Chronic microbial infections result from persistent host colonization that is not cleared via the immune response or therapeutics. Within the host, microbes can undergo adaptive evolution, whereby beneficial traits promoting persistence arise due to selection; these traits can therefore affect disease outcomes and treatment strategies. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic, fatal respiratory infections in individuals with the heritable disease cystic fibrosis (CF). The goal of this dissertation is to identify adaptations that allow P. aeruginosa to persist in the host during chronic CF lung infection. To achieve this goal, P. aeruginosa was chronologically sampled from 3 CF patients, ranging from the first infecting bacterium (the ancestor) to ~40,000 generations post-infection. By comparing gene expression profiles of ancestral and evolved isolates sampled from multiple patients, I identified 24 parallel gene expression changes that occurred over time within each lineage, suggesting that these traits are beneficial to the bacterium. Because most of these traits had unknown physiological roles, I sought to characterize their biological significance. I used a gain-of-function genetic screen and discovered that a subset of these genes enhance biofilm formation, a sessile mode of growth proposed to be important during chronic CF lung infection. I showed that enhanced biofilm formation is due to increased production of the exopolysaccharide Psl, which is traditionally viewed as less critical for maintaining chronic infections than other virulence factors. Lastly, I demonstrated that a majority (~72%) of chronic P. aeruginosa isolates produce more Psl than their corresponding ancestor, suggesting that this exopolysaccharide is important during chronic infection and an adaptive trait. / text

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

One of the hallmarks of chronic cardiac disease is the excessive formation of fibrous extracellular matrix. This inappropriate remodeling is mediated in large part by cardiac fibroblasts and phenoconverted myofibroblasts. The protooncoprotein Ski has previously been described as possessing anti-fibrotic properties within the myocardium, in addition to triggering apoptosis when overexpressed. In the current study, we found that overexpression of Ski results in a set of distinct morphological and biochemical changes within primary cardiac myofibroblasts that is indicative of apoptosis. Its upregulation is associated with the expression of pro-apoptotic factors such as Bax and Bak, as well as caspase-9 and -7. In all, our results indicate that Ski triggers a pro-death mechanism in primary rat cardiac myofibroblasts that is mediated through the intrinsic apoptotic pathway. The survival of these cells appears to be prolonged by a pro-survival autophagic response as apoptosis is hastened when autophagy is inhibited. The observed cell death response is likely working in parallel with the previously observed anti-fibrotic properties of Ski within this cell type. As myofibroblast cells are the engines of matrix expansion in heart failure, we suggest that using Ski or a projected Ski-mimetic to induce graded apoptosis in myofibroblasts within the failing heart may be a novel therapeutic mechanism of controlling cardiac fibrosis. / October 2015

No description available.

Cystic Fibrosis (CF) is a life-threatening autosomal recessive disease affecting 1:3600 children born in Canada. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The most common disease causing mutation is a deletion of residue F508, resulting in a structurally compromised protein which is retained in the endoplasmic reticulum and targeted for proteasomal degradation. Therapeutic strategies currently being pursued to alleviate the afflictions caused by this and other mutants include the use of corrector compounds to modify the surface expression of the channel, and potentiator compounds to increase cAMP-mediated channel activity. Despite the discovery of a number of small molecules affecting CFTR, much is still unknown about the nature of these interactions. This thesis contains the investigation of two potentiators: VRT-532 and VX-770, and two correctors VX-809 and C18. We assessed the consequences of interactions with these drugs on CFTR channel activity, ATPase activity and phosphorylation. We demonstrated that VRT-532 binds directly to mutant CFTR to modify its channel and ATPase activity. VX-770, known to bind directly to CFTR, can stimulate channel activity in the absence of cAMP stimulation in baby hamster kidney (BHK) cells. Correctors VX-809 and C18, based off the same molecular scaffold, are both capable of acutely augmenting cAMP-stimulated channel activity, providing evidence for potentiator activities in these compounds. Quantitative mass spectrometry (MS) techniques demonstrate a defect in phosphorylation at Ser-660 in the regulatory (R) domain in the major mutant. Treatment with C18 was unable to repair this defect. These novel findings regarding interactions between several small molecules and CFTR contributes to the understanding of the mechanism of action of these compounds, and will help identify how they may be modified for greater efficacy to improve the treatment of CF disease.

Cystic Fibrosis (CF) is a life-threatening autosomal recessive disease affecting 1:3600 children born in Canada. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The most common disease causing mutation is a deletion of residue F508, resulting in a structurally compromised protein which is retained in the endoplasmic reticulum and targeted for proteasomal degradation. Therapeutic strategies currently being pursued to alleviate the afflictions caused by this and other mutants include the use of corrector compounds to modify the surface expression of the channel, and potentiator compounds to increase cAMP-mediated channel activity. Despite the discovery of a number of small molecules affecting CFTR, much is still unknown about the nature of these interactions. This thesis contains the investigation of two potentiators: VRT-532 and VX-770, and two correctors VX-809 and C18. We assessed the consequences of interactions with these drugs on CFTR channel activity, ATPase activity and phosphorylation. We demonstrated that VRT-532 binds directly to mutant CFTR to modify its channel and ATPase activity. VX-770, known to bind directly to CFTR, can stimulate channel activity in the absence of cAMP stimulation in baby hamster kidney (BHK) cells. Correctors VX-809 and C18, based off the same molecular scaffold, are both capable of acutely augmenting cAMP-stimulated channel activity, providing evidence for potentiator activities in these compounds. Quantitative mass spectrometry (MS) techniques demonstrate a defect in phosphorylation at Ser-660 in the regulatory (R) domain in the major mutant. Treatment with C18 was unable to repair this defect. These novel findings regarding interactions between several small molecules and CFTR contributes to the understanding of the mechanism of action of these compounds, and will help identify how they may be modified for greater efficacy to improve the treatment of CF disease.

Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水野紘樹氏の博士論文として提出された

The Vasoactive Intestinal Peptide (VIP) is an agonist of the CFTR chloride channel in the human airways. In the genetic disease Cystic Fibrosis, where CFTR is defective or absent from the apical membrane of epithelial cells, VIP innervations are lost. Our group has demonstrated that VIP increases CFTR membrane stability through PKCε. However, the mechanism remained to be determined. Here we found that VIP stimulation increases the interaction of NHERF1 and P-ERMs with CFTR through PKCε phosphorylation. Moreover a reduction of the interaction between intracellular CFTR and the Golgi associated protein, CAL was observed following VIP stimulation. Silencing either ERMs or NHERF1 with siRNA prevented the VIP ability to increase CFTR surface expression and function, confirming that NHERF1 and P-ERMs are necessary for VIP regulation of the sustained activity of membrane CFTR. This study shows the cellular mechanism by which prolonged VIP stimulation of airway epithelial cells regulates CFTR-dependent secretions.

No description available.