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

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Principles of energy optimization underlying human walking gait adaptations

Author: 
Date created: 
2020-09-10
Abstract: 

Learning to move in novel situations is a complex process. We need to continually learn the changing situations and determine the best way to move. Optimization is a widely accepted framework for this process. However, little is known about algorithms used by the nervous system to perform this optimization. Our lab recently found evidence that people can continuously optimize energy during walking. My goal in this thesis is to identify principles of optimization, particularly energy optimization in walking, that govern our choice of movement in novel situations. I used two novel walking tasks for this purpose. For the first task, I designed, built, and tested a mechatronic system that can quickly, accurately, and precisely apply forces to a user’s torso. It changes the relationship between a walking gait and its associated energetic cost—cost landscape—to shift the energy optimal walking gait. Participants shift their gait towards the new optimum in these landscapes. In my second project, I aimed to understand how the nervous system identifies when to initiate optimization. I used my system to create cost landscapes of three different cost gradients. I found that experiencing a steeper cost gradient through natural variability is not sufficient to cue the nervous system to initiate optimization. For my third and fourth projects, I used the task of split-belt walking. I collaborated with another research group to analyse the mechanics and energetics of walking with different step lengths on a split-belt treadmill. I found that people can harness energy from a split-belt treadmill by placing their leading leg further forward on the fast belt, and that there may be an energy optimal gait. In my fourth project, I used computer modelling to identify that there may exist an energy optimal gait due to the trade-off between the cost of swinging the leg and the cost of redirecting the body center of mass when transitioning from step to step. Together, these projects develop a new system and a new approach to understand energy optimization in walking. They uncover principles governing the initiation of this process and our ability to benefit from it.

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

Mechanisms that relate transverse loading of muscle to changes in contractile performance

Author: 
Date created: 
2020-07-09
Abstract: 

Research has shown that factors such as the contraction of surrounding muscle, resistance to radial expansion, and external transverse loading have an effect on the way that muscle performs. Such factors are often ignored when doing muscle experiments, yet they can have significant effects on the force and power that a muscle is able to produce. The aim of this thesis is to determine to what degree external loading affects muscle force and to study the changes in muscle architecture due to external loading. The purpose of this thesis was to determine whether external transverse loading causes force reduction in humans, how external transverse loading affects muscle architecture, and describe the mechanisms involved. We applied an external transverse load to the medial gastrocnemius of prone participants as a point loaded mass. Transverse loading reduced ankle torque with higher loads resulting in greater reductions. Passive transverse loading caused a decrease in the resting pennation angle and muscle thickness, with higher loads leading to greater decreases. During activation of the muscle the pennation angle, muscle thickness, and fascicle thickness increase transiently relative to the amount of transverse loading. Alterations to the design of the experiment were made by applying a multi-directional external transverse load and changing the position of the participants from prone to seated, where sitting changed the resting length of the muscle. Our altered experiments showed no change in ankle torque. Minor differences were shown for pennation angle and muscle thickness both at passive resting values and peak active values, but not for fascicle thickness and fascicle. Fascicle thickness decrease transiently, and fascicle length increased. We replicated the external transverse loading experiments using a three-dimensional finite element model of a fibre-reinforced, non-linearly-elastic transversely isotropic composite biomaterial. The model consists of active and passive force-length properties of muscle fibre as well as passive force-length properties of base material. Results show external transverse loading causes architecture changes and reductions in muscle force. Muscle force reduction is dependent on direction of loading, initial pennation, and initial muscle length.

Document type: 
Thesis
File(s): 
Supervisor(s): 
James M. Wakeling
Department: 
Science: Department of Biomedical Physiology and Kinesiology
Thesis type: 
(Thesis) Ph.D.

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.