This dissertation involved linking a clinical cystic fibrosis (CF) data registry with administrative databases to evaluate clinical, demographic, and geographical predictors of hospitalization in CF patients living in Ontario over a 10 year period. In addition, this work assessed the ability of administrative data to identify individuals with CF using the clinical registry as the reference standard. Sex was an independent predictor of hospitalization rates for individuals with CF. Females had a significantly higher hospitalization rate compared to males even after adjusting for important clinical factors suggesting that this finding is not simply due to worse CF disease. In those between 7 and 19 years of age, the adjusted hospitalization rate was 38% higher in females (rate ratio[RR] 1.38, 95% confidence interval [CI] 1.11-1.73). Similarly in those over the age of 19, females had a 30% higher hospitalization rate compared to males (RR 1.30, 95% CI 1.06-1.59). Other significant predictors associated with higher hospitalization rates in both age groups were lower lung function, worse nutritional status, pancreatic insufficiency, and the presence of CF-related diabetes. The presence of Burkholderia cepacia complex in the sputum was a significant predictor in those over the age of 19 years (RR 1.54, 95% CI 1.26-1.89). Distance to CF centre, community size and socioeconomic status were not significant predictors of hospitalization rates in either age group. There was no significant trend in hospitalization rates over time once rates were adjusted for markers of disease severity (p=0.08). Comparing administrative data with the CF registry data, administrative data captured hospitalizations more comprehensively. Despite CF being a specific diagnosis, health administrative databases alone were insufficient to reliably and accurately identify individuals with CF unless they had been hospitalized. The reason for the gender disparity seen within this dissertation is likely multifactorial. There may be differences in outpatient management between the sexes, hormonal influences may modulate disease severity causing higher hospitalization rates, and patient and provider-level influences may affect the decision to hospitalize a patient. Further research is needed in this area to elucidate the factors contributing to this gender gap.

Acute Respiratory Distress Syndrome (ARDS) following hemorrhagic shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit” hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with increased activation of the proinflammatory transcription factor NF-κB, and augmented expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which resides in plasma membrane lipid rafts. The objective of this work is to define cellular mechanisms of macrophage priming by oxidative stress following shock resuscitation. The main hypothesis investigated is that altered cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation strategies can diminish these effects. AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface levels of TLR4, which in turn results in augmented NF-κB translocation in response to small doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy iii transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress can be diminished by early exposure to resuscitation regimens with direct or indirect systemic antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline. Hyperosmolarity was found to modulate AM TLR4 gene and protein expression. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling pathway early during shock resuscitation may represent an anti-inflammatory strategy able to ameliorate late morbidity and mortality following S/R.

The family of human kallikrein-related peptidases (KLKs) numbers 15 serine proteinases implicated in tumour progression. Despite the wide tissue distribution of KLKs and the numerous reports of their differential expression in pathological settings, the signalling mechanism(s) whereby these enzymes regulate tissue function are not yet known. Further, knowledge of the levels of their activity, as well as of their potential endogenous targets, has only been extracted from in vitro studies and cell culture systems. We hypothesized that KLKs can trigger tumour signalling via proteinase-activated receptors (PARs), a family of G-protein-coupled receptors. To test our hypothesis, we evaluated the ability of KLKs 5, 6, and 14: to activate or prevent signalling via PARs 1, 2, and 4 in cells and tissues expressing these receptors. Further, we used a novel activity-based probe approach, coupled with conventional immunoassay (ELISA), to determine the abundance of active KLK6 relative to total immunoreactive KLK6 in cancer-related biological fluids. We concluded that KLKs can regulate multiple signalling pathways triggered by PARs 1, 2, and 4, resulting in calcium release, platelet aggregation and vascular relaxation, and they can cause murine inflammation. Further, our activity-based ELISA demonstrated the presence of active KLK6 in ovarian cancer ascites fluids and cancer cell supernatants. We, therefore, suggest that tumours can produce active KLKs, which can potentially control tumour behaviour by regulating PAR activity.

Background: Traumatic musculoskeletal injury (TMsI) often leads to chronic pain and post-traumatic stress disorder (PTSD). This study examined factors of a modified diathesis-stress model in the development of PTSD symptoms following TMsI. Methods: 205 patients were recruited in this prospective, observational study. Within 14 days of injury, participants completed an in-hospital questionnaire investigating acute symptoms of anxiety, depression, pain, and PTSD. Results: Logistic regression identified multiple factors associated with symptoms of PSTD (p<.0001). Neuropathic pain (odds ratio[OR]=1.091, 95% confidence interval[CI] 1.020-1.168), general anxiety (OR=1.176, 95%CI 1.046-1.318), pain anxiety (OR=1.056, 95%CI 1.018-1.094), and pain catastrophizing (OR=1.168, 95%CI 1.016-1.348) were associated with acute symptoms of PTSD. Conclusions: The results support the modified diathesis-stress model indicating that neuropathic pain, general anxiety, pain anxiety, and pain catastrophizing are associated with symptoms of PTSD. Future studies should examine the influence of these acute factors on the development of chronic pain and PTSD following TMsI.

Master of Science / Department of Anatomy & Physiology / Deryl L. Troyer / Breast cancer ranks second as a cause of cancer death in women in the USA. Detection of early tumors and tumor-targeted treatments could decrease the problems associated with breast cancer management. Photodynamic therapy (PDT) is a cancer treatment that uses a photosensitizer and a specific wavelength of light and is currently in clinical trials for breast cancer. When tumor cells which have absorbed photosensitizer are exposed to the correct wavelength of light, reactive oxygen species are generated, resulting in tumor cell death. Poor tissue penetration of light is a major limitation in PDT, restricting its use to treatment of localized tumors. Light generation at the tumor area might increase the effectiveness of PDT. Polymorphonuclear neutrophils (PMNs) are known to often infiltrate breast adenocarcinoma, and their activatation in tumor stroma produces luminescence in the presence of luminol. Here, we hypothesized that luminol can be used as a theranostic agent for luminescence-based early tumor detection (diagnosis) and in situ PDT (treatment). BALB/c mice were transplanted with 4T1 mammary adenocarcinoma cells to establish a breast adenocarcinoma model. The early tumor detection objective was tested by daily intraperitoneal injection of luminol and in vivo luminescence imaging. To test the PDT treatment objective,the photosensitizer 5-aminolevulinic acid (ALA) and luminol were administered to mice through intraperitoneal and intravenous routes, respectively. This treatment regimen was repeated six times and ALA alone/luminol alone/saline treated tumor-bearing mice were used as controls. Results demonstrated that luminol allowed detection of activated PMNs only two days after 4T1 cell transplantation, even though tumors were not yet palpable. Relative differences in the increase of tumor volume and final tumor weights were analyzed to test the in situ PDT. Analysis of the data showed luminol treatments resulted in breast adenocarcinoma tumor growth attenuation. In conclusion this study provides evidence that luminol can be a theranostic agent for breast adenocarcinoma.

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF���s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.

An important mechanism involved in the pathogenesis of type 2 diabetes is elevation of plasma free fatty acids which induce insulin resistance and may impair both β-cell function and mass (β-cell lipotoxicity). The objective of my thesis was to investigate the role of oxidative stress in β-cell lipotoxicity, using in vivo, ex vivo, and in vitro models. I used in vivo models of 48h i.v. oleate or olive oil infusion in Wistar rats followed by hyperglycemic clamps, or islet secretion studies ex vivo, and in vitro models of 48h exposure to oleate in isolated islets. My first study showed that 48h oleate infusion decreased the insulin response to a hyperglycemic clamp, an effect prevented by coinfusion of the antioxidants N-acetylcysteine and taurine. Similar to the findings in vivo, 48h infusion of oleate decreased glucose stimulated insulin secretion (GSIS) ex vivo, and induced oxidative stress in isolated islets, effects prevented by coinfusion of the antioxidants N-acetylcysteine, taurine, or tempol. Islets exposed to oleate or palmitate showed a decreased insulin response to high glucose and increased levels of oxidative stress, effects prevented by taurine. Therefore, my data are the first demonstration that oxidative stress plays a role in the decrease in β-cell secretory function induced by prolonged exposure to FFA, in vitro and in vivo. My second study addressed downstream effects of oxidative stress involving inflammation. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the IKKβ inhibitor salicylate. GSIS in isolated islets was impaired by olive oil or oleate and restored by salicylate. These results suggest a potential role for both oxidative stress and inflammation in lipid-induced β-cell dysfunction. My third study addressed downstream effects of oxidative stress involving β-cell insulin signalling. A 48h infusion of oleate or olive oil decreased β-cell function during a hyperglycemic clamp, an effect prevented by coinfusion of the tyrosine phosphatase inhibitor bisperoxovanadate. GSIS in isolated islets was impaired by olive oil or oleate and restored by bisperoxovanadate, suggesting a role of FFA in decreasing β-cell function by induction of β-cell insulin resistance.

Degenerative disease of the lumbar spine (DDLS) is a common condition for which surgery is beneficial in selected patients. Wide variation in surgical referral and rates of surgery has been observed contributing to unequal access to care. Our objectives were to examine (1) the variation in preferences for referral and surgery among surgeons, family physicians (FPs) and patients, (2) how FP referral practices compare with preferences and guideline recommendations, and (3) how the ‘enthusiasm’ of patients and physicians influence regional variation in surgical rates. We used conjoint analysis in a mailed survey to elicit preferences based on clinical vignettes from surgeons, FPs and patients. A Delphi expert panel provided consensus guideline recommendations for surgical referral to compare with actual FP referral practices. Rates of surgery for DDLS, obtained from Ontario hospital discharge data, were used to quantify regional variation and regression models assessed the relationship with patient and physician enthusiasm. We identified significant differences in preferences for referral and surgery between patients, FPs and surgeons. Surgeons placed high importance on leg-dominant symptoms while patients had high importance for quality of life symptoms (i.e. severity, duration, walking tolerance). Surgical referral practices were poorly predicted by individual FP preferences and guideline recommendations based on clinical factors alone. Variation in Ontario surgical rates was higher than that of hip or knee replacements and was highly associated with the enthusiasm of surgeons (p<0.008), rather than FPs or patients. By appreciating the variation in preferences between patients and physicians, and exploring other non-clinical factors that influence referrals, we may be able to improve the efficiency of referrals and enhance the shared decision making process. With an understanding of the influence that surgeons have in driving variation in surgical rates, further research may allow us to direct strategies to improve access and allow for a more equitable delivery of care for patients suffering from DDLS.

Context: The study of expertise in medical education has tended to follow the traditions of describing either the analytic processes or the non-analytic resources that experts acquire with experience. We argue that a critical function of expertise is the ability to transition from the automatic mode to the more effortful mode when required – a transition referred to as ‘slowing down when you should’. Objectives: To explore the phenomenon of ‘slowing down when you should’ in operative surgical practice and its role in intra-operative surgical judgment, and to develop conceptual models of the factors involved in the display of this transition in surgical operative practice. Design: In Phase 1A, 28 surgeons were interviewed about their views of surgical judgment in general and their perceptions of the role of this phenomenon in operative judgment. In Phase 1B, a subset of surgeons from Phase 1A was re-interviewed to explore their perceptions of automaticity in operative practice. In Phase 2, observational sessions (and brief interviews) were conducted of surgeons in the operating room to explore the nature of this phenomenon in its natural environment. Results: The surgeons in this study recognized the phenomenon of ‘slowing down’ in their operative practice and acknowledged its link to surgical judgment. Two main initiators were described and observed: proactively planned ‘slowing down’ moments occurring intra-operatively initiated by critical events anticipated pre-operatively and situationally responsive ‘slowing down’ moments initiated by emergent cues intra-operatively. Numerous influences of this transition were uncovered. A control dynamic emerged as surgeon’s negotiated ‘slowing down’ moments through trainees in their supervisory academic practice. Numerous manifestations of this phenomenon were observed in the operating room and considered using a cognitive psychology attention capacity model. Conclusions: This study offers a conceptual framework for understanding the role of ‘slowing down when you should’ in operative surgical practice, providing a vocabulary that will allow more explicit consideration of what contributes to surgical expertise. Consideration of this framework with its consequent ability to make surgical practices more explicit has implications for self-regulation in practice, surgical error, and surgical training.

Familial Pancreatic Cancer (FPC) is a cancer syndrome characterized by clustering of pancreatic cancer in families, but most FPC cases do not have a known genetic etiology. Understanding genetic predisposition to pancreatic cancer is important for improving screening as well as treatment. The central aim of this thesis is to identify candidate susceptibility genes for FPC, and I used three approaches of increasing resolution. First, based on a candidate-gene approach, I hypothesized that BRCA1 is inactivated by loss-of-heterozygosity in pancreatic adenocarcinoma of germline mutation carriers. I demonstrated that 5/7 pancreatic tumors from BRCA1-mutation carriers show LOH, compared to only 1/9 sporadic tumors, suggesting that BRCA1 inactivation is involved in tumorigenesis in germline mutation carriers. Second, I hypothesized that the germline genomes of FPC subjects differ in copy-number profile from healthy genomes, and that regions affected by rare deletions or duplications in FPC subjects overlap candidate tumor-suppressors or oncogenes. I found no significant difference in the global copy-number profile of FPC and control genomes, but I identified 93 copy-number variable genomic regions unique to FPC subjects, overlapping 88 genes of which several have functional roles in cancer development. I investigated one duplication to sequence the breakpoints, but I found that this duplication did not segregate with disease in the affected family. Third, I hypothesized that in a family with multiple pancreatic cancer patients, genes containing rare variants shared by the affected members constitute susceptibility genes. Using next-generation sequencing to capture most bases in coding regions of the genome, I interrogated the germline exome of three relatives who died of pancreatic cancer and a relative who is healthy at advanced age. I identified a short-list of nine candidate genes with unreported mutations shared by the three affected relatives and absent in the unaffected relative, of which a few had functional relevance to tumorigenesis. I performed Sanger sequencing to screen an unrelated cohort of approximately 70 FPC patients for mutations in the top two candidate genes, but I found no additional rare variants in those genes. In conclusion, I present a list of candidate FPC susceptibility genes for further validation and investigation in future studies.