VictorJuiz/thesis huggingface dataset:
An Investigation of Bacteriophage Anti-CRISPR and Anti-CRISPR Associated Proteins

Phage infection poses a major threat to bacterial survival. To protect themselves, bacteria have evolved numerous defence mechanisms. The best known of these systems is CRISPR-Cas, which provides sequence-specific protection against phage infection. In turn, phages have evolved inhibitors of CRISPR-Cas systems known as anti-CRISPRS. Here, I investigated different aspects of anti-CRISPR biology. It was previously established that anti-CRISPRs must function during the process of infection to protect phages targeted by the P. aeruginosa CRISPR-Cas system; however, it was not known how they manifest during this process. I determined that anti-CRISPR genes are robustly expressed prior to any significant degradation of phage DNA by CRISPR-Cas machinery. This expression was tied to a powerful promoter found in association with anti-CRISPR loci. While the anti-CRISPR sequences are quite diverse, the organization of their locus is well conserved. Phages were found to carry one to three anti-CRISPR genes followed by a highly conserved helix-turn-helix motif encoding gene, known as anti-CRISPR associated 1 (aca1). I determined that aca1 encodes an essential repressor of anti-CRISPR transcription, thereby explaining its conservation. Additionally, I determined that other helix-turn-helix motif containing genes found in association with anti-CRISPR loci in diverse bacterial species also act as repressors of anti-CRISPR transcription. To date, the mechanism of action of less than half of the described anti-CRISPR protein families have been studied in detail. I have demonstrated that AcrIE3 from phage DMS3 is dual function anti-CRISPR with activities against the type I-E and type I-F CRISPR-Cas systems of P. aeruginosa. Additionally, AcrIE3 was found to inhibit both CRISPR-Cas systems by preventing target DNA binding. More work is needed to determine how AcrIE3 inhibits DNA binding. Together, these studies provide insight into the spread of anti-CRISPR operons in mobile genetic elements and into the arms race between phages and their bacterial hosts.

Advisor: Davidson, Alan R
The Image of the Suburbs: Planning Urbanity in the Toronto Region

This dissertation examines the discursive production of new developments and the reinvention of suburban image in the municipalities arcing around the City of Toronto, Ontario, Canada. Planning policies promoting densification, alongside rising housing prices, and persistent concerns about car-dependence, set the context for aspirations of urbanity in planning and development. Discourses analyzed include transcripts of interviews with housing and community producers (planners, municipal councillors, and developers), the planning policies produced by government, and the marketing materials produced by the development industry. Studies examining the historic planning and promotion of the suburbs have shown the prominence placed on symbolic distinction from the city with references to nature, rurality and countryside, where residents are promised a healthy retreat, privacy, safety, and an ideal place to raise children. This dissertation argues that suburban planning and housing marketing discourses in the Toronto region reveal an emerging reversal in the suburban script that downplays the urban-suburban distinction and promotes a more urbane place image, representing a case study of broader contemporary efforts to urbanize the suburbs. In the first of three empirical papers, the research shows how stacked and back-to-back townhouses are planned and promoted by intertwining discourses of suburban "evolution" and "routes to maturity" through homeownership where smaller units offer a type of fix to market constraints, and young adults are produced as suburban-re-inventor subjects. The second paper demonstrates the importance of aesthetics and modern design in the discursive production of competitive and attractive world-class growth nodes and corridors as part of the ideological-political trajectory towards post-suburbanization. The third paper examines the gap that producers describe between the promises of compact city theory and the in-practice realities of car-dependence and separated land uses. While some marketing materials draw on the long-standing discursive production of the suburbs as the "best of both worlds," producers describe the contemporary suburbs as increasingly compact in residential areas, but still car-dependent leading to concerns about being "squished-in" and stuck in traffic. Practitioner perspectives on the successes and challenges of current strategies signal the need for additional theories and policies, beyond residential densification, to resolve the challenges of the suburbs.

Keywords: Canada
discourse analysis
planning
suburbs
urbanity
Advisor: Rankin, Katharine
CELLULAR AND MOLECULAR PATHOGENESIS OF LEFT VENTRICULAR HYPERTROPHY IN RAF1- MUTANT NOONAN SYNDROME

Noonan syndrome (NS) belongs to a class of autosomal dominant disorders, termed “RASopathies”, caused by mutations in RAS/ERK pathway genes and characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1L613V/+ knock-in mice. Here, using inducible Raf1L613V expression, I show that LVH results from the interplay of cardiac cell types. Cardiomyocyte expression of Raf1L613V enhances Ca2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1L613V expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1L613V expression causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine hierarchy (TNF->IL6) from Raf1L613V-expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, post-natal TNF inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. I conclude that NS cardiomyopathy involves cardiomyocytes, ECs, and fibroblasts, TNF/IL6 signaling components represent potential therapeutic targets, and abnormal EC signaling might contribute to other forms of LVH. On the other hand, little is known about the relative contribution of ERK1 and ERK2 to the pathogenesis of LVH. Using genetic ablation of Erk1 or Erk2, globally or in a tissue-specific manner, I show that while ERK2 is the predominant ERK isoform in the heart and isolated cardiomyocytes, ablation of Erk1, but not Erk2, normalizes cardiac hyper-contractility. ERK1 and, to a greater extent, ERK2 mediates ventricular chamber size. While normalization of the increased heart mass is not observed with progressive decrease in Erk1/2 gene dosage, it is achieved with endothelial-specific knockout of Erk2. Notably, ERK1 is the major ERK isoform in cardiac endothelial cells. Taken together, these data suggest isoform-specific roles of ERK1 and ERK2 in the pathogenesis of RAF1 mutant-associated LVH.

Advisor: Neel, Benjamin G
Dynamically controlling metabolic valves to decouple and switch between phenotypic states.

Microorganisms are well positioned to address many societal concerns in areas such as health, sustainability, and energy. Rapid progress in systems biology and synthetic biology has drastically improved our ability to understand and engineer complex biological systems. Sustainable biochemical production has received significant attention over the past 30 years; however, these processes must often reach near theoretical maximum performance to become competitive with petroleum alternatives. Here, we implement synthetic biological circuits in order to gain finer control over microorganisms and surpass the theoretical limits of organisms lacking such control. To do so, we aim to identify a set of important reactions which can be dynamically controlled to redirect flux between two phenotypes: growth and production. Then, cells can rapidly accumulate in a growth stage, before a phenotypic switch to production. We have shown that this strategy can significantly improve process rates in two-stage production processes. Since the process of metabolic engineering is iterative, we tackle diverse challenges through the design-built-test-learn cycle. First, we implement and characterize a bistable transcriptional controller as a genetic memory element to drive gene expression. We show that an optimized controller can effectively decouple growth from lactic acid production and can be implemented for two-stage production. Second, we develop a strain design algorithm (MoVE) which can use a stoichiometric metabolic model to predict genetic engineering strategies using two distinct interventions: static knockouts and dynamically controlled metabolic valves. Using this algorithm, we have identified promising candidates for metabolic valves which can be used for a range of products. Finally, we develop liquid handling workflows and a data analysis framework (Impact) to allow for the rapid and thorough characterization of microbial physiology. We anticipate that these tools and techniques will expedite the implementation of efficient dynamic control strategies. Furthermore, we expect studies which improve the accuracy of metabolic models will reduce the development time to commercialization of metabolic engineering technologies.

Keywords: Dynamic control
Linear programming
Metabolic engineering
Metabolic modelling
Synthetic biology
Systems biology
Advisor: Mahadevan, Radhakrishnan
Photophysics and Photochemistry of Ions and Complexes in the Gas Phase: Investigations by Laser-Induced Photodissociation and Fluorescence

This thesis focuses on studying the intrinsic properties of some fluorescent dyes and their complexes in the gas phase, while eliminating external interactions with solvent molecules. A novel setup, which couples a trapping mass spectrometer to lasers as excitation sources, is used to perform photodissociation and fluorescence experiments on isolated, mass-selected ions. The common fluorescent dye fluorescein and halogenated fluorescein ions in the gas phase show very different fluorescence and photodissociation properties than in solution: the most fluorescent charge state in solution, the dianion, does not emit detectable fluorescence in the gas phase; in addition, the most stable monoanionic form of fluorescein in the gas phase has a protonated carboxyphenol pendant group rather than protonation on the hydroxyl group of the chromophore as found in solution. Building blocks of xanthene dyes, pyronin Y and pyronin B ions, exhibit much longer intrinsic fluorescence lifetimes in the gas phase than in solution, mostly owing to a substantial decrease in nonradiative decay rates. Gas-phase studies on more complex systems are done on gaseous noncovalent complexes of pyronin Y with a macrocyclic molecule cucurbit[7]uril and polypeptides covalently labelled by a BoDIPY dye and quencher tryptophan. Fluorescence properties of the gaseous noncovalent pyronin Y and cucurbit[7]uril complex suggest interaction of these dyes with macrocycle cucurbit[7]uril plays a relatively minor role in their photophysics compared to interactions with solvent molecules. Fluorescence properties of dye labeled polypeptides show that quenching is more sensitive to the type of repeating unit, alanine or glycine, than the presence (or location) of quencher tryptophan. Taken together, this thesis presents benchmark results on the intrinsic photophysics of key fluorophore classes and lays essential groundwork for the use fluorescence in investigations of intrinsic properties of more complex systems such as biomolecules. Overall, the work reported in this thesis serves as a proof-of-concept work demonstrating the applicability of coupling modified trapping mass spectrometer and optical spectroscopy set-ups to study the intrinsic properties of ions.

Keywords: fluorescence
gas phase
mass spectrometry
photodissociation
photophysics
quadrupole ion trap
Advisor: Jockusch, Rebecca A.
Transcranial Focused Ultrasound: New Methods and Applications

Transcranial focused ultrasound is a rapidly-advancing therapeutic modality. In recent years, clinical trials have begun for a number of neurological conditions, including Essential Tremor, Obsessive Compulsive Disorder, and Neuropathic Pain. These clinical trials follow decades of intensive laboratory research programs to understand the mechanisms behind ultrasound interaction with brain tissue and to develop clinical devices that are capable of delivering the therapeutic ultrasound to a wide range targets. Looking to the future, however, there remain many obstacles to the further implementation of focused ultrasound to a number of pathologies: limited steering range in the brain using a hemispherical array, secondary control over the spatial manifestation of the focus transcranially, and possible temperature-dependent acoustic effects. Recent clinical data of thermal lesions from treatments of Essential Tremor using MR-guided transcranial focused ultrasound have shown that in many cases the focus is oblique to the main axis of the phased array. Chapter 3 presents the pertinent clinical data and attempts to explain the phenomenon. Numerical simulations were performed on clinical data to analyze the causes of the oblique focus and determine appropriate correction methods. It was found that the focal obliquity could be replicated with the numerical simulations to within 23.2 +/- 13.6 degrees of the clinical cases. It was then found that a major cause of the focal obliquity was the presence of sidelobes, caused by an unequal deposition of power from the different transducer elements in the array at the focus. In addition, it was found that a 65% reduction in focal obliquity was possible using phase and amplitude corrections in comparison to the clinical phase corrections alone. Extending the results of Chapter 3, in Chapter 4 a technique was developed for the generalized rotation of a focus using a phased array of ultrasound transducers. In this chapter, the concept of focusing an ultrasound phased array is expanded to include a method to control the orientation of the focus using a Tikhonov regularization scheme. It is then shown that the Tikhonov regularization parameter used to solve the ill-posed focus rotation problem plays an important role in the balance between quality of focusing and array efficiency. In Chapter 5, clinical data were analyzed to indicate a reduction in the induced energy-temperature efficiency relationship during clinical treatments at higher acoustic powers; to establish its relationship with the spatial distribution of the focal temperature elevation; and to explore its cause. Computer simulations, animal experiments, and clinical system tests were performed to determine the effects of skull heating, changes in brain properties and transducer acoustic output, respectively. The reduction in the energy-efficiency relationship during treatment was found to correlate with the increase in size of the focal volume at higher sonication powers (p-value

Keywords: Focused Ultrasound
Image-Guided Therapy
Medical Physics
Neuroscience
Advisor: Hynynen, Kullervo
An Assessment of FDI in Angola: Signalling Readiness to Attract Investment

The financial system can be of extreme importance to improve capital accumulation and promote economic growth. This study, provides an assessment of the role of the financial system, specifically the banking sector, to attract FDI in Angola. Furthermore, it is also assessing how attracting FDI into the financial system can lead to the attraction of FDI into other sectors.

Advisor: Prado, Mariana M
Knowing How and Knowing Why: Integrating Conceptual Knowledge in Simulation-based Procedural Skills Training to Support Learning Transfer

In health professions education (HPE), simulation technologies are used to imitate aspects of real-life clinical skills and scenarios with goal of preparing clinical professionals for real-world practice. Though much effort has gone into incorporating simulation into HPE curricula, questions remain about simulation-based instruction can be designed to support trainees’ ability to transfer skills learned in simulation to novel contexts. Instructional designs that promote trainees’ conceptual understanding have shown to enhance their ability to transfer across a variety of domains but has yet to be operationalized the context of healthcare simulation. In this dissertation, I investigate the relationship between instructional design, conceptual understanding, and skill retention and transfer in the context of simulation-based procedural skills training with novice medical trainees. Building on research in education, cognitive psychology, and clinical reasoning, this work assesses the impact of instructional designs that seek to integrate two types of knowledge that underlie procedural flexibility and expertise: conceptual knowledge (i.e., knowing why) and procedural knowledge (i.e., knowing how). In three randomized, controlled experiments, I test the cognitive mechanisms of effective integration of conceptual and procedural knowledge by manipulating the availability and presentation of these knowledges in instructional material. Results show the integrated instruction increases in trainees’ conceptual knowledge, which in turn mediates improvements in trainees’ skill retention and transfer. Study 1 establishes the benefits of video-based instruction that integrates conceptual knowledge (in addition to procedural knowledge) for trainee’s skill retention and transfer. Study 2 demonstrates that video-based integration is most effective when it encourages trainees to create causal linkages between procedural and conceptual knowledges at the level of cognition, a process called cognitive integration. In Study 3, we replicate previous findings and attempted to bolster cognitive integration by designing simulators that make the causal relationships between procedural and conceptual knowledge visible and interactive. Taken together, the dissertation operationalizes cognitive integration in simulation-based procedural skills training and provides evidence that integrated instruction can enhance simulation-based skill retention and transfer. Hence, lower fidelity training that emphasizes developing an integrated understanding of procedural and conceptual knowledge may be superior to repeated practice with more realistic simulations.

Keywords: Expertise
Instructional Design
Knowledge Integration
Procedural Skills
Simulation
Transfer of Learning
Advisor: Moulton, Carol-anne
Brydges, Ryan
An In Vivo Screen for Genes Involved in Central Nervous System Control of Obesity Identifies Diacylglycerol Kinase as a Regulator of Insulin Secretion

The increasing prevalence of obesity as well as its association with many chronic diseases has turned obesity into a major health concern worldwide. Obesity has many underlying environmental and genetic factors that disturb the balance between energy intake and energy expenditure, which is controlled by the central nervous system. The goal of my project is to help further our understanding of these CNS mechanisms by using the powerful tools available in Drosophila melanogaster to identify neuronal genes involved in energy homeostasis. Using the neuronal fru-Gal4 driver we performed an RNAi screen with 1748 genes and assayed for obese or lean phenotypes defined as increases or decreases in triacylglycerol (TAG) levels. After 3 rounds of screening I identified 25 hits that were reproducible and confirmed these phenotypes with independent RNAi lines. One of these hits was Diacylglycerol kinase (Dgk) whose mammalian homologues have been implicated in genome-wide association studies for metabolic defects. Manipulation of neuronal Dgk levels affects TAG and carbohydrate levels but these effects donâ t seem to be mediated through Dgkâ s regulation of DAG, PA levels or PKC activity. I hypothesized that Dgk acts in the insulin-producing cells (IPCs), a set of neurosecretory neurons that secrete Drosophila insulin-like peptides (dILPs) and are involved in the regulation of dILP secretion. Knockdown or overexpression of Dgk within the IPCs reproduces the same TAG, glucose and glycogen phenotypes seen with fru-Gal4. Moreover, overexpression of kinase-dead Dgk, but not wild-type, decreased circulating dILP2 and dILP5 levels resulting in lower insulin signalling activity. Conversely, despite having higher circulating dILP levels, Dgk RNAi flies have decreased pathway activity suggesting that they are insulin-resistant. Thus, after screening over 1700 genes in vivo I identified 25 genes as potential neuronal mediators of energy homeostasis. My results have also shown that one of these genes, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila.

Keywords: Central Nervous System
Diacylglycerol kinase
Obesity
Advisor: Boulianne, Gabrielle L
The role of Norrie Disease Pseudoglioma (NDP) signaling in glioblastoma

Norrin is a WNT ligand that binds Frizzled-4 (FZD4) and Low-density lipoprotein receptor-related protein (LRP5/6) receptor complex to activate canonical WNT/ β-Catenin signaling. Norrin/FZD4 signaling is involved in the regulation of vasculature in several tissues including retina, inner ear and for blood-brain barrier function. The role of Norrin in cancer is not very well characterized. Here, we show that NDP is expressed in a wide range of cancer types, with a particular enrichment in glioblastoma (GBM) and lower grade glioma (LGG). Kaplan-Meier survival analysis of publicly available datasets revealed a significant correlation between NDP expression and survival in GBM, LGG and neuroblastoma. To investigate the function of NDP in GBM, we performed a set of NDP and FZD4 gain and loss of function experiments in patient-derived GBM stem cell (GNS) lines. Recently ASCL1 expression was shown to stratify GNS lines into two cohorts with different tumorigenic, proliferation and differentiation dynamics. Surprisingly, we found that NDP manipulation resulted in opposite effects in ASCL1hi versus ASCL1lo lines. NDP inhibited proliferation and sphere formation in ASCL1lo lines through WNT- dependent mechanisms, while it stimulated proliferation and sphere formation in ASCL1hi lines through WNT-independent mechanisms. Immunocytochemistry staining for proliferation markers indicated that NDP affects cycle kinetics and cell cycle exit in both cohorts. Interestingly, RNA-Seq analysis of NDP knockdown ASCL1hi and ASCL1lo lines revealed a remarkable effect of NDP knockdown on cell cycle controlling genes. In addition, the library revealed a significant number of uniquely expressed genes in each cell line, consistent with the divergence of NDP molecular functions between the two lines. Collectively, our results indicate that NDP is involved in the regulation of GBM progression, and that NDP function in GBM stratifies with ASCL1 expression.

Keywords: ASCL1
GBM
Glioblastoma
NDP
Norrin
WNT
Advisor: Wallace, Valerie A