Biomedical Physiology and Kinesiology - Theses, Dissertations, and other Required Graduate Degree Essays

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Production of hiPSC-derived atrial cardiomyocytes to study the contribution of the KCNN3 variants to lone atrial fibrillation

Author: 
Date created: 
2019-05-28
Abstract: 

Atrial fibrillation (AF), is the most common cardiac arrhythmia worldwide. AF increases the risk of stroke five-fold and heart failure three-fold. Over a quarter of AF patients suffer from lone AF which has been found to have a significant genetic component. Recently, a number of GWAS studies have found KCNN3, the gene expressing a Ca2+-activated K+ channel SK3, to be associated with lone AF. AF is a complex disease that is difficult to study with current experimental models. The advent of pluripotent stem cell (PSC) derived cardiomyocytes (hPSC-CMs) has revolutionized the field of cardiac research. For the first time, we are able to study human disease in human models while avoiding the challenges of obtaining biopsy tissue. Additionally, we are able to study a patient’s disease in a personalized manner by the use the patient-derived induced pluripotent stem cells (hiPSCs). Current differentiation protocols result in a mixed cardiac population that consists of nodal, atrial, and ventricular cells. This makes the study of chamber-specific diseases, like atrial fibrillation (AF), difficult. As such, the development of atrial-specific differentiation protocols is vital. Using retinoic acid, we optimized a protocol to selectively differentiate hiPSC-derived atrial cardiomyocytes (hiPSC-aCMs). We found that the addition of retinoic acid from days 4 – 6 at a concentration of 0.75 µM resulted in a predominantly atrial phenotype at a transcript, protein, and functional level. We then used CRISPR-Cas9 genome editing technology to insert an early stop codon into exon 7 of the KCNN3 gene to knockout its expression. In the future, we hope to differentiate these cells into hiPSC-aCMs to determine the contribution of SK3 to cardiac function and potentially AF.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Glen Tibbits
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

Development and evaluation of an in vitro model of exercise for studying AMPK signaling dynamics in skeletal muscle

Author: 
Date created: 
2020-03-13
Abstract: 

Exercise promotes AMP-activated protein kinase (AMPK) signaling in skeletal muscle, where it functions to enhance the expression of fitness-promoting genes. The magnitude of the adaptations depends in part on the dynamics of AMPK signaling; however, the time course of AMPK signaling remains poorly characterized. The purpose of my thesis was to develop and evaluate electrical stimulation of cultured C2C12 myotubes as a method to study AMPK signaling dynamics. I confirmed that differentiation resulted in contractile C2C12 myotubes, that AMPK signaling was detectable, and that electrical stimulation increased cellular oxygen consumption. In response to three hours of electrical stimulation, AMPK signaling increased. Upon cessation of stimulation, AMPK signaling decreased. However, the magnitude of signaling was marginal, such that further work is required to define experimental conditions that lead to robust AMPK signaling. I conclude that electrical stimulation of C2C12 myotubes is a promising means to study AMPK signaling dynamics.

Document type: 
Thesis
Senior supervisor: 
David Clarke
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

Investigating the function and pharmacology of human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs)

Author: 
Date created: 
2019-04-30
Abstract: 

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia that causes the irregular and uncoordinated contractions of the atrial chambers. Current first-line pharmacological treatments are limited in efficacy with side effects including ventricular proarrhythmia. Thus, it is imperative to find novel treatments for better management of the disease. However, current preclinical assays such as heterologous expression and animal models do not recapitulate the entirety of human cardiac physiology. As such, the ability to generate hiPSC-derived atrial-like CMs (hiPSC-aCMs) and ventricular-like CMs (hiPSC-vCMs) can provide a more robust physiological system to assess drug effects for AF treatment in vitro. The objective of this thesis is to develop a preclinical assay system using optical mapping technique and human induced pluripotent stem cells (hiPSCs). Here, I characterized the function of hiPSC-aCMs and demonstrated the sensitivity and specificity of the assay system in capturing the effects of atrial-selective compounds.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Glen F. Tibbits
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

Spatial and temporal gaze decisions during walking: role of uncertainty, task priority, and motor cost

Date created: 
2020-01-14
Abstract: 

We continuously use vision to navigate the cluttered environment in which we live. To accomplish this, we adapt the location and timing of gaze shifts to gain environmental information to achieve a behavioural goal. However, despite the growing interest in eye tracking research during natural behaviours, the factors that guide gaze behaviour to accurately navigate and interact with our environment still remain unclear. The goal of this thesis is to determine the relationship between environmental, cognitive, and biomechanical factors in the control of gaze during visually-guided walking. In the first study, I sought to understand how environmental uncertainty influences gaze behaviour to accurately perform a motor action. To test this, I used a visually-guided walking task where I manipulated the visual uncertainty associated with stepping targets. Using different task instructions to manipulate the value assigned to foot-placement accuracy, I found that environmental uncertainty increases gaze time on visual targets when having to step accurately. In the second study, I tested if motor cost, a factor that influences the way we move, is integrated into the decision of when to shift gaze to upcoming stepping targets. I found that the cost associated with redirecting foot placement onto a target modifies how gaze is allocated; when the cost to move the body increases, gaze strategies shift from one that focuses on the planning of future steps to one that prioritizes online visual control of the current action. After identifying how uncertainty, motor cost, and task priority influence gaze behaviour, in the third study, I aimed to understand how these factors interact to decide where to look when facing multiple choices for foot placement. Using a forced-choice walking paradigm, I showed that when facing a decision conflict, where two targets compete for gaze allocation, people sample the environment using different strategies that lead to differences in walking decisions. This suggests that, during walking, individuals assign a different priority to information and motor cost. Taken together, my thesis provides a novel perspective on the factors that guide gaze strategies during walking.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Daniel Marigold
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.

Effects of troponin cardiomyopathy mutations on the calcium binding properties of the troponin complex and reconstituted thin filaments

Author: 
Date created: 
2019-04-18
Abstract: 

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease that could result in sudden cardiac death. Mutations in the genes encoding sarcomeric proteins, including the thin filaments, are the most common cause of HCM. Thin filaments are an integral part of the cardiac muscle contractile unit, composed of actin, tropomyosin, and troponin (Tn) complexes which contain troponin C (TnC), troponin I (TnI) and troponin T (TnT). HCM molecular mechanisms remain unclear, partially due to the lack of a high-resolution thin filament structure and the complex molecular interactions between each component. My first goal was to investigate the effects of three TnT mutations, I79N, F110I and R287C, in human reconstituted thin filaments (RTF), using steady-state and stopped-flow fluorometry to determine Ca2+ sensitivity (Kd) and Ca2+ dissociation rates (koff), respectively. Our data showed that I79N and R278C mutations significantly decreased Kd by lowering koff, and all three mutations attenuated the functional effects of phosphomimetic TnI, suggesting an important role in impaired relaxation with HCM. My second goal was to investigate the effects of the I79N TnT mutation and the fetal cardiac R37C TnI mutation in their corresponding adult/fetal RTF. The I79N TnT mutation did not change the Ca2+ binding properties in fetal RTF but significantly decreased the Kd in adult RTF. In contrast, the R37C TnI mutation significantly increased the Kd in fetal RTF, yet its corresponding mutation, R68C TnI in adult RTF, exhibited reverse Ca2+ binding properties. My third goal was to use cryo-electron microscopy (EM) to solve the RTF structure. Optimal buffer conditions were found using negative-stain EM to ensure Tn binding on RTF with a periodicity of 38.5 nm; however, unexpected challenges arose during cryo data collection. Persistent filament aggregations obscured most of the cryo-images. Suggestions on how to address this problem are provided in the last chapter. In summary, using cardiac thin filaments as a physiologically relevant biochemical model allows us to investigate how HCM mutations alter Ca2+ binding properties. The resulting studies provide a better understanding of HCM molecular mechanism and can potentially help develop specific therapies that address the underlying causes of the disease.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Glen F. Tibbits
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.

Voltage-gated sodium channels and their role in prostate cancer

Author: 
Date created: 
2019-12-09
Abstract: 

Voltage-gated sodium channels (VGSC) increase invasiveness and metastatic potential in prostate cancer and may be a novel drug target for castration resistant prostate cancer. VGSC isoforms expressed in prostate cancer differ depending on cell type, and laboratory growing conditions Immunocytochemistry experiments suggest that VGSC localize in invadopodia, structures required for invasiveness of cancer cells. VGSC significantly colocalize with vimentin, a marker of invadopodia (p<0.0001) and display polarized expression patterns on the cell membrane. Functional invasion experiments using a variety of VGSC blockers such as tetrodotoxin, lidocaine, and cannabidiol demonstrate that VGSC inhibition significantly reduces cancer invasiveness (p<0.0001). These results suggest that VGSC plays a functional role in invadopodia and VGSC inhibition reduces invasiveness and metastatic potential in prostate cancer.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Dr. Peter Ruben
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

Elevated blood pressure and hypertension in South Asian children: A mixed-methods analysis exploring associated factors and behavioural influences

Author: 
Date created: 
2019-10-16
Abstract: 

The overarching objective of this PhD thesis was to develop a better understanding of how broad-based (physiological, lifestyle, social and cultural) factors, influence blood pressure (BP) in South Asian children in Canada. This was done using a mixed-methods approach that included: a systematic review which informed the direction of the study; multivariate regression analyses to estimate the correlates of BP and hypertension in South Asian children; receiver operating characteristics curve analysis to estimate the validity of adiposity metrics in estimating adverse risk of hypertension in South Asian children, including the appropriate risk thresholds; and semi-structured qualitative interviews to explore attitudes towards healthy behaviours in South Asian children and their parents. From the systematic review, I identified a range of physiological, social and lifestyle factors that were associated with elevated BP and hypertension in children. These variables were subsequently investigated in a sample of 762 South Asian children in Canada. The results suggested that for these children, physiological variables provided better explanatory capacity regarding the risk of elevated BP and hypertension than social or lifestyle factors. Age, sex, BMI z-score, heart rate and weight accounted for 30% of the variance of sBP z-score, while age, BMI z-score, heart rate and daily fast food intake accounted for 23% of the dBP z-score variance. The prevalence of hypertension was found to be high at 12%. The area under the curve (AUC) values for the adiposity measures for boys and girls ranged from 0.74–0.80, suggesting that the adiposity measures were fair in their ability to estimate hypertension risk. Yet, sex-stratified cut-offs associated with adverse risk of hypertension for South Asian boys and girls suggested that these children might be at high risk of hypertension at levels of adiposity considered normal. Last, my interview findings documented the range of influences on healthy behaviour in South Asian children and their parents including school, peer, social media and cultural dynamics. Taken as a whole, my thesis provides vital information for healthcare practitioners in identifying and treating at-risk South Asian children, and for public health practitioners and policymakers in informing the development of effective intervention strategies aimed at preventing hypertension and CVD risk in this population.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Scott Lear
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.

Pediatric syncope diagnosis and management: Validation of continuous blood pressure monitoring and alternatives to 24-hour urine sodium sampling

Author: 
Date created: 
2019-10-10
Abstract: 

Syncope, or fainting, is common and has a devastating impact on quality of life. Diagnosis and management of syncope is challenging. In pediatric populations the current diagnostic gold standard, a tilt test, cannot be safely or properly performed, because the necessary non-invasive beat-to-beat blood pressure monitoring is not validated for children. In addition, low sodium intake is common in syncope patients, and salt supplementation is recommended. However, standard assessments of sodium from urine collections are difficult and unpleasant. This thesis demonstrated that: (i) continuous non-invasive finger blood pressure monitoring provides a novel, comfortable, and accurate approach for use in children compared to an intra-arterial catheter, (ii) Quantab test strips provide a valid alternative to flame photometry for the determination of 24-hour urine sodium levels, and corrected spot sample averages offer an acceptable and convenient alternative to 24-hour urine sampling. These advancements in diagnostic tools for syncope will enhance quality of life for affected individuals.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Victoria Claydon
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

Evaluation of an exercise program delivered prior to hemodialysis

Author: 
Date created: 
2019-06-25
Abstract: 

Although it is broadly accepted that exercise offsets poor health outcomes experienced by hemodialysis patients, incorporating exercise into patients’ lives remains a challenge. This study aimed to (1) determine the effects of a pragmatically-designed exercise program on physical performance, mental health, and quality of life of hemodialysis patients, and (2) explore patient and staff experiences with the exercise program at five dialysis units. Intervention participants were offered a thrice-weekly, 12-week exercise program at the unit prior to hemodialysis. Health-related quality of life (EQ-5D-5L Index Value) improved significantly for the exercise participants compared to control. Physical performance (Short Physical Performance Battery) and mental health (Center for Epidemiological Studies Depression Scale-Revised) did not change significantly. The majority of participants and staff reported positive feedback and benefits from the exercise program. In conclusion, a pragmatically-designed exercise program delivered before hemodialysis improved patients' quality of life and was well-received by patients and staff.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Dawn C. Mackey
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

The effect of sudden drop in partial pressure of oxygen during ascent on heart function

Author: 
Date created: 
2019-08-12
Abstract: 

Background: Cardiovascular disease is the second leading cause of diving deaths. Scarcity of 12 lead ECG recordings during dives leaves many questions unanswered about cardiac function during ascent. I hypothesized that decreased oxygen partial pressure (PpO2) initiates cardiac arrhythmia on ascent. I examined heart rate variability (HRV), rhythm and circulating markers of cardiac damage in the blood in response to submersion, increased pressure and reduction in the partial pressure of oxygen during the ascent. Methods: Experiments were performed in the wet pot of the hyperbaric chamber. Participants (N=19) were occupational and scientific divers, age 39.3 years, BMI 26.5 kg/m2, 3 females. Each completed two dives in a drysuit while swimming in 8oC water outfitted with a 12 lead ECG holter recorder. A 30-minute swim was performed at ambient pressure followed by a dive to 5 atmospheres absolute (ATA) with a direct ascent to surface pressure. The experimental exposure held the PpO2 at 1.0 ATA for the ascent. Blood samples were drawn at baseline, immediately after the dive and one-hour post dive. ECG analysis was performed for 5 epochs of 5 minutes each. Results: Diving increased heart rate and decreased HRV (p<0.05). The change in heart rate variability time domain was increased on ascent with oxygen clamping over the control (P=<0.004). Diving increased markers of cardiac vagal tone (P=<0.02) and decreased markers of sympathetic tone (P=<0.003). Diving caused QTc prolongation, particularly in the control (P=0.021) on ascent. No ST depression was observed, and ST elevation was present in the anterior leads with no differences between epochs or conditions. Diving increased the number of atrial ectopics (PAC) particularly with oxygen clamped on ascent (P=0.002). There was no troponin (cTnI) or significant change in pH or Lactate however there was a significant increase in B-Type natriuretic peptide (BNP) production with the oxygen clamped on ascent (P=<0.0001). Discussion: This is the first study to examine the effect of submersion and diving on HRV using 12 lead ECG while exercising in a controlled study, and unlike previous studies without exercise I saw an increase in HR and a significant decrease in heart rate variability. This agrees with the effect of exercise alone. The effect of clamping oxygen on the ascent eliminated the reduction of HRV from control. Submersion and diving both increase markers of cardiac vagal tone unlike the effect of exercise. Markers of sympathetic tone were decreased during submersion but not during the 5 ATA dive. This suggests an autonomic conflict not observed when no exercise is present during a dive. The ST segment elevation showed the typical “early repolarization syndrome” of young, athletic, healthy, males with physically demanding jobs. Under conditions of high oxygen pressure an increase in the QT interval from baseline was observed along with a significant increase in PACs and BNP levels. Its relation to the observed PAC’s and QTc is unclear.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Peter Ruben
Michael Koehle
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.