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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

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

The separation, identification, and characterisation of the proteins present in a tissue or biological sample is called ‘proteomics’. This technique can be used for example to identify biomarkers and investigate signalling pathways. Increasingly, proteomics is being applied to the analysis of virus related samples; here two such examples are described. Presently there is no reliable non-invasive way of assessing liver fibrosis. Here a novel 2D-PAGE based proteomics study was used to identify potential fibrosis biomarkers. Serum from patients with varying degrees of hepatic scarring induced by infection with the hepatitis C virus (HCV) was analysed. Several proteins associated with liver scarring and/or viral infection were identified. The most prominent changes were observed when comparing serum samples from cirrhotic patients with healthy controls: Expression of inter-α-trypsin inhibitor heavy chain H4 fragments, α1 antichymotrypsin, apolipoprotein L1 (Apo L1), prealbumin and albumin was decreased in cirrhotic serum, whereas CD5 antigen like protein (CD5L) and β2 glycoprotein I (β2GPI) increased. In general, α2 macroglobulin (a2M) and immunoglobulin components increased with hepatic fibrosis whereas haptoglobin and complement components (C3, C4 and factor H-related protein 1) decreased. Novel proteins associated with HCV-induced fibrosis include the inter-alpha-trypsin inhibitor heavy chain H4 fragments, complement factor H-related protein 1, CD5L, Apo L1, β2GPI and the increase in thiolester cleaved products of a2M. The relationship between these changes is discussed. One of the accessory genes of the HIV viral genome encodes for the Nef protein. Nef is present in lipid rafts and increases viral replication within infected host cells by binding to a guanine nucleotide exchange factor, Vav. This leads to activation of a GTPase, Cdc42, however, the signalling pathway is poorly understood. 2D-PAGE based proteomics was used to identify differentially expressed raft-associated proteins by comparing T cells in the presence and absence of Nef. A ubiquitin conjugating enzyme UbcH7, which acts in conjugation with c-Cbl, was absent from the rafts of Nef-transfected cells. Vav ubiquitination was also absent from these rafts. In collaboration with Dr. Alison Simmons and Prof. Andrew McMichael the absence of UbcH7 in rafts was found to be caused by β-Pix forming a ternary complex with c-Cbl and activated Cdc42. Vav ubiquitination was restored and viral replication was diminished when β-Pix was knocked down providing a new candidate target for inhibiting HIV replication. This thesis demonstrates the use of proteomics in providing novel information for virus related samples. This influential technology benefits in both biomarker discovery to aid clinicians with early diagnosis of diseased individuals and in the elucidation of novel signalling pathways in infected cells to provide new candidate targets.

Introduction: Non-invasive assessment of steatosis and fibrosis is of growing relevance in non-alcoholic fatty liver disease (NAFLD). 1H-Magnetic resonance spectroscopy (1H-MRS) and the ultrasound-based controlled attenuation parameter (CAP) correlate with biopsy proven steatosis, but have not been correlated with each other so far. We therefore performed a headto- head comparison between both methods. Methods: Fifty patients with biopsy-proven NAFLD and 15 healthy volunteers were evaluated with 1H-MRS and transient elastography (TE) including CAP. Steatosis was defined according to the percentage of affected hepatocytes: S1 5-33%, S2 34–66%, S3 $67%. Results: Steatosis grade in patients with NAFLD was S1 36%, S2 40% and S3 24%. CAP and 1H-MRS significantly correlated with histopathology and showed comparable accuracy for the detection of hepatic steatosis: areas under the receiveroperating characteristics curves were 0.93 vs. 0.88 for steatosis $S1 and 0.94 vs. 0.88 for $S2, respectively. Boot-strapping analysis revealed a CAP cut-off of 300 dB/m for detection of S2-3 steatosis, while retaining the lower cut-off of 215 dB/m for the definition of healthy individuals. Direct comparison between CAP and 1H-MRS revealed only modest correlation (total cohort: r = 0.63 [0.44, 0.76]; NAFLD cases: r = 0.56 [0.32, 0.74]). For detection of F2–4 fibrosis TE had sensitivity and specificity of 100% and 98.1% at a cut-off value of 8.85 kPa. Conclusion: Our data suggest a comparable diagnostic value of CAP and 1H-MRS for hepatic steatosis quantification. Combined with the simultaneous TE fibrosis assessment, CAP represents an efficient method for non-invasive characterization of NAFLD. Limited correlation between CAP and 1H-MRS may be explained by different technical aspects, anthropometry, and presence of advanced liver fibrosis.

The objective of this study was to investigate the extent to which vitamin D and K are associated with bone health in pediatric cystic fibrosis (CF) patients. We hypothesized that: (1) the prevalence of vitamin D and K deficiencies would be high despite routine vitamin therapy, (2) bone health would be reduced and (3) vitamin K and D status would be associated with bone health. / Our results showed poor bone mineral mass in these CF children despite mild disease and good nutritional status. Neither vitamin K nor D was a predictor of bone health but weight and height Z-scores, fat-free mass, physical activity and lung function were all consistent predictors. / These results indicate that nutritional status as well as physical activity are key determinants of bone health in CF children and offer a unique opportunity in the prevention of CF-related bone disease. Further vitamin intervention research needs to be done in this population.

Genetic studies of lung disease in Cystic Fibrosis are faced with the challenge of identifying a severity measure that accounts for chronic disease progression and mortality attrition. Further, combining analyses across studies requires common phenotypes that are robust to study design and patient ascertainment. This thesis uses data from the North American Cystic Fibrosis Modifier Consortium (Canadian Consortium for CF Genetic Studies (CGS), Johns Hopkins University Twins and Siblings Study (TSS), and University of North Carolina/Case Western Reserve University Gene Modifier Study (GMS)), to calculate two novel phenotypes using age-specific CF percentile values of FEV1 (Forced Expiratory Volume in 1 second), with adjustment for CF age-specific mortality. The normalized residual, mortality adjusted (NoRMA) was designed for population based samples, while KNoRMA, using Kulich percentiles, is robust to sample ascertainment; both account for the effects of age-related disease progression and mortality attrition. NoRMA was computed for 2122 patients representing the Canadian CF population. KNoRMA was computed for these 2122 patients and also 1137 extreme phenotype patients in the GMS study and 1323 patients from multiple CF sib families in the TSS study. Phenotype was distributed in all three samples in a manner consistent with ascertainment differences, reflecting the lung disease severity of each individual in the underlying population. The new phenotype was highly correlated with the previously recommended mixed model phenotype1; 2, but computationally much easier and suited to studies with limited follow up time. As an example of its use, KNoRMA was used to test the association between locus variants in a previously published candidate gene, Transforming Growth Factor β1(TGFβ1), and lung function in CF, in an attempt to provide insight into discrepant results in the literature. A disease progression and mortality adjusted phenotype reduces the need for stratification or additional covariates, increasing statistical power and avoiding possible interpolation distortions.

Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.

Myocardial infarction activates the renin-angiotensin system, consequently upregulating aldosterone production that may stimulate pathological cardiac fibrosis via mineralocorticoid receptor (MR) activation. Results presented in this thesis were derived from an in vitro experimental model using cultures of human cardiac fibroblasts to study the effect of aldosterone on elastin production. They first confirmed that treatment with 1-50 nM of aldosterone leads to a significant increase in collagen type I production via MR activation. Most importantly, we discovered that treatment with 1-50 nM of aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition. Strikingly, pretreatment with MR antagonist spironolactone did not eliminate aldosterone-induced increases in elastin production. Interestingly, while cultures treated with elevated aldosterone concentrations (100 nM and 1 µM) showed a further increase (~3.5-fold) in collagen and (~3-fold) in elastin mRNA levels, they demonstrated subsequent increases only in the net deposition of collagen but not elastin. In fact, cultures treated with elevated aldosterone concentrations displayed a striking decrease in the net deposition of insoluble elastin, which could be reversed with spironolactone or with MMP inhibitors doxycycline or GM6001. Most importantly, we discovered that the pro-elastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) and that the IGF-IR kinase inhibitor AG1024 or an anti-IGF-IR neutralizing antibody inhibits both IGF-I- and aldosterone-induced elastogenesis (Bunda et al., Am J Pathol. 171:809-819, 2007). Furthermore, we showed that the PI3 kinase signaling pathway propagates the elastogenic signal following IGF-IR activation and that activation of c-Src is an important prerequisite for aldosterone-dependent facilitation of the IGF-IR/PI3 kinase signaling. Results of explorative microarray analysis of 1 hour aldosterone-treated cultures revealed that aldosterone treatment upregulated expression of a heterotrimeric G protein, Gα13, that activates the PI3 kinase signaling pathway. We additionally demonstrated that aldosterone treatment transiently increases the interaction between Gα13 and c-Src and that siRNA-dependent elimination of Gα13 inhibited the pro-elastogenic effect of aldosterone. In summary, aldosterone, which stimulates collagen production in cardiac fibroblasts through the MR-dependent pathway, also increases elastogenesis via a parallel MR-independent pathway involving the activation of Gα13, c-Src, and IGF-IR/PI3 kinase signaling.