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.

abstract: Introduction: Cystic fibrosis (CF) is the most common life-shortening autosomal recessive genetic disease affecting Caucasians. The disease is characterized by a dysfunctional cystic fibrosis transmembrane regulator (CFTR) protein and aberrant mucus accumulation that subsequently alters the physicochemical environment in numerous organ systems. These mucosal perturbations have been associated with inflammation and microbial dysbiosis, most notably in the lungs and gastrointestinal (GI) tract. Genistein, a soy isoflavone and dietary polyphenol, has been shown to modulate CFTR function in cell cultures and murine models, as well exert sex-dependent improvement of survival rates in a CF mouse model. However, it is unknown whether dietary genistein affects gut microbiome diversity and community structure in cystic fibrosis. This study sought to examine associations between dietary genistein treatment and gut microbiome diversity and community structure in a murine model of CF. Methods: Twenty-four male and female mice homozygous for the DF508 CFTR gene mutation were maintained on one of three diet regimens for a 45-day period (n=11, standard chow; n=7, Colyte-treated water and standard chow; n=6, 600 mg dietary genistein per kg body weight). One fecal pellet was collected per mouse post-treatment, and microbial genomic DNA was extracted from the fecal samples, quantified, amplified, and sequenced on the Illumina MiSeq platform. QIIME 2 was used to conduct alpha- and beta-diversity analyses on all samples. Results: Measures of alpha-diversity were significantly decreased in the dietary genistein group as compared to either standard chow or Colyte groups. Measures of beta-diversity showed that community structure differed significantly between dietary treatment groups; these differences were further illustrated by distinct clustering of taxa as shown by principal coordinates analysis plots. Conclusion: This 3-arm parallel experimental study showed that dietary genistein treatment was associated with decreased microbial diversity and differences in microbial community structure in DF508 mice. / Dissertation/Thesis / Masters Thesis Nutrition 2019

Gene delivery via lentiviruses can yield long term expression of transgenes. Specificity of host cell targeting by viral vectors occurs primarily through viral glycoprotein (GP)/cellular receptor interactions. Ebola virus (EBOV) GP has broad tropism for a variety of cell types making this viral GP a potentially useful reagent for delivery of gene therapy. However, titers of EBOV GP pseudotyped lentiviruses are insufficient for practical use in clinical applications. Enhancement of EBOV-GP pseudotyped titers by as little as half a log might yield clinically applicable titers. In an alanine scanning study, we identified 19 residues in EBOV-GP1 that increased transduction efficiency two to three fold. When mapped onto the crystal structure of EBOV GP, these residues were primarily located at the interface of GP1/GP2 suggesting these residue substitutions may confer conformational changes in the protein structure thereby enhancing transduction efficiency. To determine if combinations of these alanine substitutions might further enhance transduction, we have introduced the changes into EBOV GP in a stepwise manner. To date, introduction of some combinations of alanine substitutions resulted in as much as an eight-fold increase in transduction over WT GP, this being our super mutant combination, whereas other combinations eliminated transduction. Identification of 5 additional mutations via 3D modeling of the glycoprotein uncovered an additional mutation in GP2, located at the GP1/GP2 interface, which also enhances EBOV GP transduction. Transduction of cell lines important for gene therapy including hepatocytes and porcine airway cells confirmed an enhancement in transduction as well. Other cell populations, specifically fibroblasts and renal cells, were also transduced but enhanced transduction was not observed indicating this phenomenon may be cell type specific. The in vivo studies were inconclusive because no expression was detected from any of the EBOV GP pseudovirions. Even expression of the positive control, GP64 particles, waned after 3 weeks post inoculation indicating insufficient quantities or poor quality pseudovirions were used. These EBOV GPs should prove useful for future gene therapy studies by providing an alternate glycoprotein that is as effective as GP64 at producing high titer lentiviral vectors.

Doctor of Philosophy / Cystic fibrosis (CF) is the most common inherited lethal disorder among Caucasian populations. Chronic pulmonary infections, particularly from Pseudomonas aeruginosa, are the major determinant of the morbidity and mortality of people with CF. It is generally accepted that people with CF acquire this pathogen independently from their surrounding environment, and that individual CF patients carry unique strains different from others. The spread of this pathogen from patient to patient is thought to be rare and occurs particularly among closely contacted cases such as CF siblings. However, over the past decade, there have been several reports of an emergence of clonal P. aeruginosa strains commonly found infecting a number of CF patients. One such report is from the CF paediatric clinic at the Royal Children’s Hospital in Melbourne in which more than half of the patients were infected with a single strain or clone, subsequently called Australian epidemic strain 1 or AES-1. A preliminary survey showed that AES-1 had spread extensively along the Australian eastern seaboard among CF patients attending other CF centres in Melbourne, Sydney and Brisbane, including adult patients at the Royal Prince Alfred Hospital (RPAH), Sydney. Another clonal strain, subsequently called AES-2, was identified in both CF adults and children at the Prince Charles Hospital and the Royal Children’s Hospital, in Brisbane. The total extent of prevalence of the AES-1 and AES-2 strains at the RPAH as well as the clinical status of patients who carried these strains was unknown. Moreover, the pathogenicity of these two clonal strains had not been investigated. The studies presented in this thesis investigated the prevalence of these clonal strains among CF patients attending the adult CF clinic at RPAH, Sydney by using pulsed-field gel electrophoresis. Overall, 50% of 112 patients with P. aeruginosa were found to be infected with clonal strains. The AES-1 and AES-2 strains were identified in 38% and 5% of the patients respectively. Two new clonal strains, called Sydney-1 and Sydney-2, were also identified. Patients with clonal strains had a significant increase in their number of exacerbations and hospitalisation days, and tended to have lower pulmonary functions when compared to patients infected with non-clonal strains. By using a variety of bioassays to examine the pathogenicity of the clonal and non-clonal strains, it was found that both AES-1 and AES-2 produced more virulence factors and were more resistant to antibiotics when compared to the non-clonal strains. AES-1 and AES-2 were associated with increased production of proteases, including elastase, alkaline protease and protease IV. Overall the results presented in this thesis suggest that there may be a link between virulence and transmissibility of this pathogen. The studies presented in this thesis also compared the biofilm forming capacities of the AES-1 and non-clonal isolates. AES-1 was shown to have greater biofilm-forming capacity than the non-clonal strains, when they were grown on a glass surface, suggesting a possible association between clonality and biofilm formation. A model for the study of bacteria grown in conditions similar to CF sputum was also developed. P. aeruginosa grown in this model was found to develop into clumps which may be comparable to the biofilm structure in the CF lung. This model was shown to be beneficial for transcriptomic and proteomic studies which are underway within the research group. AES-1 was also found to have phenotypic variations between isolates. By applying the amplified fragment length polymorphism technique, more subtypes of this clone were revealed. However, these detected subtypes did not correlate with the different phenotypes, suggesting minor mutations such as single point polymorphisms may be responsible for the phenotypic diversity within the clone. The final part of this thesis was devoted to examining the safety of a novel CF treatment: hypertonic saline (HS) inhalation. HS was shown to increase airway mucociliary clearance, while increased osmolarity associated with the use of HS was also shown to have an inhibitory effect on the formation of biofilms. Findings in this study proved that there was no evidence of strain selection in patients who received the long-term treatment with HS. The study also demonstrated that AES-1 was significantly more persistent in the CF lung than the non-clonal strains. The present thesis not only defines the clonal strains of P. aeruginosa and their implications for infected patients, but also provides a general understanding into the pathogenesis of both clonal and non-clonal strains infecting CF lungs.

Bibliography: leaves x-xxii / xvii, 285, xxii leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1987

INTRODUCTION Antioxidant micronutrients may help alleviate oxidative stress in cystic fibrosis (CF) lung disease. To determine treatment effect, systematic reviews (SR) synthesize available evidence. Cochrane SRs are known for being methodologically rigourous, however, may have limited generalizability. OBJECTIVES To assess effectiveness of antioxidant micronutrients in CF lung disease using Cochrane and non-Cochrane SR methodology; to determine whether Cochrane SRs trade relevance for rigour METHODS The first SR followed Cochrane-preferred methods, while the non-Cochrane SR employed a broader search strategy and nclusion criteria. Reviews were contrasted regarding yield of search, treatment effect (efficacy and safety) and risk of bias. RESULTS Neither SR had enough data to support or refute efficacy or safety of antioxidant supplementation in CF lung disease. Compared to the Cochrane SR, the non-Cochrane SR had four more included studies, more precise estimates of efficacy, additional harms data and a similar risk of bias. CONCLUSION Broader search strategies and inclusion criteria may improve relevance of Cochrane SRs without compromising rigour. / Clinical Epidemiology

This study documented the creation and initial validation of the Perceived Adolescent Social Support: Cystic Fibrosis (PASS-CF) inventory. The inventory was developed from semi-structured interviews of adolescents with cystic fibrosis (CF) and measured both supportive and non-supportive behaviors provided to adolescents by their family and friends. This study reports the findings from these interviews, results of the pilot testing of the measure, exploratory analyses of the utility of individual items, and the relationships between supportive and non-supportive behaviors and important clinical outcomes, such as treatment adherence, health-related quality of life (HRQoL), and other health outcomes. In particular, the study compared two measurement models suggested by popular definitions of social support. The "perceived support" model emphasized adolescents' cognitive appraisals of the support provided to them by family and friends, and the functional support model emphasized the utility of specific behaviors in managing CF. Results provided support for both models and provided insights into important next steps in the study of social support in adolescents with CF.

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause loss function of the CFTR channel on the apical surface of epithelial cells. ΔF508-CFTR, the major mutation in patients, is misfolded, retained in the endoplasmic reticulum (ER) and degraded. Small molecule corrector compounds partially rescue the trafficking defect of ΔF508-CFTR by allowing escape from the ER and trafficking to the plasma membrane where it exhibits partial function. These compounds may bind directly to the mutant protein and rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis by evaluating the consequence of corrector compound on the conformation of each nucleotide-binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. We found that VRT-325 was capable in partially restoring compactness only in NBD1. In comparison, ablation of the arginine framed peptide sequence: R553XR555 (ΔF508-KXK-CFTR) modified the protease resistance of NBD1, NBD2 and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together these interventions restored processing of ΔF508-CFTR to near wild-type levels. Importantly however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. This defect in channel activation can be fully corrected by addition of the potentiator: VX-770. The experiments performed partly elucidated ii the molecular mechanism of action for drug therapy and suppressor mutation. It is important to understand these basic concepts in hopes to layout a blue print for future drug design.

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause loss function of the CFTR channel on the apical surface of epithelial cells. ΔF508-CFTR, the major mutation in patients, is misfolded, retained in the endoplasmic reticulum (ER) and degraded. Small molecule corrector compounds partially rescue the trafficking defect of ΔF508-CFTR by allowing escape from the ER and trafficking to the plasma membrane where it exhibits partial function. These compounds may bind directly to the mutant protein and rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis by evaluating the consequence of corrector compound on the conformation of each nucleotide-binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. We found that VRT-325 was capable in partially restoring compactness only in NBD1. In comparison, ablation of the arginine framed peptide sequence: R553XR555 (ΔF508-KXK-CFTR) modified the protease resistance of NBD1, NBD2 and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together these interventions restored processing of ΔF508-CFTR to near wild-type levels. Importantly however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. This defect in channel activation can be fully corrected by addition of the potentiator: VX-770. The experiments performed partly elucidated ii the molecular mechanism of action for drug therapy and suppressor mutation. It is important to understand these basic concepts in hopes to layout a blue print for future drug design.

Thesis (M.S. )--Marshall University, 2009. / Title from document title page. Includes abstract. Document formatted into pages: contains 155 p. Includes bibliographical references p. 151-152.