Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies. / text

Lactic acid, which induces pH depression, leads to ATP efflux from muscle to extracellular space: it was reported that CFTR was involved in this process. However, the mechanism by which lactic acid activated CFTR and brought about the ATP release is still unknown. This study was performed to investigate (1) what channels may be involved or even conduct ATP release, and (2) how lactic acid activated CFTR. Expression of the possible channels that may conduct ATP release in L6 cells was investigated using RT-PCR: ClC-2, ClC-3, ClC-7, CACC, VDAC, connexin 40, connexin 43 and pannexin 3 were expressed in L6. Incubation of cultured L6 cells with lactic acid (10 mM) increased the extracellular ATP from 0.6 ± 0.06 to 1.1 ± 0.09 nM (P ? 0.05), indicating that lactic acid stimulated ATP efflux in vitro. The non-specific chloride channel inhibitor, DIDS, failed to abolish the lactic-acid-induced ATP release, suggesting that DIDS-sensitive chloride channels were not involved in the ATP efflux. Among the non-specific inhibitors of connexin channels, gadolinium inhibited acidosis-induced ATP efflux, but carbenoxolone failed to inhibit it, and so the role of connexins remains uncertain. The specific inhibitor of CFTR, CFTRinh-172, and the non-specific open-channel blocker of CFTR, glibenclamide, both abolished the acidosis-induced ATP release, but another specific inhibitor of CFTR, GlyH-101, which blocks CFTR from the external side, failed to abolish the ATP release, suggesting that acidosis-induced ATP is dependent on CFTR-activation, but does not involve ATP moving through the CFTR chloride channel. We hypothesize that, at low pH, the Na+/H+ exchanger (NHX) extruded H+ out of the cell and the resulting intracellular Na+ was transported out by Ca2+/Na+ exchanger (NCX); the localized increase in Ca2+ activated adenyl cyclase (AC), thus elevating intracellular cAMP; cAMP-activated-PKA then phosphorylated CFTR, which regulated an ATP release channel. KT-5720, an inhibitor of PKA, abolished the acidosis-induced ATP release, and forskolin, an agent that elevates cAMP, stimulated it, suggesting that the cAMP/PKA pathway was involved. The specific inhibitor of NCX, SN-6 and KB-R7943, both abolished the acidosis-induced ATP release, supporting a role for NCX in mediating this process. However, amiloride, the non-specific inhibitor of NHX failed to abolish ATP efflux. The whole cell Cl- currents were studied in L6 cells: lactic acid increased the whole cell currents from 2.33 ± 0.10 to 3.54 ± 0.34 nA (P ? 0.05), and this lactic-acid-induced increase in Cl- current could be inhibited by CFTRinh-172, suggesting that the CFTR Cl- channel was opened at low pH. Moreover, forskolin increased whole cell Cl- currents, which supported a role for the cAMP/PKA pathway in the lactic-acid-induced increase in CFTR current. These data confirm that CFTR is involved in the lactic-acid-induced ATP release from L6 cells. The roles of the NCX and cAMP/PKA pathway in activating CFTR at low pH are supported, but further studies are required to determine whether the NHX is involved in CFTR activation and whether connexins participate in ATP release. / published_or_final_version / Physiology / Master / Master of Philosophy

Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions. Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions. Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction. These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction. / published_or_final_version / Physiology / Master / Master of Philosophy

When a child is diagnosed with a chronic life threatening illness there is a significant impact on the entire family. Siblings are at risk for psychological adaptation problems because of their unique relationship with the ill child and the effect of that illness on family functioning. Few studies have been reported which examine the impact of chronic life-threatening illnesses in children on healthy siblings.The purpose of this study was to investigate the predisposing risk and protective factors that affect the psychological adaptation of healthy siblings of a child with Cystic Fibrosis (CF). A descriptive study was conducted using a convenience sample from two CF centers. The relationship between several variables was explored using non- parametric correlations.In this sample, significant negative correlations were found between parental stress and their reported financial well being and emotional or behavioral problems in the well siblings prior to the diagnosis of CF was made in the sick sibling and perceived parental support.For adolescent siblings, The Behavioral Symptoms Index (BSI) was correlated to reported stress in their parents and negatively correlated to the parental perceived support. The Emotional Symptoms Index (ESI) was correlated to the BSI. A significant negative correlation was also noted between age and Internalizing Behaviors. These relationships were not significant for the child siblings in this sample.There were no significant relationships among gender, maternal education, and caretaker for the well sibling when the child with CF was hospitalized and their Internalizing and Externalizing Behaviors. The health care teams in these clinics seldom discussed CF with the well sibling. However, for the children in this study, there was a significant negative correlation with this discussion and their Externalizing Behaviors.Findings from this study suggest that the family environment, especially parental stress and perceived social support, may affect the adaptation of the well sibling. Adolescent siblings were more at risk for this environmental influence than their younger counterparts.

Studies were conducted using cultured immortalized wild type (non-CF) and mutant (CF) DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) tracheal epithelial cells on the anti-inflammatory impact of agents that may alter ceramide and glutathione (GSH) metabolism. The CF cells demonstrated abnormally high levels of GSH and glutathione disulfide (GSSG), which could diminish intracellular production of ceramide, a key modulator of inflammation and apoptosis. Hence, additional cell culture studies were carried out with a known inducer of in situ ceramide synthesis, N-4(4-hydroxyphenyl) retinamide (fenretinide) on interleukin (IL)-8 release, intracellular ceramide content, and cellular proliferation in both the basal state and following the inflammatory stimuli of tumor necrosis factor (TNF) -alpha. Fenretinide treatment was associated with a dose-dependent increase in the cellular content of ceramide in both CF and non CF cells. Also, an inhibition of IL-8 release in the inflammatory condition of TNF-alpha treatment was observed following fenretinide treatment in the CF cells. As hyperbaric treatment of whey proteins was previously associated with improved survivability and higher GSH content in a Pseudomonas aeruginosa murine model of cystic fibrosis (CF), the anti-inflammatory role of low molecular weight peptides (< 1kDa) generated from enzymatic hydrolysis of native and pressurized whey protein isolates (WPI) was examined. Pressure treatment of WPI was associated with an enhanced protein digestibility and an altered peptide profile following in vitro digestion. The whey peptides were tested CF and non-CF lung epithelial cells to identify for their effects on GSH metabolism. The impact of the combined treatment of fenretinide and WPH was also tested in terms of apoptosis and cytokine release in cell culture. As opposed to non-CF cells, CF cells showed a strong downtrend in release of IL-8 following the combined fenretinide and whey peptide treatment. In addition, whey peptides protected wild type epithelial cells from the apoptotic effect of fenretinide. Our results suggest the usefulness of these agents as a pharmacological treatment in CF.

Cystic Fibrosis (CF) is caused by mutations in CFTR, a protein for chloride efflux in epithelial cells. VIP is a peptide that activates CFTR and improves membrane stability; VIP has 3 receptors VPAC1, VPAC2 and PAC1 that can cause CFTR phosphorylation. VIP-knockout (VIPKO) mice experience inflammation and reduced CFTR membrane localization comparable to CF phenotypes, that’s reversible after 3 weeks of VIP treatment (VIPKOT). In this thesis western blotting showed VPAC1 and VPAC2 expression increased in VIPKO and VIPKOT lung and duodenum tissues. The expression and maturation of CFTR was unchanged in both VIPKO and VIPKOT tissues. The results showed absence of VIP caused increased receptor expression in VIPKO mice, after VIP treatment VIPKO mice maintained increased receptor expression. VIP treatment reduces inflammation and restores existing CFTR membrane localization in VIPKO mice. VIP receptor expression may be important for future treatment of CF for CFTR localization and reducing tissue inflammation.

The pulmonary infection with Pseudomonas aeruginosa is considered as one of the main causes of health deterioration in cystic fibrosis patients (CF). Efficient management of P. aeruginosa in CF remains difficult mainly with the emergence of multidrug-resistant strains leading ultimately to death. There is a pressing need for new approaches to control these Pseudomonal infections. Current studies on the antimicrobial efficacy of liposomal antibiotics have shown conflicting results. We sought to assess whether the incorporation of clarithromycin into liposomes could improve its antibacterial activity against clinical isolate of Pseudomonas aeruginosa from CF patients. Different formulations of liposomal clarithromycin were prepared, characterized and their antibacterial activities against resistant strains of P. aeruginosa were investigated. These formulations reduced the biofilm formation, the virulence factors production and the bacterial motilities compared to free drug. The therapeutic importance of liposome containing macrolides in the management of experimental pseudomonal lung infection in animals is warranted.

No description available.

This thesis is concerned with families and chronic illness in childhood. The specific focus of the study is the nursing contribution to support of the family. A longitudinal study of the experience of four families caring for a child with cystic fibrosis was carried out using the ethnographic approach. Analysis of the four case studies provides insight to the effect of cystic fibrosis on each family member and on family interaction. The psychosocial transition by which the families moved from seeing themselves as healthy families to accepting the reality of a long-term health problem is described. The complexity of the effect of the genetic aspects and the grim prognosis is explored. The importance of finding meaning in suffering, the experiences of crisis and the chronic burden of care during the long-term adaptive stage of the illness are described. The support networks used by the families, and the nurse's contribution to that support are analysed. The research arose from practice and raises issues for nursing - issues relating to the level of interpersonal skill and the emotional investment required. The theoretical underpinning of nursing interaction is elaborated and the utility of systems theory in the understanding of the nursing situation is discussed. The importance of synchrony in the parents' adaptation to the child's illness is an emergent theme. The implications of the findings for nursing practice, management and research are discussed. The case for the development of a concept of family nursing to meet contemporary health care needs is argued.

Medical advancements and research initiatives in the last two decades have changed the experience of growing up with a chronic illness. Young people living with Cystic Fibrosis (CF), a chronic, life-threatening, life-limiting, genetic disease, have benefitted from these advances and are living fuller, healthier, longer lives than previously thought possible. Literature exploring the experiences of young people living with CF has traditionally relied on information from caregivers and health care practitioners. It does not reflect the diverse experiences of young people today, or explore the subjective meanings constructed from experiences. Using a social constructionist and narrative inspired methodology, this study explores illness narratives and identity constructions among three young women living with CF. Their narratives are broad and diverse. Shared elements include; making meaning of their illness, and constructing a multi-faceted, relational, layered and flexible sense of self. The layered experiences of CF are one of many important factors influencing their unfolding identity. Relational processes and socially constructed norms and expectations of illness, health, and gender also influence participants’ unfolding sense of self. This study demonstrates the value of rich conversations exploring identity construction and illness narratives, and the complexities and nuances within individual experiences. / Graduate / 0758