VictorJuiz/thesis huggingface dataset:
Selective Rescue of NMDA Receptor Function in Dopaminergic Neurons of NMDA Receptor Deficient Mice

Dysfunction in both the dopaminergic and glutamatergic systems has been implicated in the pathophysiology of schizophrenia. Schizophrenia is associated with a state of subcortical hyperdopaminergia, and NMDA receptor (NMDAR) hypofunction. The goal of our study is to assess which behavioural deficits present in NMDAR deficient mice are due to lack of NMDAR specifically in dopamine neurons. GluN1 knockdown (GluN1KD) mice express low levels of NMDARs resulting in global NMDAR hypofunction, leading to schizophreniaâ like behavioural endophenotypes, and an altered dopamine system. Schizophrenia-like behaviours observed in GluN1KD mice could be a result of the dysfunction of the dopamine system in these animals. To address this issue, a new mouse line was generated allowing for selective rescue of NMDARs in dopamine neurons using Cre recombinase. These rescue mice (GluN1DATâ Cre) have a global reduction of NMDARs except in dopaminergic neurons where a BAC transgene under the dopamine transporter promoter expresses Cre recombinase. In Aim 1, we assessed the neurochemistry and homeostasis of the dopamine system by measuring tyrosine hydroxylase activity and dopamine metabolism. In GluN1DAT-Cre mice, tyrosine hydroxylase activity was reduced compared to WT mice, and unchanged compared to GluN1KD mice. Additionally, in these rescue mice, dopamine catabolism was increased compared to both WT and GluN1KD mice indicating that neurochemical deficits are not improved by rescuing NMDARs in dopaminergic neurons. In Aim 2, we evaluated which schizophrenia-relevant behaviours result from lack of NMDARs in dopaminergic neurons. We assessed the following behavioural paradigms: locomotor activity, repetitive behaviours, sensorimotor gating, working memory, cognitive flexibility and executive function, sociability, and conditioned learning. GluN1DAT-Cre mice were fully rescued in sensorimotor gating and Pavlovian approach learning, and partially rescued in locomotor activity and repetitive behaviours. Unexpectedly, the rescue animals were more impaired in conditioned place preference compared to GluN1KD mice. Finally, similar behavioural abnormalities were observed in GluN1KD and GluN1DAT-Cre mice in working memory, cognitive flexibility and executive function, sociability, and fear conditioning compared to WT mice. These results indicate that rescuing NMDAR function selectively in dopaminergic neurons does not normalize neurochemical dopamine dysfunction, and ameliorates some, but not all the schizophrenia-relevant behaviours observed in GluN1KD mice.

Advisor: Salahpour, Ali
Variation in Smoking and Nicotine Metabolism Among American Indians: Novel Influences on In Vivo and In Vitro Nicotine Metabolism Phenotypes

Tobacco use and associated disease risk vary widely among different populations and ethnic/racial groups. While American Indian and Alaska Native populations collectively have the highest prevalence of smoking, recent studies have highlighted disparities in smoking and disease risk across different American Indian/Alaska Native populations. The Northern Plains American Indian tribal populations of South Dakota have a much higher smoking prevalence, smoke more cigarettes per day, and have higher lung cancer risk than the Southwest American Indian tribe of Arizona. Underlying reasons behind the differences in smoking and lung cancer risk between these tribes remain unknown. We sought to biochemically characterize the level of tobacco exposure among smokers and non-smokers in the Northern Plains and Southwest tribes, using cotinine as a biomarker. We demonstrated that despite both tribes being relatively light smoking populations, secondhand tobacco smoke exposure is high among non-smokers, highlighting a potential source of increased risk for tobacco-related disease. As variation in the rate of nicotine metabolism (i.e. CYP2A6 activity) has been associated with differences in smoking behaviours and lung cancer risk in other ethnic/racial groups, we then evaluated variation in the CYP2A6 gene and the rate of nicotine metabolism in the Northern Plains and Southwest tribes. We identified distinct patterns of CYP2A6 genetic variation across the two tribes, and an overall faster rate of nicotine metabolism in the Northern Plains compared to the Southwest tribe. We then used a human liver bank to explore additional sources (both genetic and non-genetic) of variation in CYP2A6 activity. We identified several independent and shared sources of variation that could translate to variable hepatic clearance of nicotine, and may impact smoking behaviour. Lastly, we searched for CYP2A6 gain of function genetic variants in order to identify sources of faster CYP2A6 activity. Through investigation of novel uncharacterized variation at the CYP2A6 locus using next-generation sequencing, we identified seven novel high frequency CYP2A6 genetic variants and a five-SNP diplotype, which were associated with faster CYP2A6 activity in vitro and in vivo. Overall, our findings provide insight into the patterns of tobacco exposure, CYP2A6 genetic variation, and nicotine metabolism rate in American Indian tribal communities, and expand on our knowledge of genetic and non-genetic sources of variation in nicotine metabolism rate. These findings could improve our understanding of lung cancer risk in these American Indian populations, and help to inform future tobacco cessation efforts.

Keywords: Cotinine
CYP2A6
Genetic variation
Nicotine
Nicotine Metabolism Rate
Smoking
Advisor: Tyndale, Rachel F
Understanding Expectations: The Influence of Educator and Child Expectations on Kindergarten Childrenâ s Self-Regulation, Early Reading, and Vocabulary Outcomes

This dissertation is comprised of two studies. Study 1 is a qualitative study exploring the factors that may contribute to the formation of educator expectations in Kindergarten. This study presents emerging themes from an educator questionnaire that asked Early Childhood Educators and teachers to describe their teaching philosophies, roles, teaching practices, and other interactions that support students. Findings revealed key distinctions in educator responses to the questionnaire. Early Childhood Educators responded with a child-centred focus that was reflected in multiple themes. A teacher-directed, structured lens was evident in the themes that emerged from the teachers. This study presents the necessary background information to understand the differences in the two groups in terms of how their expectations may be formed and in turn may affect processes leading to child outcomes. Study 2 is an empirical study examining the influence of educator and child expectations on children’s self-regulation, early reading and vocabulary outcomes. Study 2 compared the expectation levels of the Early Childhood Educators and teachers. Results indicate that Early Childhood Educator and teacher expectations were congruent at Time 1 but dissonant at Time 2, with ECEs having significantly higher expectations for children’s self-regulation, early reading and vocabulary outcomes at Time 2. Furthermore, findings revealed that Early Childhood Educator and child expectations had significant positive direct effects on outcomes. The only significant direct effect from teacher expectations to child outcomes was for vocabulary, and this was a negative effect. Differences in professional knowledge base, educator roles, and the amount of time spent with children are potential explanations for these findings. Overall, the findings provide insight into the factors that may contribute to the formation of educator expectations in Kindergarten and begins to address the gaps in the expectation field by exploring the influence of educator and child expectations on children’s outcomes in the early years.

Keywords: child expectations
educator expectatations
Advisor: Pelletier, Janette
Push & Pull: Social determinants of the use of smoking cessation support in Ontario

Tobacco use continues to undermine the health of the Ontarian population. In addition, socio-economic disparities in smoking rates persist in Ontario, despite public health care and universal tobacco control policies. Public health strategies require a detailed understanding of how people are actually quitting smoking in order to develop appropriate interventions and strategies. This thesis used existing evidence and data from the Ontario Tobacco Survey (OTS) to explore unassisted and assisted quitting. The first manuscript estimated the proportion of adult smokers who report attempting to quit unassisted, without the use of pharmacological and/or behavioural assistance, by systematically reviewing population-based studies. A majority of quit attempts were unassisted; however, across and within countries over time, a trend toward lower prevalence of unassisted quit attempts was identified. The second manuscript estimated the prevalence of unassisted quitting and the reach and pattern of use of assisted methods in Ontario. Unassisted quitting was the dominant method reported (58.8%, 95% CI: 56.0-61.6). For assisted methods, pharmaceutical support was the most common (92.9%, 95% CI: 91.1-94.6). Use of behavioural support either alone (2.5%, 95% CI: 1.5-3.6) or in combination with pharmaceutical support (4.6%, 95% CI: 3.3-5.9) was rare. The third manuscript examined the associations between socioeconomic status (SES) and access to care measures and use of cessation strategies using multinomial regression models. Smokers living in areas with the lowest ethnic concentration were more likely to make an assisted quit attempt compared to unassisted quitting (RR=1.67; 95% CI=1.08-2.60) or making no quit attempt (RR=1.65; 95% CI=1.14-2.41). Smokers who reported visiting a doctor in the previous 6 months were more likely to quit with assistance versus unassisted, whether they were advised (RR=1.96, 95% CI=1.49-2.59) or not advised to quit (OR=1.37, 95% CI=1.04-1.82). Together, these studies make a substantial contribution to understanding how smokers are choosing to quit smoking.

Keywords: smoking cessation
socioeconomic status
unassisted quitting
Advisor: Bondy, Susan J
Elucidating the Reprogramming Mechanism: Selection Pressures Direct a Poised Subpopulation of Somatic Cells to an Induced Pluripotent Stem Cell State

Through the overexpression of key transcription factors – Oct4, Klf4, c-Myc, and Sox2 (OKMS) – mouse somatic cells can be reprogrammed to an induced pluripotent stem cell (iPSC) state, enabling them to give rise to all cell types of the body. However, the process of reprogramming has remained elusive and it is unclear whether all somatic cells traverse the same trajectory following OKMS transgene overexpression. The reprogramming process has been mapped by conducting ‘omics analyses of bulk reprogramming culture over time. These population-averaged datasets describe reprogramming as a stepwise process where somatic cells transition through a (transgene-dependent) pre-iPSC state to finally stabilise in a (transgene-independent) iPSCs state. On the other hand, single mouse somatic cells reprogrammed in isolation exhibit stochastic reprogramming latencies. As a result, cells experiencing asynchronous reprogramming dynamics are expected to give rise to heterogeneity in bulk reprogramming culture. Here, we aim to consolidate the apparent dichotomy between stepwise cell state changes observed in population reprogramming with the stochasticity of isolated clonal reprogramming by investigating heterogeneity in mouse reprogramming culture. We first develop a surface marker strategy to track transgene-dependent and -independent iPSCs, enabling us to assess heterogeneity with ease. This marker strategy also shows utility in separating naïve and primed pluripotent states in mouse and human culture. We next combine this surface marker-based approach with clonal tracking and mathematical modeling to investigate the reprogramming trajectory of individual reprogramming clones in bulk culture. In doing so, we uncover clonal competition as a novel driver of population reprogramming dynamics. In fact, we show that a subset of poised somatic cells gives rise to clones that have an elite propensity to dominate bulk reprogramming culture and contribute to the iPSC fraction. We also show, for the first time, that a few clones overtake population reprogramming culture, thus making population-averaged data clonally biased. This work provides novel insights into the role of cell competition in driving heterogeneity in reprogramming culture, providing a new lens with which to interpret population-averaged ‘omics datasets. Taken together, we show that not all reprogramming clones exhibit equivalent behaviour in multicellular contexts, underscoring the need to consider the competition potential of iPSCs.

Keywords: Cellular competition
Cellular fitness
Cellular heterogeneity
Induced pluripotent stem cell
Reprogramming
Systems biology
Advisor: Zandstra, Peter W
Malignancy and Solid Organ Transplantation: Evaluating Cancer Mortality, Pretransplant Malignancies, and Cancer Screening

This thesis explores the intersection of malignancy and solid organ transplantation. First, establishing that solid organ transplant recipients (SOTRs) are at risk of cancer mortality and cancer is the second leading cause of death in this population. Second, exploring pretransplant malignancies (PTM): our systematic review identified a lack of clear and consistent recommendations for cancer evaluation of transplant, with only one in every four clinical practice guidelines (CPGs) including recommendations for cancer evaluation. Most recommended cancer screening as per the general population, which might be not adequate to ensure transplant candidates they are free of occult neoplasms. We also found that recipients with PTM in remission are at increased risk of all-cause mortality compared to recipients without prior history of cancer, with increased risk not only of cancer-specific mortality but also non-cancer death. However, when stratified by type of PTM, only those with high-risk PTM had worse outcomes. Lastly, this dissertation explores recommendations for cancer screening in SOTRs and their uptake. A systematic review of CPGs identified several weaknesses in the available recommendations for cancer screening, including variable screening recommendations, lack of recommendations for non-kidney recipients, and paucity of involvement of relevant stakeholders. Our population-based study found that the uptake of cancer screening uptake in SOTR was low; most SOTR had periods when they were non-adherent to screening recommendations during follow-up, and in fact, many recipients were never screened. In addition, we identified that recipients with visits to primary care physicians were more likely to become up-to-date with cancer screening. Overall, the results of this thesis provide compelling evidence that SOTRs are at increased risk of dying from cancer and highlight the role of cancer evaluation of transplant candidates and cancer screening recipients as a strategy to reduce the burden of cancer in this population.

Keywords: malignancy
mortality
screening
transplantation
Advisor: Baxter, Nancy N
Identity Politicking: New Candidacies and Representations in Contemporary Canadian Politics

This dissertation centres on the candidacies and leaderships of three politicians—Justin Trudeau, Olivia Chow, and Kathleen Wynne. It examines the ways in which gender, race, sexuality, and other salient aspects of politicians’ identities are strategically negotiated and mobilized by politicians, political actors, the media, and the grassroots. The cases herein question the extent to which identity matters in Canadian electoral politics at the municipal, provincial, and federal levels, bridging sociological understandings of power and authority with feminist analyses of identity. The project engages broadly with qualitative methods—discourse analysis, media analysis, participant observation, and interviewing. The research contributes to understandings of: (1) the durability of masculinity in Canadian electoral politics; (2) dispositional requirements for leaders; (3) the compensatory labour that minority politicians perform; (4) alignments and allegiances between politicians and grassroots movements.The first case of the dissertation examines media coverage of a charity-boxing match between Liberal Member of Parliament Justin Trudeau and Conservative Canadian Senator Patrick Brazeau. It offers the concept recuperative gender strategies to describe how political leaders work to restore their public gender identities. The second case is focused on the candidacy of visible minority Toronto mayoral candidate, Olivia Chow. The case offers three concepts that illuminate forms of identity work that minority politicians navigate on the campaign trail: dispositional requirements, ideological alignment, and political compensatory labour. The final case analyzes social movement actors’ assessments of Premier Kathleen Wynne—the first woman and openly lesbian premier of Ontario—and presents a typology of words and deeds to map the terms under which social movement actors judge progressive politicians.

Keywords: Canadian Politics
Gender
Identity
Political Leadership
Advisor: Taylor, Judith
Investigating the Cellular Effects of Mitochondrial DNA Damage Using Targeted Chemical Probes

Mitochondria are compartments within eukaryotic cells that produce energy to power cellular metabolism. Uniquely amongst mammalian organelles, mitochondria contain a small amount of their own genetic material (mtDNA), the proper maintenance and expression of which is essential for mitochondrial function. Mitochondrial DNA damage has been linked to several pathophysiological processes, but the cellular effects of mtDNA damage remain poorly understood. In order to better understand mtDNA damage, our group has pioneered the development of selective mitochondria-targeted DNA damaging agents using positively charged, hydrophobic delivery vectors known as mitochondria-penetrating peptides (MPPs). In my first study, an analogue of the commonly used anticancer drug cisplatin was conjugated to an MPP, yielding a mitochondria-targeted platinum DNA-damaging agent (mtPt) that induced specific damage to mtDNA. Using mtPt, I showed that selective mtDNA platinum damage induced apoptotic cell death associated with reactive oxygen species production, demonstrating that mtDNA damage is sufficient to mediate the cytotoxic activity of a platinum drug. In another work, I aimed to use mitochondria-targeted DNA damaging agents in a screen to characterize novel factors in mtDNA replication and repair. By knocking down expression of known DNA repair genes, and monitoring synergistic effects of gene knockdown on the activity of an mtDNA-oxidizing agent, I identified a series of genes with novel roles in mtDNA repair and replication. Included amongst these hits was DNA Polymerase Î¸ (PolÎ¸), a novel mtDNA polymerase. In a further study, I found that PolÎ¸ plays an essential role in facilitating mtDNA replication under conditions of oxidative stress. Finally, I conducted a genome-wide CRISPR screen to comprehensively identify genes whose knockout sensitizes cells to mitochondria-localized DNA damage. The hits of this screen were heavily enriched for genes involved in mitochondrial translation, implying the importance of mitochondrial protein synthesis in mediating an adaptive response to mtDNA damage. These studies demonstrate the application of mitochondria-targeted DNA damaging agents to the study of a wide range of significant areas in mitochondrial biology. Going forward, my thesis work shows mtDNA-damaging chemical probes to be essential tools for achieving better understanding of mtDNA damage in health and disease.

Advisor: Kelley, Shana O
Development and Evaluation of a High Fidelity Cleft Palate Simulator for Surgical Training and for Development of a Robotic Approach to Infant Cleft Palate Surgery

Cleft palate is one of the most common birth defects requiring surgical repair for proper speech development and feeding. The surgical procedure is challenging due to the small confines of the infant oral cavity, as well as the delicate tissue handling and dissections required. Surgical access, visualization and instrument manipulation is limited. As a result, cleft palate repair is both difficult to learn with limited teaching opportunities as well as technically challenging to perform. A simulator that incorporates the fidelity necessary to capture the critical steps of cleft palate surgery is a valuable training tool providing a safe environment to practice. In addition, a surgical robot provides improved access, visualization and precision within small workspaces that may overcome the challenges of using traditional instruments. The objectives of this work are to develop and evaluate a high fidelity cleft palate simulator to enhance cleft palate surgery training and to develop a robotic approach to infant cleft palate repair using the simulator as a test bed. The simulator was evaluated and found to be an effective, anatomically accurate and valuable training tool that enhances training metrics such as technical performance. The simulator was utilized to perform infant robotic cleft palate surgery using the latest da Vinci速 robotic surgical systems. Overall, the robotic procedures provide superior visualization and access. The da Vinci速 testing guided development of a novel instrument wrist mechanism with more optimal design characteristics for the infant oral cavity workspace and cleft palate surgery.

Keywords: Cleft palate surgery
Surgical learning curve
Surgical robotics
Surgical simulation
Advisor: Forrest, Christopher R
Drake, James M