-6 weeks) term, was then established in study two. V ̇O2max was repeatedly determined with no learning effect over 48 h (typical error (TE): ∆150 mL; ∆9.3%) and 4-6 weeks (TE: ∆160 mL; ∆13.3%). Supplementary maximal and submaximal CPET parameters should be incorporated for a comprehensive evaluation of a patient, however they are characterised by greater variability over time. The influence of mild-to-moderate CF on aerobic exercise function and the matching of muscle O2 delivery-to-O2 utilisation during ramp incremental exercise to exhaustion were then examined in study three. Aerobic function was impaired in CF, indicated by very likely reduced fat-free mass normalised V ̇O2max (mean difference, ±90% CI: -7.9 mL∙kg-1∙min-1, ±6.1), very likely lower V ̇O2 gain (-1.44 mL∙min-1∙W-1, ±1.12) and a likely slower V ̇O2 mean response time (MRT) (11 s, ±13). Arterial oxygen saturation was lower in CF, supporting the notion that centrally mediated O2 delivery may be impaired during ramp incremental exercise. Although a faster rate of fractional O2 extraction would be expected in the face of reduced O2 delivery, this was not observed, suggesting additional impairment in O2 extraction and utilisation at the periphery in CF. The fourth study then demonstrated the clinical utility of CPET to assess the response to 12 weeks treatment with Ivacaftor, using a case-based design. Whilst one patient with relatively mild disease demonstrated no meaningful change in V ̇O2max, the second demonstrated a 30% improvement in V ̇O2max, due to increased O2 delivery and extraction. Furthermore, changes in aerobic function were detected earlier than spirometric indices of pulmonary function. This study demonstrated that CPET represents an important and comprehensive clinical assessment tool and its use as an outcome measure in the functional assessment of patients is encouraged. Study five investigated the V ̇O2 kinetics in this patient group. During moderate intensity cycling, the phase II V ̇O2 time constant (τ) (p = 0.84, effect size (ES) = 0.11) and overall MRT (p = 0.52, ES=0.33) were not slower in CF. However, both were slowed during very heavy intensity cycling (p = 0.02, ES = 1.28 and p = 0.01, ES = 1.40, respectively) in CF. Cardiac output and muscle deoxygenation dynamics were unaltered in CF, however, the arterial-venous O2 content difference (C(a-v ̅)O2) was reduced (p=0.03) during VH and ∆C(a-v ̅)O2 correlated with the phase II τ (r= -0.85; p=0.02) and MRT (r = -0.79; p=0.03) in CF. This study showed that impaired oxidative muscle metabolism in this group is exercise intensity-dependent and mechanistically linked to an intrinsic intramuscular impairment, which limits O2 extraction and utilisation. In conclusion, this thesis has provided guidelines for a valid and reproducible CPET protocol for children and adolescents with mild-to-moderate CF, demonstrated the utility of CPET as clinical outcome measure and furthered our understanding of the factors responsible for impaired aerobic exercise function in this patient group.

Introduction: Emerging pathogens (EP) in cystic fibrosis (CF) include organisms that have infected individuals with CF for many years e.g. Burkholderia multivorans and Mycobacterium abscessus and more recently identified potential pathogens in CF e.g. Pneumocystis jirovecii and Pandoraea spp. The clinical implications of infection with these organisms are emerging but much remains unknown. Current evidence suggests that infection with some EP is associated with a worse prognosis. This thesis aimed to investigate the epidemiology, prevalence and clinical impact of EP in adults with CF.Methods: (1) The prevalence of P. jirovecii was determined in adults attending Manchester Adult Cystic Fibrosis Centre (MACFC) who were clinically stable versus those experiencing an acute pulmonary exacerbation (PEx). (2) The prevalence of M. abscessus at MACFC was determined, isolates of M. abscessus were strain typed, and cross infection risk was assessed. The clinical impact of Gram-negative EP was assessed by: (3) assessing their prevalence and determining if any patients attending MACFC harboured identical strains and had opportunities for cross infection to occur, and by (4) following these patients longitudinally and comparing outcome with age, gender and FEV1 matched Pseudomonas aeruginosa infected controls. Results: (1) P. jirovecii was detected via sputum PCR in 10 (4.4%) of 226 samples tested from 111 patients. P. jirovecii was more likely to be detected in samples taken from an acute pulmonary exacerbation compared with samples taken from stable patient visits (7 (9.2%) of 76 exacerbations samples versus 3 (2%) of 150 stable visit samples, p = 0.033). (2) Prevalence of M. abscessus was stable at ≤3.6% from 2010 to 2015. 21 patients (91.3%) with a positive culture for M. abscessus since 2010 were infected with M. abscessus subsp abscessus. 2 clusters of 7 and 6 patients harboured strains with identical variable number tandem repeat profiles and some of these patients had opportunities for cross infection to occur. 28.6% of patients developed M. abscessus pulmonary disease, 38.1% were persistently culture positive with no related pulmonary disease, and 33.3% spontaneously cleared M. abscessus from their sputum. (3) Prevalence of Gram-negative EP ranged from 1.9% (Ralstonia spp.) to 6.2% (B. multivorans). Small numbers of patients shared strains of B. multivorans; Stenotrophomonas S. maltophilia and Achromobacter; Ralstonia and Pandoraea species. Epidemiological connections consistent with possible cross infection were found in patients infected with Pandoraea and Ralstonia species. (4) Patients with B. multivorans; S. maltophilia; Ralstonia spp. and Pandoraea spp. had higher antibiotic requirements than P. aeruginosa infected matched controls. B. multivorans; Achromobacter spp.; Ralstonia spp. and Pandoraea spp patients had median FEV1 (% predicted) values ≥10% (absolute) lower than the overall median FEV1.Conclusion: Prevalence of all EP investigated at MACFC was low. P. jirovecii was approximately 5 times more likely to be detected in patients with acute PEx compared with stable patients suggesting it may be a cause of PEx. Results suggest that some patients attending MACFC may have acquired infection with M. abscessus subsp abscessus, Pandoraea spp. or Ralstonia spp. through cross infection. Patient numbers are too small to establish this with certainty and a common environmental source is possible. Gram-negative EP other than Achromobacter spp. were associated with higher acute antibiotic requirements than P. aeruginosa matched controls suggesting these EP are associated with an increased risk of PEx. The fact that many Gram-negative EP were associated with lower median lung function may indicate that these EP cause accelerated lung function decline or that patients with more advanced disease are at most risk of acquiring EP.

Viral respiratory infections (VRI) are common in children with cystic fibrosis (CF) and are associated with significant clinical deterioration. Little previous research has been conducted on VRI in adults with CF. This thesis describes a prospective study to determine the epidemiology and clinical impact of VRI among 100 adults with CF.The incidence of identifiable VRI was 1.66 cases/patient-year. Rhinovirus accounted for 72.5% of viruses. Identifiable VRI was associated with increased risk of pulmonary exacerbation, increased respiratory symptoms and higher C-reactive protein levels. Changes in the climate and seasons affected the incidence of identifiable VRI. Rhinovirus was most common in autumn and other viruses predominated during winter. Warmer ambient temperatures were associated with increased risk of rhinovirus infection while other viruses were more common in colder temperatures. Genetic sequencing of a subset of 42 rhinoviruses identified during the study showed that rhinovirus A accounted for 69% of cases and was associated with more severe respiratory symptoms and higher C-reactive protein levels than rhinovirus B.The impact of identifiable VRI on changes to bacterial communities within the lungs of patients with CF was investigated. Ribosomal intergenic spacer analysis (RISA) was developed as a tool to profile the bacterial diversity of CF sputum and was compared with standard culture and 16S rRNA gene pyrosequencing. No consistent effect of identifiable VRI on the microbial diversity of CF sputum was detected with any of these methods in longitudinal analysis of a subset of 18 patients.

Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.

Cystic Fibrosis (CF) is a degenerative disorder that is often associated with chronic lung disease. CF is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel, which lead to defective chloride and sodium ion movement across epithelia. Subsequent dehydration of the airway surface liquid (ASL) on airway epithelia, is associated with poor mucociliary clearance and chronic lung infection. The monogenic nature of CF, along with the accessibility of the lung, makes the disease amenable to gene replacement therapy. Gene therapy clinical trials have focused on replacing the mutated CFTR with a functional copy, which has led to improved chloride transport, but has shown no significant effects on sodium transport. An alternative strategy for CF gene therapy therefore, could be to reduce the expression of the epithelial sodium channel (ENaC) in the lung, using RNA interference (RNAi), combined with CFTR delivery. Developing a dual-function gene transfer vector could potentially restore chloride and sodium levels in the ASL and help alleviate CF lung disease. The aim of this thesis was to develop a recombinant lentivirus delivery system capable of simultaneously delivering CFTR expression and knocking down ENaC expression in the airways. A modular HIV vector genome plasmid was developed to allow simple insertion of various promoter elements, transgenes and knockdown sequences, for subsequent virus production. Insertion of the CFTR transgene and a short-hairpin RNA (shRNA) sequence targeting the ENaC alpha subunit (ENaCα) resulted in significant knockdown of human ENaCα and simultaneous expression of CFTR in A549 (human lung carcinoma) cell culture. Replacement of the ENaCα shRNA with an shRNA targeting the transcription factor BACH1 resulted in target gene knockdown and concomitant HMOX1 up-regulation, confirming specific knockdown effects, and demonstrating that the dual-function rLV vector could mediate target gene knockdown irrespective of the target. Attempts were made to knock down BACH1 in primary cultures of human bronchial epithelial cells grown at the air-liquid interface (ALI), but improved transduction efficiencies from the apical surface will be required to generate successful knockdown in this experimental model. These studies provide proof-of-principle for the utility of this versatile dual-function prototype virus. The dual function vector not only has the potential for treatment of CF lung disease, but could be readily altered to target other lung diseases where combinations of prolonged target gene knockdown and gene expression/up-regulation could collectively provide an appropriate therapy. In conclusion, the focus on the rational design of gene transfer vectors for specific therapeutic effects will aid the development and translation of gene therapy approaches to human studies.

As depicted in Chapter I, 2-oxoglutarate- (2OG) dependent oxygenases are ubiquitous in living systems and display a wide range of cellular functions, spanning metabolism, transcription, and translation. Although functionally diverse, the 2OG oxygenases share a high degree of structural similarities between their catalytic sites. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity presents a rather challenging task for the development of selective small molecules for functional studies in vivo. The non-selective metal chelator 8-hydroxyquinoline (8HQ) was used as a template for the generation of tool compound I for the KDM4 subfamily of histone demethylases via application of the Betti reaction. Structural analogue II was used as the corresponding negative control (Figure A). These compounds were characterised in vitro against a range of 2OG oxygenases and subsequently used for studies in cells. I displays selectivity for KDM4 and increases the level of the H3K9me3 histone mark in cells. It has an effect on the post-translational modification pattern of histone H3, but not other histones, and reduces the viability of lung cancer cells, but not normal lung cells, derived from the same patient. I also stabilises hypoxia-inducable factor HIF in cells via a mechanism which seems to be independent from prolyl hydroxylase inhibition. This work is described in Chapters II and III. The chemical biology research in epigenetics is complemented by qualitative analysis conducted in the social sciences at Said Business School. With a global view on how innovation occurs and may actively be fostered, Chapter IV focuses on the potential of epigenetics in drug discovery and how this process may actively be promoted within the framework of open innovation. Areas of focus include considerations of incremental and disruptive technology; how to claim, demarcate, and control the market; how knowledge brokering occurs; and insights about process, management, organisation, and culture of open innovation. In contrast to the open-skies approach adopted for the development of a tool compound in Chapters II and III, a focused-library approach was taken for the generation of a tool compound for the OGFOD1 ribosomal prolyl hydroxylase. The development of a suitable in vitro activity assay for OGFOD1 in Chapter V enabled the development of lead compound III in Chapter VI. III is selective for OGFOD1 against the structurally closely related prolyl hydroxylase PHD2.

Over the past several decades, the rate of pulmonary nontuberculous my- cobacterial (PNTM) disease has been increasing. PNTM patients gener- ally consist of lean and tall women presenting with symptoms in the sixth decade of life. They have a de nitive morphophenotype, but no consistent immunological abnormalities despite extensive investigation. I hypothesized that respiratory epithelial dysfunction might play a critical role in PNTM disease predisposition because diseases with defects of mucociliary transport have high rates of PNTM disease that increase with age, suggesting a direct connection between airway epithelial function and PNTM disease. I found that PNTM patients have a distinct respiratory epithelial phenotype ex vivo and decreased nasal nitric oxide levels in vivo. The PNTM ex vivo phenotype consists of an abnormally low resting ciliary beat frequency (CBF) and abnormal CBF response to toll-like receptor (TLR) agonists. The depressed baseline CBF response in PNTM patient cells can be normalized ex vivo by augmenting the nitric oxide-cyclic guanosine monophosphate pathway without appreciable e ect on CBF in healthy controls. In healthy controls, bacterial TLR agonists increase CBF and viral TLR agonists decrease CBF. In PNTM patients these responses are impaired and are not normalized with the normalization of the resting CBF rate. Inhibitor-induced disruption of signalling pathways associated with CBF regulation demonstrated that the majority of the CBF response to TLR agonists involves the PI-3K pathway and PKC. Inhibition of the PI-3K pathway (PI-3K , Akt1, and PDK1) closely mimicked the ex vivo phenotype seen in PNTM patient respiratory epithelia. These data identify a novel aspect of PNTM disease with in vivo and ex vivo correlates that suggest that PNTM infection is associated with abnormal function at both the CBF and TLR response levels. This phenotype is novel, reproducible, and provide a foundation with which to determine the genetic basis of PNTM infection.

The incidence of CF related diabetes is on the rise as patient life expectancy continues to improve. Sugars elevated in diabetics include glucose, fructose, and mannose. These sugars, in addition to mannitol (recently approved as an inhaled osmolyte) are the basis for this study, aimed at assessing the impact these clinically relevant sugars have on virulence in Burkholderia multivorans. B. multivorans is a member of the Burkholderia cepacia complex (Bcc), and is the most frequent cause of Bcc infection in CF patients. Using an exopolysaccharide-deficient knockout in macrophage and Galleria mellonella infection models, biofilm formation, and adhesion assays, this study has identified exopolysaccharide-dependent and -independent phenotypes. Sequencing of B. multivorans C1576, a CF outbreak isolate, identified three putative adhesins in clinical isolate C1576 but not present in the sequenced environmental strain ATCC17616. Mannitol promoted adhesion and enhanced expression of these adhesins. This study characterised these adhesins and assessed the distribution within other clinical and environmental isolates of B. multivorans and the Bcc. Additionally, transcriptomic profiling of B. multivorans assessed the sugar response and EPS regulation during growth on clinically relevant sugars. Where possible, links were made between phenotypic studies and transcriptome data. B. multivorans EPS derived from fructose and mannitol was subjected to composition analysis using mass spectrometry, and assessed for biological activity. Still relevant to CF related diabetes, the ability of some members of the Bcc to bind insulin was assessed. Results indicated that a minority of strains bound insulin. Furthermore, by using flow cytometry cell sorting and fluorescence microscopy, results also showed only a small number of cells within a given population that bound insulin. In all, this study has added to the knowledge base of B. multivorans but more work is needed to fully understand virulence strategies exploited by this CF pathogen.

Background and aims: Cystic Fibrosis (CF) is the most common genetic, life threatening disorder in the UK (Cystic Fibrosis Trust, 2010). Given the trajectory of the illness, adolescence may be a particularly challenging period, during which young people become more aware of differences from peers, and are faced with the task of balancing increasing illness demands with the drive to aspire to developmental goals. Nevertheless, little research specifically explores how young people with CF reconcile their illness experiences with the emerging sense of self. In an attempt to address this gap in the literature, this study sought to hear the narratives of young people with CF with reference to the local and broader contextual factors influencing their construction, with the aim to further understanding, inform clinical practice and improve support for young people with CF. Methodology: A qualitative approach was employed. A purposive sample of six participants diagnosed with CF and aged between 12 and 16 years was recruited. Participants were asked to take photographs of their experiences of life as a young person with CF which were used alongside a semi-structured topic guide in individual interviews to explore the young person’s narratives. The interviews were audio-recorded, transcribed, and analysed using a narrative approach to explore both what was said and how it was told.Analysis and Findings: The researcher’s global impressions of each person’s narratives, along with details of the local context of the interview were presented. Following this, similarities and differences across the narratives were considered with particular attention to how the main storylines were interwoven with participants’ emotional experiences, the identity work taking place through the narrative, and the broader narratives available to them. It emerged that (i) CF was perceived as part of participants’ normality which they had grown accustomed to over time, (ii) participants drew on cultural narratives to position themselves as normal teenagers, to maintain a positive sense of self, though also leading them to minimise difficulties and distress, and (iii) participants continued to position themselves within the norm as they talked of their futures, describing similar hopes to their peers, and again played down concerns about how CF might impact on their futures. These findings are discussed with reference to the clinical implications, strengths, and limitations of the methodology, and directions for future research.