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

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Design and validation of genetically encoded probes for the analysis of neuronal catecholamine and ATP co-transmission

Author: 
Date created: 
2019-07-31
Abstract: 

BACKGROUND: Sympathetic nerves co-release several neurotransmitters, including adenosine-5'-triphosphate (ATP) and norepinephrine (NE). Our studies are aimed at understanding how these nerves provide automatic regulation of blood vessel diameter and therefore blood pressure. Relatively little is known at the molecular level about how these nerves control the release of multiple neurotransmitters. Using immunofluorescence microscopy, we recently showed that clusters of vesicles containing ATP and NE are segregated within sympathetic nerve terminals. METHODS: To assess the mechanisms of ATP and NE release, we developed genetically encoded reporters of the vesicular monoamine transporter VMAT2 (SLC18A2) and the vesicular nucleotide transporter VNUT (SLC17A9) tagged with pH-sensitive fluorescent proteins to monitor the release of NE and ATP containing vesicles with molecular specificity and high spatial resolution. RESULTS: First, we characterized the dopaminergic Neuro-2a (N2a) cell line as a model to study catecholamine and ATP co-release. N2a cells express VMAT2 and VNUT, and we found that their expression is upregulated upon differentiation, induced by retinoic acid (RA) and serum deprivation. We optimized retinoic acid and serum concentrations to drive neurite outgrowth while minimizing cell death. Following differentiation, cells exhibited release of VMAT2-pHuji, evoked by field stimulation and the calcium Ionophore 4-Bromo-A23187. Second, we tested whether ATP and NE localize to separate vesicles in N2a cells. Nearest-neighbour colocalization analysis showed that VMAT2 and VNUT are located in common varicosities but in separate vesicles. VNUT and VMAT2 appear to traffic independently, and they appear to be localized into vesicles with pH <6.0 and >7.0, respectively. CONCLUSIONS: Our results corroborate reports that NE and ATP are stored in separate vesicles but segregated into separate pools within the varicosity. The N2a cell line is a promising model to further identify fundamental aspects of differential trafficking and release of VMAT2 and VNUT containing vesicles, while VMAT-pHuji and VNUT-pHluorin permit simultaneous detection of catecholaminergic and purinergic vesicle release.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Damon Poburko
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) M.Sc.

Sex differences in coordinated brain activity in clinical child populations

Date created: 
2020-07-08
Abstract: 

A disruption of normal brain development during early stages of life has been associated with higher male vulnerability expressed by male preponderance among affected individuals and/or more severe impairments in males for developmental disorders. Although this phenomenon is frequently acknowledged by the scientific community, its neurophysiological underpinnings remain largely unclear. In this thesis I investigate male vulnerability in very preterm children and individuals with Autism Spectrum Disorder (ASD). Both clinical child populations entail early developmental adversity leading to behavioural and cognitive alterations, believed to be elicited, in part, by disrupted communication between brain areas. Therefore, I examine resting state whole-brain connectivity and its developmental changes in these clinical populations using fMRI and MEG and test the hypothesis of sex-specific connectivity differences between males and females resulting in male disadvantage. In the first study I investigate sex differences in interhemispheric homotopic connectivity and its developmental trajectories in participants with ASD as well as in typically developing individuals. Our findings demonstrate differences in developmental trajectories rather than connectivity. Both females and males with ASD deviate from typical female trajectories while expressing similar developmental trajectories to typical males. In the second study I examine local connectivity and its age-related changes using a similar cohort of participants. Group and sex differences are observed in both local connectivity and its developmental trajectories. Females with ASD are characterised by more robust alterations. Lastly, in the third study I test the hypothesis that male vulnerability in very preterm children can be detected as more pronounced alterations in inter-regional connectivity in boys compared to girls. Our results confirm this hypothesis suggesting that connectivity alterations might contribute to male disadvantage reflected in long-term behavioural and cognitive outcome. Overall, this thesis highlights that disruptions in brain connectivity and/or its developmental trajectories differ between males and females with altered early development supporting the existence of female protective features preventing females from developing pathological outcome.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Sam Doesburg
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.

Altered structure-function relationships in children born preterm and in autism spectrum disorder

Date created: 
2020-07-06
Abstract: 

Sensation, perception, cognition and behavior depend on complex neural processes carried by the coordinated function of brain structures. This coordination is achieved through oscillatory activity and synchronization, and the main pathways can be captured with resting-state activity. Structural alterations might affect the functional coordination with other brain structures, and a structure-function approach can provide a better understanding of the underlying neural mechanism that characterizes human populations. This thesis will study altered structure-function relationships in Autism Spectrum Disorder and in children born preterm at school age. Functional activity is assessed with MEG or fMRI resting-state data and structural characteristics with MRI. Both populations present brain oscillatory and structural alterations related to the thalamic-cortical system. Recent evidence indicates that the development of brain networks connectivity is altered in ASD and in very preterm born children. Evidence remains scant, however, regarding the relationship between atypical brain network connectivity and altered structure-function relationships in these groups. In ASD, there is contradictory evidence on the nature of such alterations with some studies suggesting increased or decreased functional connectivity involving particular structural areas. In very preterm children, evidence regarding the overall nature of structure-function network alterations remains scarce. Both populations present structural alterations and atypical oscillatory activity, and this research will investigate how structure-function relationships in brain networks are altered in ASD and in very preterm children and their association with developmental difficulties. Specifically, in ASD it will be assessed the functional brain networks spatial maps variability and atypical structural developmental trajectories of cortical thickness, and in preterm, atypical oscillatory activity and synchrony, and altered thalamic structural measures.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Sam Doesburg
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.

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): 
Supervisor(s): 
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
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
Scott Lear
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.