VictorJuiz/thesis huggingface dataset:
Structural and Functional Insights into the Molecular Mechanisms of Viral-cell and Cell-cell Fusion
Keywords: fertilization
Fusion
glycoprotein
Membrane
structural biology
Virus

Membrane fusion is an essential step in various developmental, physiological, and pathological processes in eukaryotes. Specific fusion proteins catalyze the merger of two lipid bilayers. The molecular mechanisms of many membrane fusion reactions still remain unclear. Here, we characterized the molecular details of various adhesion and fusion proteins involved in viral-cell, placental trophoblast, and sperm-egg fusion events to better understand their mechanisms. In total, nine crystal structures of these human and viral glycoproteins are presented. Furthermore, structural comparisons combined with mutagenesis, biochemical, biophysical, and cell culture experiments allowed us to investigate the implications of specific residues of these proteins in mediating membrane fusion. Structures of class I viral fusion proteins revealed that electrostatic interactions are critical for post-fusion stability in viruses that fuse at neutral pH, whereas the salt bridges do not play a stabilizing role in fusion proteins that proceed through low pH. Instead, hydrophobic residues stabilize the fusion core, and histidine/arginine residues in the chain reversal region stabilize a helix-dipole moment. Moreover, the structure of human syncytin-1 fusion protein unveiled a striking overall structural similarity to class I viral fusion proteins, and the presence of salt bridges within the fusion subunit zipper the inner and outer helices. These electrostatic interactions stabilize the post-fusion structure and provide the energetics for trophoblast cell fusion in placentation. While class I fusion proteins share similar structural and functional features, the mechanism of sperm-egg fusion requires the involvement of sperm Izumo1 and egg Juno proteins. Atomic resolution structures of Izumo1 and Juno display an interface stabilized through extensive interactions and a major conformational change within Izumo1 upon Juno binding. Moreover, mutational studies at the Izumo1-Juno interface revealed the structural determinants required for binding. We provide a comprehensive analysis that now identifies general trends and signatures important in viral-cell and cell-cell fusion events.
High-throughput Analysis of Alternative Splicing Regulatory Networks that Control Cell Fate
Keywords: alternative splicing
cell fate control
gene regulation
high-throughput screening
RNA biology

Advances in high-throughput profiling technologies have revolutionized our view of transcriptome complexity and regulation. Alternative splicing (AS) is increasingly recognized as a widespread mechanism for gene regulation and is responsible for greatly expanding transcriptome and proteome diversity in metazoans. It is often controlled in a precise spatiotemporal manner by an intricate RNA-protein network. Given the extensive roles of AS in development and disease, understanding the molecular mechanisms underlying AS regulation is of critical importance. The main focus of my thesis is to develop and apply high-throughput strategies to systematically discover AS networks that control cell fate, as well as the factors and mechanisms that regulate these AS networks. By combining high-throughput RNA sequencing and splicing code analyses, I describe the identification of the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved negative regulators of a large program of alternative exons that are differentially spliced between embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) and diverse non-ESCs/tissues. My results from this study provided the first evidence that AS regulatory networks play a central role in the core circuitry required for ESC pluripotency and iPSC reprogramming. Next, I present the development of a new multiplexed functional genomics screen whereby thousands of gene perturbations can be simultaneously assayed for effects on dozens of endogenous AS and gene expression events of interest. Using this system, I revealed a large network of novel positive and negative factors from multiple gene layers that regulate distinct subsets of AS events linked to ESC pluripotency, neural differentiation, and transitions in reprogramming. In summary, these studies have significantly broadened our knowledge of the global regulatory networks that determine cell fate decisions, and provided powerful tools for large-scale investigation of the regulation of AS and other RNA metabolic pathways in diverse cell types.
Determinants of Disability and Disablement in Ontario Long-term Care Residents
Keywords: activities of daily living
disability
Disablement Process
long-term care
nursing homes

Purpose: Disability is difficulty with or dependence on others to conduct activities of daily living, such as bathing, eating and dressing; disablement is worsening disability measured over time. Among long-term care residents, disability and disablement lower quality of life and increase health care costs. Understanding the determinants of disability and disablement in this population is critical to guide clinical practice and accountability policies in long-term care homes. Methods: This thesis is theoretically grounded in the Disablement Process Model. It consists of a literature review and two retrospective studies done using Ontario health administrative data. Study 1 features a critical literature review and analytic framework of the determinants of disability and disablement in older adults. Study 2 examines the relative effect of long-term care home versus resident characteristics in explaining residentsâ disability. Study 3 focuses on the association between disability and geriatric syndromes present at admission and disablement experienced by long-term care residents over time. Hierarchical linear regression models were used in both Studies 2 and 3. Implications: The conceptually-grounded, evidence-based analytic framework from Study 1 can be used to advance future research on disability and disablement in older adults, whether or not they live in the community or long-term care. Study 2 demonstrates that the majority of variation in disability among Ontario long-term care home residents is explained by residentsâ geriatric syndromes, not characteristics of the homes in which they live. Study 3 shows that residents with lower disability at admission become disabled more rapidly over the course of their stay; our exploration of possible mechanisms for this finding is relevant to frontline providers and researchers alike.
Palladium Catalyzed C H Functionalization: Application in Multicomponent Reactions and Natural Product Synthesis
Keywords: catalysis
multi-component reactions
multi-metal reactions
palladium
total synthesis

This thesis describes the development and application of palladium catalyzed multicomponent and multicatalytic reactions, along with medicinal chemistry. The contents are divided into four chapters. Chapter one describes the application and advances of the Catellani reaction. The first project outlines the pathways undertaken in the total synthesis of a natural product. Using the Catellani reaction, linoxepin was synthesized in 8 steps from commercial materials without the use of protecting groups. The second project adds to the methodology by incorporating secondary alkyl halides. 1,2,3 tri-substituted aromatic compounds were synthesized in good to excellent yields. The reaction was shown to be scalable on up to 5 mmol scale. The effect of β-heteroatoms was explored using enantioenriched secondary alkyl iodides. Chapter two focuses on dual metal catalysis. The first project attempts to combine rhodium catalyzed isomünchone formation with palladium catalyzed [3+2] cycloaddition, with the ultimate goal of developing a multimetal-catalyzed asymmetric aryne reaction. A second ongoing project features ruthenium catalyzed C – H insertion followed by palladium catalyzed asymmetric allylation. Oxindoles with chiral quaternary centres were synthesized with good yields and ee’s. Chapter three describes the combination of palladium and copper in a multicomponent multicatalytic reaction, (MC)2R. An organic azide, terminal alkyne and an internal alkyne are combined in a chemoselective process initiating with a copper-catalyzed azide alkyne cycloaddition (CuAAC). Following this, a palladium catalyzed C – H functionalization is achieved with the incorporation of an internal alkyne. A novel class of polycyclic triazoles were synthesized in good yields. Chapter four illustrates an on-going project in collaboration with the Structural Genomics Consortium and AbbVie. Probes for the CECR2 enzyme are being synthesized based on the benzimidazole core. Routes to differentially substituted cores were optimized. The final compounds were tested for activity by SGC.
Strong Electron Donor Ligands and their Catalytic Applications
Keywords: Carbenes
Carbodicarbenes
Gold complexes
Hydrogenation Catalysis
Iron complexes

The coordination chemistry of several strong electron donor ligands, including anionic imidazol-4-ylidenes, cyclic bent allenes, carbodiphosphoranes, and carbodicarbenes were explored with transition metals including Au, Ag, Ru, Rh, Ir, and Fe. Chapter 2 explores the transition metal coordination chemistry of an anionic imidazol-4-ylidene. Ru complexes of this ligand were accessed by transmetalation of an anionic imidazol-4-ylidene Ag complex - the first instance of Ag mediated transmetalation of an anionic imidazolylidene. A coordinatively unsaturated Ru complex of this ligand was shown to decompose through a unique metal assisted proton transfer reaction. Rh complexes of this ligand have revealed its overall moderately strong donor ability. Chapter 3 details the synthesis of a family of Ru-hydrido species bearing strongly donating ‘cyclic bent allenes’ and reveals their very high activity in olefin hydrogenation catalysis. Chemoselective and diastereoselective hydrogenations, as well as hydrogenations of sterically hindered olefins are achieved at exceptionally low catalyst loadings. Chiral modifications of the cyclic bent allene ligands have been explored in Ir based systems. Chapter 4 explores the Fe chemistry of cyclic bent allenes and carbodiphosphoranes. These strong electron donors are shown to stabilize Fe in a rare highly unsaturated three-coordinate geometry in several instances. Chapter 5 details the synthesis of a new type of carbodicarbene, a ‘carbodicyclopropenylidene’, which is the first example of a carbodicarbene without heteroatomic stabilization. The main group and transition metal coordination chemistry of this ligand is investigated. The carbodicyclopropenylidene is shown also to be a strong electron donor.
Organotellurium Reagents for Multiparametric Interrogation of Dynamic Biology
Keywords: Cancer
Dynamic biology
Hypoxia
Isotopologues
Mass cytometry
Tellurium

Population-averaged measurements (PAMs) of cells have advanced understanding of innumerable biochemical processes and continue to be routinely used in the laboratory. Despite this success, PAMs are limited in their ability to report on the precise state of important individual members of a population. The recent emergence of mass cytometry (MC) has offered a new solution to this problem: the ability to simultaneously measure a large number of parameters on a single cell. Unlike traditional fluorescence-based flow cytometry, which suffers from spectral overlap of fluorophore reporters, MC allows for simultaneous measurement of a massive number of parameters—limited only by the number of unique heavy isotopes on the periodic table. Since its inception, MC has relied on high molecular weight metal-chelating polymer antibody-conjugates (MCPACs) to quantify meaningful static biomarkers. Although very useful, MCPACs are unable to report on active biochemical processes. In response to this limitation, we have developed a modular, bio-compatible organotellurium “tag” with a mass less than 250 Da that can be incorporated into a variety of activity-based scaffolds. The work presented in this thesis outlines our efforts to develop a MC-compatible activity-based probe using this tag that is able to report on cancer cell oxygenation levels. We demonstrate that this molecule can be used to discriminate single cells based on their exposure to oxygen, both in vitro and in vivo. By synthesizing “isotopologous” variants of this probe (molecules that are structurally identical but differ in mass) and administering them to human pancreatic tumour models in a temporally-spaced dosing regime, we demonstrate that tumour microenvironments experiencing temporal fluctuations in oxygenation can be highlighted reliably at the single cell level for the first time. By coupling this approach with recently developed imaging mass cytometry technology, we present multiplexed images of specific regions within pancreatic tumours that exhibit fluctuating hypoxia with spatiotemporal resolution.
The Effect of Omega-3 Polyunsaturated Fatty Acids on the Resolution of Inflammation in the Rodent Brain
Keywords: fat-1 mouse
microglia
neuroinflammation
omega-3 polyunsaturated fatty acids
resolution
specialized pro-resolving lipid mediator

Resolution of inflammation in the periphery is believed to be mediated by omega- 3 polyunsaturated fatty acids (n-3 PUFA) derived specialized pro-resolving lipid mediators. However, the resolution of neuroinflammation, and the role of n-3 PUFA and their specialized pro-resolving lipid mediators in the resolution of neuroinflammation have yet to be studied. Moreover, while ischemia induces the production of various mediators in the brain, this effect has yet to be demonstrated for specialized pro-resolving lipid mediators. The first objective of this thesis was to develop a lipidomic approach to measure the rodent neurolipidome without the effect of ischemia using head-focused microwave fixation. Once a lipidomic approach was developed, we attempted to develop a selfresolving model of neuroinflammation and to determine the effect of increasing brain docosahexaenoic acid (DHA) on resolution of neuroinflammation. We demonstrated that microwave-fixation inhibits ischemia-induced production of bioactive mediators, including specialized pro-resolving lipid mediators, and changes in various intact lipid species. We then developed a self-resolving model of neuroinflammation using intracerebroventricular injections of lipopolysaccharide (LPS). Following LPS injection, microglia activation peaked at 5 days and returned to baseline by 21 days. Using a microarray, we illustrated that various markers had varying time courses of inflammation. Interestingly, no neutrophil infiltration was detected. Since neutrophils carry the lipoxygenase enzyme, which produces specialized pro-resolving lipid mediators, we also did not detect specialized pro-resolving mediator production following LPS injection as measured by our new lipidomic approach combined with microwave fixation. In order to increase brain DHA, we compared a wildtype mouse fed a safflower diet deficient in n-3 PUFA to the fat-1 mouse and a wildtype mouse fed a fish oil diet high in n-3 PUFA. Increasing brain DHA resulted in modest increases in resolution of microglia activation and cyclooxygenase (COX)-2 mRNA expression. However, many other inflammatory markers were unaffected by the increased brain DHA. In conclusion, we illustrated that microwave fixation inhibits the ischemiainduced changes on the rodent neurolipidome and that n-3 PUFA have small proresolving properties in a self-resolving model of neuroinflammation. These appear to be independent of specialized pro-resolving mediator production.
Unmasking Novel Epigenetic Mechanisms of Medulloblastoma Pathogenesis
Keywords: Cancer Genomics
Epigenetics
HIC1
Medulloblastoma
Methylation
Pediatrics

The leading cause of cancer-related death in children is due to brain tumours. Medulloblastoma is the most common malignant paediatric brain tumour, representing 20% of childhood brain malignancies. Despite current multimodal therapies, there remains significant treatment-induced morbidity. We therefore require a greater understanding of medulloblastoma pathogenesis to further stratify those patients that require aggressive treatment and those in which milder regiments can be implemented. The aim of this thesis is to unravel the molecular underpinnings of childhood medulloblastoma. To date, our lab has re-conceptualized the genetic landscape of medulloblastoma by demonstrating that it is a heterogeneous disease with multiple distinct molecular subgroups. These different subgroups are currently treated the same, but have vastly different prognosis and patient characteristics. In the first half of this thesis, I attempt to address the question whether medulloblastoma subgroups remain stable between the primary and metastatic compartments. This has significant implications on clinical management given the advent of targeted, subgroup-based therapies. This investigation represents the largest reported primary-metastatic paired cohort profiled to date and provides a unique opportunity to evaluate subgroup-specific molecular aberrations within the metastatic compartment. Our findings further support the hypothesis that medulloblastoma subgroups arise from distinct cells of origin, which are retained during metastatic progression. Given the paucity of recurrent somatic mutations found to date using sequencing technologies, much effort has gone into understanding the epigenetic mechanisms that drive medulloblastoma formation. In the latter half of this thesis, I present data to support the identification of a previously well studied hypermethylated locus on 17p13.3 harbouring a newly described bidirectional promoter. This locus is frequently methylated and rarely mutated in cancer, suggesting a clonal selection towards hypermethylation. Further characterization of this locus reveals a novel tumor suppressor microRNA cluster miR-212/132 in the pathogenesis of medulloblastoma. Re-expression of this endogenously methylated locus using RNA-guided gene activation with CRISPR-Cas9 technology decreased tumour proliferation in vitro and in vivo; this approach may represent new therapeutic options in the fight against cancer.
The Economic Burden of Clostridium difficile infection
Keywords: C. difficile
cost of illness
health economics

Clostridium difficile infection (CDI) leads to significant burden on the healthcare system (e.g., lengthy hospital stay, readmissions). The economic impact associated with CDI in Canada is poorly understood, as only three studies have quantified the burden with results primarily based on expert opinion. This thesis is comprised of three studies to further the understanding of CDI costs. The first study was a systematic review of CDI costing literature. This study identified 45 cost of illness studies that found an economic burden associated with CDI. Mean attributable CDI costs ranged from $10,961 to $36,960 per hospitalized patient (2014 Canadian dollars, range derived from six studies with similar patient populations). The costing methods across the 45 studies were heterogeneous and some studies did not follow standard cost of illness methodology (e.g., provide a justified study viewpoint, conduct sensitivity analyses). The second study examined the economic burden of hospital-acquired CDI using linked health administrative data and a population-based propensity-score matched cohort design. This study evaluated costs unadjusted and adjusted for survival, with the latter estimating costs using the phase of care approach. The results were stratified by elective (e.g., planned) and non-elective admissions. Mean attributable 1-year costs unadjusted for survival were $48,029 (95% confidence interval [CI], $43,315-$52,871) for elective admission subjects and $40,889 (95%CI, $39,768-$42,010) for non-elective admission subjects. Mean attributable 1-year costs adjusted for survival were $32,151 (95% CI, $28,192-$36,005) for elective admission subjects and $21,909 (95%CI, $21,221-$22,609) for non-elective admission subjects. The third study examined the economic burden of community-onset CDI also using a population-based propensity-score matched cohort design and evaluating costs unadjusted and adjusted for survival. Mean attributable 1-year costs were $13,217 (95%CI, $12,062-$14,388) unadjusted for survival and $10,700 (95%CI, $9,811-$11,645) adjusted for survival. Both the second and third studies were from the Ontario healthcare payer perspective. Overall, CDI is associated with considerable economic burden. Decision makers can use the absolute and relative results of the three studies to assess CDI-related interventions. This body of work contributes estimates to the economic burden of CDI and demonstrates the use of health administrative data to estimate the costs of infectious diseases.
Teachers' Responses to Educational Research: A Hermeneutic Inquiry
Keywords: Hermeneutics
Knowledge mobilization
Reader-response
Research utilization
Teacher development

This dissertation explores teachers' responses to educational research. The study is situated in the growing area of inquiry known as knowledge mobilization. The existing literature shows that teachers face a variety of barriers to reading and utilizing educational research, including insufficient institutional support and a lack of time. Other challenges come with research itself. For instance, Denzin and Lincoln (2005) describe a triple crisis of representation, legitimation, and praxis in research. First, it is not possible for the researcher to sufficiently capture the lived experiences of research-participants. Second, the validity of research findings is always contested. Third, it is a challenge to make ethical choices and act upon them. These three crises constitute the conceptual framework for understanding how teachers address them while reading educational research. Twelve in-service language teachers joined a wiki-based discussion forum, whose purpose was to form a community of readers built upon the principles of reader-response criticism. The participants then read a peer-reviewed journal article and exchanged opinions through threaded discussions. These discussions were the first source of data for this study. The second source of data was individual in-depth interviews conducted with the participants at the end of the wiki project. The collection and interpretation of data were guided by Hans-Georg Gadamerâ s philosophical hermeneutics. In this dissertation, I present a number of ways of reading that the participants employed in their interpretations of the research text. Their ways of reading are discussed as a recognitive approach to the crisis of representation, an interpretive-ecological approach to the crisis of legitimation, and an imaginative-ethical approach to the crisis of praxis. Based on the insights gleaned from these ways of reading, I propose that the field of knowledge mobilization take an audience-oriented approach to disseminating and utilizing educational research.