Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

Most cystic fibrosis (CF) sufferers will die before reaching their mid thirties as a result of life long bacterial lung infection. Pseudomonas aeruginosa is the most important respiratory pathogen in CF patients; factors implicated in its pathogenesis include alginate over production, pyocyanin, and extracellular proteases. It is also capable of synthesising hydrogen cyanide, but the role of this potent inhibitor of cellular respiration has not yet been assessed in CF infection. Cyanide concentration was measured in sputum from CF and non-CF bronchiectasis patients with and without P. aeruginosa lung infection using a cyanide ion sensing electrode. Cyanide was detected in sputum from 19/25 patients with current P. aeruginosa infection, whereas it was not detected in any of the 10 patients without this organism (p<O.01). Maximum levels were 130IJM (mean Ç'ñSE: 72 Ç'ñ 6.6 IJM). Lung function data w~s examined for 21 P. aeruginosa-infected CF patients; the group with measurable sputum cyanide (n=11) had significantly poorer lung function than those without (FEV1% predicted: 26.8Ç'ñ3.8% versus 46.0Ç'ñ6.7%, p<O.01; FVC% predicted: 44.4Ç'ñ4.9% versus 60.1Ç'ñ7.7, p<O.05). The switch of P. aeruginosa to an alginate over producing, mucoid phenotype is a pivotal event in CF lung infection. Mucoidy usually results from loss of regulation of the alternate sigma factor AlgU via mutation in mucA, encoding an anti-sigmp factor. It is possible that this affects the expression of other virulence factors in addition to alginate. Thirteen mucA mutants were constructed in three wild type backgrounds and their level of cyanide, pyocyanin and elastase production was found to be 1/3 that of wild type strains. In addition, in contrast to wild type strains, mucA mutants showed a lack of suppression of cyanide and pyocyanin production in later stationary phase. The Burkholderia cepacia complex (Bcc) is another group of important CF pathogens. It was hypothesised that, like P. aeruginosa, they might also be cyanogenic. All 9 species of the Bcc complex were 'able to make cyanide at comparable levels to P. aeruginosa, but only when grown as surface attached colonies on agar plates or during biofilm growth on glass beads. In contrast to P. aeruginosa and other cyanogenic bacteria, cyanide was not detected during planktonic growth of Bcc strains. The data suggests that cyanide production by P. aeruginosa, and possibly members of the Bcc, is important in CF lung infection and that the CF relevant switch to mucoidy via mucA mutation affects the production of cyanide and other virulence factors in addition to alginate.

Poor nutritional (underweight) status in cystic fibrosis (CF) is associated with poorer health outcomes and ultimately a decreased survival rate. Eating / disorders or disturbances in eating attitudes and behaviours (DEABs) may contribute to poor nutritional status in people with CF. Existing measures of DEABs developed for the general population are unsuitable for use in CF due to the different meaning of some of these items in a CF population and a failure to assess CF-related eating attitudes and behaviours (EABs). This study aimed to develop a measure of EABs likely to be associated with poor nutritional status specifically for the CF population. The study had three parts: firstly, the formal evaluation of a draft CF-eating attitudes and behaviours (CFEAB) measure's content validity via an expert evaluation of the items and a literature review and secondly, the piloting of an amended CFEAB measure with participants with CF using a cognitive interviewing procedure to assess the measure's practical ease of use and understanding. Finally, a further amended CFEAB measure was administered to a large development sample of people with CF attending routine clinics. The final CFEAB measure resulting from this displayed adequate psychometric properties with a three- factor structure ('Eating-Disorders' 'CF-related EAB' (provisionally named) and 'Appetite') with good internal consistencies for the subscales (0.92, 0.84, 0.83) and whole measure (0.90). The final CFEAB measure displayed some initial evidence of construct validity in its relationships with related variables including an existing measure of DEABs. Some ambiguities regarding the interpretation of the CFEAB measure's subscales need to be clarified, as well as further evidence for its construct validity sought, in future studies. The 3 ~ .. ----------------------------------- CFEAB measure looks promising as a tool to highlight disturbances in EABs that will benefit from further assessment and targeted interventions, as appropriate.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a CI channel found in the apical membrane of epithelial cells. CFTR activity is tightly controlled by complex regulation. However, CFTR overactivity or loss-of-function mutations in CFTR are both disease causing conditions. The aims of my research were to investigate the mechanisms Of action of small-molecule CFTR modulators that regulate CFTR function. Loop diuretics are inhibitors ofNa+-K+-2Cr-cotranspOlter isofOlm 1 (NKCC1) located in the basolateral membrane of epithelial cells. I demonstrated that loop diuretics also inhibit CFTR cr channels, with lower potency, as open-channel blockers to occlude the CI permeation pathway in both recombinant cells and epithelia. Therefore, loop diuretics inhibit cr secretion both by inhibiting NKCC1 and CFTR in the basolateral and apical membranes, respectively. Like FS08del, AS61E is a CFTR processmg mutation that causes cystic fibrosis. I demonstrated that surface expressed AS61 E-CFTR cr channels have a severe gating defect with shortened mean burst duration and prolonged interburst interval. I also showed that the activity of surface expressed AS61E-CFTR is enhanced by the CFTR potentiator UCcF-8S3 , but not by UCcF-180. These data suggest that UCcF-8S3 is non-specific for FS08del-CFTR. Finally, to target the root cause of the commonest CF mutation, FS08del, I studied a group of dual-acting (corrector-potentiator) small molecules. I demonstrated that EPIX 107813 acts as a CFTR dual-acting modulator to, first, rescue cell surface expression of FS08del-CFTR; second, to partially restore wt-CFTR cr channel gating behaviour to FS08del-CFTR; and third, to improve cell surface thelmal stability of FS08del-CFTR CI channels. Therefore, EPIX 107813 acts as both CFTR corrector and potentiator. In conclusion, this study elucidated the mechanism of action of CFTR modulators and demonstrated their direct interaction with CFTR. Knowledge of how these modulators work will be of value in the development of new therapeutics that target CFTR directly.

The Na2+-Ca2+ Exchanger (NCX), all important Ca2+ handling plasmalemmal protein, is expressed widely in cardiac tissues and plays important roles in physiological and pathophysiological cardiac function . A key aim of this study was to determine whether or not B-adrenoceptor/PKA stimulation modulates NCX current (lNCX), measured as Ni2+ sensitive current, independent of potential contamination from a PKA-activated CI current (lCIPKA), encoded by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Pharmacological effects of a 'selective' CTFR blocker, GyH-101 were also tested. Additional work was also conducted on human atrial INCX and NCX expression. Methods: Rabbit ventricular and atrial and human atrial myocytes were used in whole-cell patch-clamp experiments at 37°C. Selective conditions for NCX and CI currents were used as appropriate. NCX protein was measured by Western Blotting. Results and conclusions: Under NCX•selective recording conditions 10 mM Ni2+ blocked isoprenaline activated current, but the Nil+• sensitive ventricular INcx activated by forskolin was similar to that in control. In rabbit atrial tissues, which lack ICLPKA, forskolin did not increase the Ni2-•sensitive current component. External but not internal Ni 21 inhibited lc1PKA, with a sub• millimolar IC50, when this was activated by isoprenaline but not forskolin suggestive of a Nil+ action upstream to adenylyl cyclase, likely directly on the B1• adrenoceptor. Thus, the apparent increase rabbit ventricular INt:x with isoprenaline in NCX recording conditions is likely to be attributable to overlapping Ni2+•sensitivc IctPKA, rather than INCX activation by B-adrenoceptor/PKA stimulation. The CFTR inhibitor GlyH•-1Ol produced voltage• dependent inhibition of cardiac IC1PKA, but additionally also inhibited I and IKI . Consequently this compound is not entirely CFTR•selective in respect of cardiac electrophysiology. Human atrial INt:x density and its current voltage relationship were very similar to those of rabbit atrial myocytes. In the human atrial samples examined, there was no significant difference in NCX protein expression between patients in sinus rhythm and those in chronic atrial fibrillation.

Cystic fibrosis (CF) is the most common genetic disorder amongst Caucasians and is associated with pulmonary infection and respiratory failure leading to a shortened life expectancy. Chronic respiratory infection by PseudomonaTaeruginosa contributes significantly to the morbidity and mortality associated with CF. hi the UK, I number of transmissible strains have been identified, the most common of which is the Liverpool Epidemic Strain (LES) of P. aeruginosa, which has been associated with causing greater morbidity of CF patients, superinfection, and the infection of non-CF parents of a CF patient. The aim of this project was to identify some of the characteristics of this successful strain and determine the factors that contribute to its success.

Viridans group streptococci (VGS) are commensal bacteria inhabiting the human oral-nasopharynx, gastrointestinal and urogenital tracts. Recent studies have revealed various aspects of VGS, including possible pathogenesis in patients with cystic fibrosis (CF). This thesis aims to investigate molecular identification techniques for the VGS thus allowing the population structure of VGS to be determined in adult patients with cystic fibrosis, as well as their molecular characterisation in terms of fluoroquinolone and macrolide resistance. The thesis consists of 10 chapters, including a literature review and eight experimental chapters and general discussion. Chapter 1 provides an up-to-date review of the current literature involving the identification and characterisation of VGS organisms. The first experimental theme is the development of a molecular technique for the reliable speciation of members of the VGS into distinct species, as well as an exploration of other novel molecular markers, including the development of a novel PCR assay which uses clustered regulatory interspaced short palindromic repeats (CRlSPRs)-like sequence. Having established methods to identify these organisms, these techniques formed the foundation for a study describing the population structure of VGS in adults patients with CF. Possible pathogenic mechanisms and molecular epidemiology of the resulting VGS populations were examined, as well as the frequency of antibiotic resistance in VGS organisms. Emergence of antibiotic resistance in VGS is major concern as seen in other pathogenic bacteria. Antibiotic resistance in VGS was further characterised in terms of its specific.

Antibiotic treatment of lung infection has been a major contributor to increased life expectancy amongst cystic fibrosis (CF) patients in recent years. Diverse polymicrobial communities including bacteria from the genus, Prevotella, are detected in respiratory samples from CF patients. However, the role of Prevotella in disease progression and the effect of high antibiotic consumption on resistance in this genus are unknown. The primary aim of this study was to determine and compare antimicrobial susceptibility and mechanisms of resistance in Prevotella spp. isolated from CF and non-CF patients. All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam while metronidazole resistance was rare. In contrast, resistance to amoxicillin, ceftazidime and tetracycline was apparent. CF isolates were more resistant to azithromycin, clindamycin and co-amoxiclav compared to non-CF isolates. Azithromycin resistance was also associated with current prescription of the antibiotic in CF patients. More than 50% of isolates weref3-lactamase positive with an association between β-lactamase production and presence of cfxAlcfxA2. Extended spectrum f3-lactamase (ESf3L) production was detected in 69% of isolates tested and was associated with resistance to ceftazidime and amoxicillin. Isolates positive for ermF and tetQ were more resistant to azithromycin/clindamycin and doxycycline/tetracycline, respectively with ermF detected more commonly in the CF isolates. The nim-type gene was rarely detected but a metronidazole resistant subpopulation with a stable phenotype could be selected for in a nim-positive isolate following exposure to a subinhibitory concentration of the antibiotic in vitro. This study profiles in vitro antibiotic susceptibility of Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF can affect all exocrine organs, CF lung disease is the major cause of morbidity and mortality in CF patients. In addition to the chronic infection and inflammation found in the CF airways, there are some publications looking at apoptosis in CF epithelial cells although the findings from these studies are unclear. In this work, I examined the relationship between the ~F508 CFTR mutation, ER stress activation and ER-stress related apoptosis in CF airway epithelial cells. However, there was no evidence of ER stress in our CF cells and therefore no suggestion of ER stress-induced apoptosis as evidenced by an absence of caspase-4 activation. However, caspase-3 and caspase-8 were found to have upregulated activity in CF cells compared to non-CF controls and this upregulation was demonstrated to be associated with CFTR mutation. Meanwhile, studies focusing on the extrinsic pathways revealed that upregulated Fas at both mRNA arid protein level might contribute to the increased caspase-3 & -8 activation in CF cells. However, low level of DNA fragmentation found in CF cells indicated no increased apoptosis in these cells and subsequent activation with a Fas activating antibody showed significantly increased apoptosis in CF cells compared to non-CF cells. Measurement of several apoptotic regulators including lAPs, Bcl-2 and c-FLIP suggested a cell-type dependent, but not CFTR mutation dependent, inhibition of apoptosis in CF tracheal and bronchial cells. Further studies are required to elucidate the entire signalling pathway in CF cells from Fas activation to caspase-3 activation and in addition further work is needed to show why caspase activation in basal cells does not lead to downstream activation of apoptotic mediators beyond caspase-3.

Cystic fibrosis (CF) lung disease is characterised by progressive destruction and dilatation of the airways. This occurs in the setting of chronic infection, particularly with Pseudomonas aeruginosa (Pa), and is associated with loss of elastin and cartilage, with progressive submucosal fibrosis. Matrix Metalloproteinases (MMPs) can degrade all components of the extracellular matrix, and are inhibited by the Tissue Inhibitors of Metalloproteinases (TIMPs). High MMP-9 activity has been identified in CF sputum, and correlates inversely with lung function (FEV1). The source and regulation of MMP-9 in CF is unknown. Direct Pa infection of epithelial cells resulted in destruction of TIMP-1, the major inhibitor of MMP-9. Pa infection of inflammatory cells" neutrophils, monocytes and monocyte derived macrophages (MDMs); resulted in an unopposed increase in MMP-9. Conditioned media from Pa infected neutrophils (coNPa), monocytes (coMPa) and MDMs (coMDMPa) drove further secretion of MMP-9 from epithelial cells. This was shown to be regulatecl by Mitogen activated protein kinase (MAPK) pathways. The functional, matrix degrading effects of MMPs produced following conditioned media stimulation were examined and revealed that stimulated epithelial cells were able to invade through a matrigel membrane and drive an invasive phenotype, potentially characterising the involvement of MMPs in extracellular matrix breakdown in CF. Further, MMP-9 secretion in conjunction with cytokines and growth factors found in the CF airway, acted on epithelial cells resulting in loss of epithelial cell contact with basement membrane and subsequent invasion of epithelium into the mesenchyme- a process known as Epithelial to mesenchymal transition (EMT). In summary, infiltrating leukocytes and the lung parenchyma are potential sources of excessive MMP-9 activity in CF, and may contribute to the airway destruction that occurs in response to Pa infection.