Antimicrobial therapy has been a major contributor to the increased life expectancy of CF patients since CF was first describe<! in 1938. However, the high antimicrobial consumption in this patient population means that resistance has become a major problem in CF. As such, there is a need for new antimicrobials or combinations of antimicrobials for use in CF patients. It is also apparent that there are anaerobic niches in the lungs of CF patients; therefore, a new antibiotic which retains its effect under such conditions would be particularly beneficial. The aim of this study was to investigate the activity of a new antibiotic combination consisting of fosfomycin and tobramycin in a 4: I (w/w) ratio (F:T) under aerobic and anaerobic conditions by susceptibility, synergy, resistance development and microarray studies. Results of this study have shown that F:T has good activity against P. aeruginosa and MRSA cultured from CF patients, with enhanced activity under anaerobic conditions. The combination was more likely to be synergistic under anaerobic conditions where it exhibited synergy against 45% of isolates tested compared to 20% under aerobic conditions. F:T also prevented or delayed the emergence of resistance compared to either fosfomycin or tobrarnycin alone under aerobic and anaerobic conditions. In addition, F:T remained active against isolates which developed resistance to either fosfomycin or tobramycin alone. F:T \\-Wi also found to downregulate nitrate reductase genes essential for the anaerobic growth of P. aeruginosa and was able to reverse nitrate utilisation in this organism. As increased nitrate utilisation confers a selective advantage to P. aeruginosa in the Cf lung, this may be particularly beneficial in CF patients. Overall, this study has shown that F:T is a potentially promising new treatment option for CF lung infections caused by P. aeruginosa and MRSA.

Cystic fibrosis (CF) is the commonest autosomal recessively inherited condition leading to reduced survival in the Caucasian population. It is caused by defective chloride regulation, resulting in premature death, predominantly from respiratory failure secondary to chronic infection and inflammation. Over the past 70 years, there have been vast improvements in survival in CF due to advances in early diagnosis, antibiotics, airway clearance, nutritional support and infection control. Accordingly, researchers and clinicians have endeavoured to identify prognostic indicators and outcome measures that can accurately assess response to treatment. Over 20 years ago, females and individuals of lower socioeconomic status were found to have a poorer prognosis than males or those of higher socioeconomic status. The first part of this thesis describes an epidemiological study that showed that these socioeconomic and gender disparities are still present today. The natural history of CF is a slow decline in lung function punctuated by episodes of increased respiratory symptoms, termed pulmonary exacerbations. These exacerbations are associated with reduced lung function, lower quality of life and increased mortality. The second part of this thesis describes the investigation of clinical and sputum factors that may influence the time between pulmonary exacerbations. The study showed that female gender, increased bacterial load in a patient's sputum and shorter duration of infection with the bacterium Pseudomonas aeruginosa are independent predictors for a shorter time to next the exacerbation. The third part of this thesis focuses on respiratory infection with P. aeruginosa, a key pathogen in CF. These bacteria thrive in the warm, moist environment of the CF airways where they form antibiotic resistant biofilms. As a community, the bacteria co-operative with each other to sense and respond to changes in environmental stimuli using a cell-to-cell communication system, known as quorum sensing (QS). QS 'signal' molecules have been detected in the sputum and plasma of patients with CF but little is understood about their clinical significance in the human host. This thesis describes a prospective observational clinical study showing that: (1) QS signal 2 molecules can be detected in the sputum, plasma and urine of patients with CF; (2) QS signal concentrations in the sputum, plasma and urine are correlated with quantitative measures of P. aeruginosa in the lung and (3) QS signal molecules have significant potential as biological markers for lung infection with P. aeruginosa. Overall, this thesis highlights several clinical factors associated with increased morbidity and mortality in CF patients and the potential use of biological markers of infection to measure and improve respiratory outcomes in future. It concludes by reflecting on the clinical significance of these observations and considering future areas of research that may increase our understanding of the biological processes involved. 3

Malnutrition remains a major clinical problem in Cystic Fibrosis (CF). As the degree of underweight correlates closely with reduced survival, interventions are needed which optimise nutritional outcomes. The focus of this thesis was on developing a home based behavioural nutrition education programme for adults with CF and assessing its effectiveness on nutritional status, knowledge and other psychosocial measures using a randomised control study design. Chapter 2 describes the development of the "Eat well with CF" programme, which used a framework of Social Cognitive Theory. The next investigations aimed to test the programme both with consumers and with peers. The results showed that adults with CF would be motivated to take part and felt they would learn from the programme. The peer review demonstrated that the programme was rated highly with regard to content, accuracy and information. In Chapter 3 the effectiveness of "Eat well with CF" was tested in a randomised trial (n=74) using a control group who received standard care. The results demonstrated a trend towards an increase in weight. After 6 months the average weight gain in the intervention group was 0.57 kg compared to control weight gain of 0.09 kg (p=0.545, 95%CI -1.07-2.0). Subjects undertaking the "Eat well with CF" programme had significantly increased their self-efficacy to cope with their diet, (p=0.003, 1.19-5.67), their specific nutritional knowledge (p < 0.001, 4.05-7.38) and their reported dietary fat intake (p=0.014, 0.76-6.50) compared to the control group. At 12 months, the average weight gain was 0.02 kg in the control group and 1.14kg in the intervention group with no statistical differences between the two groups. The intervention group continued to show a marked and significant improvement in CF specific nutritional knowledge and self-efficacy score. Chapter 4 examines the reasons for subject non- participation in the study, which led to the development of an audio version of "Eat well with CF". The positive results of the process evaluation detailed in chapter 5 highlight the significant personal enjoyment and benefit received by the participants. These studies combine to demonstrate the utility, acceptability and efficacy of "Eat Well with CF". In addition they challenge traditional dietetic practice. We suggest this novel behavioural education approach could enhance current dietetic practice, to improve outcomes and lead to life long maintenance of optimal nutritional status for adults with CF.

Cystic fibrosis transmembrane conductance regulator (CFTR) is activated by cAMP-dependent phosphorylation, and functions as an ATP-dependent chloride channel involved predominantly in the movement of chloride ions to maintain cellular ionic homeostasis. Mutations in this channel cause cystic fibrosis, the most common lethal autosomal recessive disease in caucasians. The regulation of CFTR forms a large body of research; this thesis investigated the role of three potential components of the regulatory machinery – nucleoside diphosphate kinase B (NDPK-B), tyrosine phosphorylation and G proteins. This thesis aimed to: 1. Describe the functional relationship between NDPK-B and CFTR. 2. Describe the effect of altered tyrosine phosphorylation in 16HBE14o- cells on CFTR function. 3. Identify the tyrosine phosphatases involved in CFTR regulation. 4. Explain how alterations in tyrosine phosphorylation change CFTR function. 5. Describe the effect of G protein stimulation on CFTR channels which have already been activated by an increase in cellular cAMP. The whole cell patch clamp technique was used to examine ion channel function in two cell types - human bronchial epithelial cells (16HBE14o-) and baby hamster kidney cells (BHK-21). Due to alterations in the function of cultured cells, it was not possible to describe the functional relationship between the histidine kinase NDPK-B and CFTR. Further work is required in this area to elucidate the role of this protein in CFTR regulation. The use of general tyrosine phosphatase inhibitors resulted in a significant decrease in the CFTRinh-172-sensitive conductance in 16HBE14o- cells, and specific inhibitors ruled out the involvement of PTP1B and Shp1/2 phosphatases. Further work is required to explain how tyrosine phosphatase inhibition alters CFTR function. Finally, G protein stimulation in 16HBE14o- after increasing intracellular cAMP had no significant effect on channel function. This suggested that the cAMP-dependent activation of the channel is the predominant mechanism for stimulating channel function.

In contrast to the neutrophilic inflammation found in the airway lumen, lymphocytes predominate in the airway wall of Cystic fibrosis (CF) patients. The Th17 pathway could play an important role in CF, as IL-17, secreted by Th17 lymphocytes, is proinflammatory, stimulates neutrophil recruitment and has been detected in CF sputum. This thesis investigates the hypothesis that T lymphocytes, in particular Th17 lymphocytes, are present in the CF airway wall and activation of the Th17 pathway is CF specific. Lymphocyte populations were characterised in endobronchial biopsies of children with CF, non-CF bronchiectasis and controls and related to clinical status, microbiological cultures and inflammation (cells and soluble factors) in bronchoalveolar lavage fluid (BALF). In parallel, lymphoid aggregates seen in the nasal mucosa of CF mice were investigated. Immunohistochemistry was performed on these endobronchial biopsies, staining for CD4, CD8 and IL-17. Cellular sources of IL-17 were determined following development of a novel immunofluorescence double staining protocol. Cells and cytokine levels were measured in BALF. Flow cytometry analysis of the nasal mucosa of CF and wild type mice was performed. Significant differences in T helper (CD4+) cells but not cytotoxic (CD8+) T cells were found between patient groups. Established CF and non-CF bronchiectasis samples had higher numbers of IL-17+ cells than controls; newly diagnosed CF patients had intermediate counts. Double staining confirmed there were Th17 lymphocytes in the submucosa of these biopsies. IL-17+ neutrophils, !"T cells and natural killer T cells were also identified. CF mice were also shown to have CD4+IL-17+ cells in their nasal mucosa. In conclusion, Th17 lymphocytes are present in the airway submucosa in CF and represent one of a number of sources of IL-17. Non-CF bronchiectasis patients had similar numbers of IL-17+ cells, suggesting activation of the Th17 pathway is not CF specific.

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