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<p>This work used biotechnology to invent new caloxins - allosteric peptide inhibitors of plasma membrane Ca²⁺pumps (PMCA) needed to understand the Ca²⁺signalling in coronary artery.</p> <p>PMCA are encoded by genes PMCA1-4. Defects in PMCA expression have been associated with several pathologies. The major objectives of my thesis were to determine the expression of PMCA isoforms in the smooth muscle and the endothelium of coronary artery and to invent high affinity and specificity caloxins for the isoforms present in these tissues.</p> <p>In Aim 1 it was determined that the total PMCA protein and activity was much greater in smooth muscle than in endothelium. Both tissues expressed only PMCA1 and PMCA4, with PMCA4 > PMCA1 in smooth muscle and PMCA1 > PMCA4 in endothelium. Therefore, the search for PMCA1 and 4 selective caloxins using phage display technique was conducted.</p> <p>Aim 2 was to invent PMCA1 selective inhibitors. Caloxin 1b3 was invented as the first known PMCA1 selective inhibitor. It inhibited PMCA1 Ca²⁺-Mg²⁺-ATPase with higher affinity than PMCA2, 3 or 4. Aims 1 and 2 were consistent with the greater potency of caloxin 1b3 than a known PMCA4 selective caloxin 1b1 in increasing cytosolic Ca²⁺ concentration in endothelial cells.</p> <p>Aim 3 was to obtain ultrahigh selectivity and affinity PMCA4 bidentate inhibitor using the previously invented PMCA4 selective caloxins 1c2 and 1b2. In the first step the affinity of caloxin 1b2 was improved by limited mutagenesis to obtain caloxin 1c4. Caloxin 1c4 had 5-6 times higher affinity than caloxin 1b2 for inhibiting PMCA4 activity. Optimization of the bidentate caloxins from caloxin 1c2 and 1c4 was also attempted.</p> <p>The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.</p> / Doctor of Philosophy (PhD)

<p> Meta-analysis is a statistical method to summarize the overall evidence of effects on intervention by systematically combining outcomes from available studies in the literature which are homogeneous in research methodology and research interest. The objective of this project is to evaluate the treatment effects of preoperative aspirin on bleeding and other cardiovascular outcomes from 11 randomized control trials (RCT) and 19 observational (non-RCT) studies. Both Bayesian meta-analysis and classical (frequentist) meta-analysis were applied to continuous and binary outcomes, and the results were compared.</p> <p> The robustness of the Bayesian approach is assessed by examining the performances of different likelihood functions and priors. We also discuss strategies on dealing with zero-event studies for binary outcomes, and the implementation of multiple imputation (MI) technique to missing data for continuous outcomes.</p> <p> Most results of primary analysis agree between the Bayesian and classical approaches. We suggest that the final conclusion of a meta-analysis should be based on the comparison of the results from both Bayesian and classical approaches.</p> / Thesis / Master of Science (MSc)

<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

Background: Coronary Artery Disease (CAD) represents the leading cause of mortality and morbidity worldwide despite declines in the prevalence of environmental risk factors. This trend has drawn attention to the risk conferred by genetic variation. Twin and linkage studies demonstrate a profound hereditary risk for CAD, especially in young individuals. Rare genetic variants conferring high risk for extreme disease phenotypes can provide invaluable insight into novel mechanisms underlying CAD development. Methods: Whole exome sequencing was performed to characterize rare protein-altering variants in 52 early-onset CAD (EOCAD) patients encompassing the DECODE study. The enrichment of Mendelian dyslipidemias in EOCAD was assessed through interrogation of pathogenic mutations among known lipid genes. The identification of novel genetic CAD associations was conducted through case-only and case-control approaches across all protein-coding genes using rare variant burden and variance component tests. Lastly, beta coefficients for significant risk genes from the European population in the Early-onset Myocardial Infarction (EOMI) cohort (N=552) were used to construct calibrated, single-sample rare variant gene scores (RVGS) in DECODE Europeans (N=39) and a local European CAD-free cohort (N=77). Results: A 20-fold enrichment of Familial hypercholesterolemia mutation carriers was detected in EOCAD cases compared to CAD-free controls (P=0.005). Association analysis using EOMI Europeans revealed exome-wide and nominal significance for two known CAD/MI genes: CELSR2 (P=1.1x10-17) and APOA5 (P=0.001). DECODE association revealed exome-wide and nominal significance for genes involved in endothelial integrity and immune cell activity. RVGS based upon beta coefficients of significant CAD/MI risk genes were significantly increased in DECODE (z-score=1.84; p=0.03) and insignificantly decreased among CAD-free individuals (z-score=-1.61; p=0.053). Conclusion: Rare variants play a pivotal role in the development early CAD through Mendelian and polygenic mechanisms. Construction of RVGS that are calibrated against population and technical biases can facilitate discovery of single-sample and cohort-based associations beyond what is detectable using standard methods. / Thesis / Master of Science (MSc)

The functional capacity of patients with coronary artery disease is often limited by breathlessness. The intensity of breathlessness is primarily determined by respiratory muscle effort, which increases as the pressures generated by the inspiratory muscles approach maximum capacity. Exercise training can potentially decrease breathlessness by inducing adaptations that reduce the ventilatory demand and increase the maximum capacity of the respiratory muscles. This study examines the extent to which the physiological adaptations occurring with exercise training contribute to reductions in breathlessness in patients with coronary artery disease. The pre- and post-training results were compared in 21 patients participating in the McMaster University Cardiac Rehabilitation program. Respiratory parameters were measured at rest and during a progressive maximal exercise test. The intensity of breathlessness was measured by psychophysical techniques using the Borg scale. Following training, the capacity to generate maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and maximum inspiratory flow (Vimax) improved by 22%, 18% and 5%, respectively, with no significant change in vital capacity, forced expiratory volume in one second and maximum expiratory flow. Exercise capacity improved by 14%, with an 11% decrease in the maximum intensity of breathlessness experienced. Although maximum ventilation (Ve) did not change significantly, Ve per unit workload decreased by 10% and breathlessness per unit Ve decreased by 13%. At the highest similar workload between the 2 tests (850 kpm), breathlessness decreased by 34%, with a 19% reduction in Ve. Breathing frequency, mean inspiratory flow (Vt/Ti), inspiratory flow generated during exercise in relation to maximum inspiratory flow (Vi%max), and time of inspiration in relation to total duty cycle (Ti%Ttot) decreased significantly by 20%, 15%, 20% and 4%, respectively. The decreases in breathlessness were significantly related to the reduction in ventilation associated with training. A significant decrease in breathlessness remained after removing the variance accounted for by Ve, reflecting the improvements in respiratory muscle performance. The decreases in breathlessness were better correlated with improvements in the dynamic measure of Vimax than the static measure of MIP. The decreases in breathlessness were best correlated to the changes in Vi%max, which accounts for decreases in ventilatory demand and increases in respiratory muscle performance. Decreases in breathlessness primarily occurred in patients who achieved a training effect. Exercise training decreases breathlessness in patients with coronary artery disease largely through decreases in Ve, with improvements in respiratory muscle performance contributing to a lesser degree. / Thesis / Master of Science (MSc)

Health-related quality of life (HRQL) measures taken before and after coronary artery bypass grafting (CABG) aid in determining meaningful patient-perceived outcomes associated with alternative clinical interventions. This study compared performance of the Medical Outcomes Study Short Form-36 (MOS SF-36) subscales for Physical Functioning (PF), Role Physical (RP), Mental Health (MH), and Role Emotional (RE) against two other questionnaires, i.e. the Veteran's Specific Activity Questionnaire (VSAQ: self-efficacy for vigorous physical activity) and the Beck Depression Inventory (BDI-II: mental-emotional functioning). Seventy-one patients (59-M; 12-F; age, Mean + SD = 63 ± 8.6 years) were administered these three questionnaires just before and 3 months following CABG surgery. Score distributions were evaluated for the pre- and post-surgery measurements, as were change scores after CABG. All measures except the MOS SF-36 subscales for RP and RE showed statistically significant change after CABG (p<0.01). Only the subscales of RP and RE demonstrated substantial ceiling (21.0% and 56.3%) and floor effects (49.3% and 16.9%). Evaluation of individual change scores after CABG indicated that 59% and 62% of the patients, respectively, had clinically meaningful increases in the two measures of physical capability, i.e. PF and VSAQ. In contrast, 60% and 72% of patients, respectively, showed no clinically meaningful changes in the two measures of emotional functioning, i.e. RE and BDI-II scores. Chi-square analyses revealed that use of scales with similar definitional constructs resulted in significantly different surgical outcomes for the following: PF vs VSAQ (p<0.001), RP vs VSAQ (p<0.02); and MH vs BDI-II (p<0.0001). These findings illustrate the limitations in performance of the MOS SF-36 for assessing changes of importance in HRQL after CABG. The VSAQ and BDI-II, two simple measures of physical and emotional functioning that are fundamentally similar to those contained in the MOS SF-36, appear to be sensitive markers for detecting changes in these important outcomes after CABG surgery. / Master of Science

A patient monitoring system for cardiopulmonary bypass surgery has been developed. This monitoring system uses a SWAN 286-10 computer (fully IBM PC/AT compatible) and a DT280l-A Input/Output board to monitor seven surgical parameters. This system monitors six temperatures, the hemoglobin content, the arterial oxygen saturation, the venous oxygen saturation, the oxygen consumption, and the blood flow rate through the cardiopulmonary bypass circuit. Additionally, there are three individual timers available. Details and the evaluation of the hardware and software design of this monitoring system are presented. Also, recommendations for clinical use are discussed. / Master of Science / incomplete_metadata