VictorJuiz/thesis huggingface dataset:
The Role of Tumour Necrosis Factor Receptor Family Members CD137 (4-1BB) and CD30 in Antiviral T Cell Responses

The tumour necrosis factor receptor (TNFR) superfamily is a collection of immunomodulatory molecules that aid in the fine-tuning and regulation of immunity through stimulatory or inhibitory signalling functions at various stages of the T cell response. In this thesis, I investigate the roles of TNFR family members CD137 (4-1BB) and CD30 in antiviral T cell responses, most notably during influenza virus infection. Using a prime-boost vaccine immunization regimen in which mice are infected with mild influenza and then boosted intranasally with a replication defective adenovirus containing the influenza nucleoprotein (NP) and CD137L (4-1BBL) (Ad-NP-4-1BBL), I find that this supraphysiological administration of 4-1BB signals induces a robust long-lived circulating effector memory (Tem) population and a persistent lung tissue-resident memory (Trm) population, providing protection against lethal heterotypic influenza challenge. Ad-NP-4-1BBL acts directly on 4-1BB expressed by CD8 T cells to induce this expansion, and accumulation of memory CD8 T cells in the lung is dependent on local immunization with both antigen and 4-1BBL, recruitment of circulating effector cells into the tissue, and mTOR signalling. Moreover, we find endogenous 4-1BB to be a critical regulator of effector CD8 T cell accumulation in the lung tissue after influenza infection, which correlates with Trm establishment. This implicates 4-1BB as a potent regulator of both local and systemic T cell responses to influenza and may be a potential therapeutic target in future vaccine design. On the other hand, CD30 has no discernable roles in CD4 or CD8 T cell responses during acute influenza or chronic lymphocytic choriomeningitis virus (LCMV) clone 13 infections. Rather, CD30 regulates the age-dependent expansion of the T memory compartment, which may have implications for the role of CD30 in human lymphoproliferative diseases. Taken together, we have identified novel functions of two TNFR members in regulating T cell responses during infection and at steady state. A better understanding of the post-priming mechanisms of these molecules can provide insight into future clinical interventions for viral infection and cancer malignancies.

Keywords: 4-1BB
aging
CD30
co-stimulation
influenza
tissue-resident memory
Advisor: Watts, Tania H
Evolution and Molecular Mechanisms of Photoreceptor Transmutation in Reptiles

Natural light levels vary drastically, and to deal with this variance vertebrates typically utilize a duplex retina that contains rod photoreceptors for dim-light vision and cone photoreceptors for bright-light vision. Squamate reptiles, however, are unique in the predominance of simplex retinas that contain only rods or cones. Evolutionary transitions between rods and cones, termed photoreceptor transmutations, have been proposed to explain the evolution of these simplex retinas, but little previous work has focused on the molecular evolutionary underpinnings of the observed morphological changes. The goal of this thesis is to expand knowledge of the evolution and molecular mechanisms of photoreceptor transmutation. In the first study, I provide strong support for the hypothesis that the morphologically all-cone retina of diurnal colubrid snakes evolved through transmutation of the rods to resemble the appearance, and function, of cones. In the second, I developed a new method of targeted sequence capture that enables efficient sequencing of complete coding regions across divergent taxa, which further provided data for the final two studies. In the third, I analyzed the effect that photoreceptor transmutation and snake origins had on phototransduction gene evolution. I found results consistent with a strong effect of transmutation, including positive selection on cone-specific genes that may indicate adaptation during the evolution of rod-like cones. Furthermore, the low degree of gene loss in snakes, and a lack of relaxed selection early during their evolution, support a dim-light ancestor that lacked strong fossorial adaptations. In the final study, I used whole eye transcriptome sequencing to demonstrate that geckos do not utilize only cone phototransduction machinery as previously thought, and instead appear to co-express both rod and cone genes in cone photoreceptors. I also expand upon the third study to show that geckos experienced a similar shift in selective pressures as snakes that are also associated with transmutation. As a whole, this thesis provides the first molecular evidence for photoreceptor transmutation in snakes, produces a new methodology for efficiently producing sequence data relevant for molecular evolutionary studies, revises our view of transmutation in geckos, and provides the first evidence for molecular changes associated with photoreceptor transmutation.

Keywords: lizards
molecular evolution
selection
snakes
vision
Advisor: Chang, Belinda SW
Controlled Generation of Progenitor T-cells from Hematopoietic Stem Cells and Pluripotent Stem Cells

The thymus integrates multiple niche molecules including cytokines, ligands and extracellular matrix to drive T-cell development in vivo. We hypothesized that engineering technologies to integrate essential thymic niche molecules required for progenitor T-cell development would enable defined in vitro generation and quantification of T-cells. We optimized a defined 2D serum-free culture system using Notch ligand Delta-like-4 (DL4) for producing progenitor T-cells from mouse Sca-1+cKit+ hematopoietic stem and progenitor cells (HSPCs). Serum-free culture enabled us to define assay design criteria such as input cell density, DL4 presentation and cytokine concentrations. The fully defined engineered in vitro niche revealed synergistic interactions between DL4 and vascular cell adhesion molecule-1 (VCAM-1), and its role in enhancing downstream Notch pathway activation, progenitor T-cell migration and differentiation. The engineered niche also enabled scalable differentiation of human umbilical cord blood-derived CD34+ HSPCs to CD7+ progenitor T-cells, yielding 25-fold total cell expansion with 59.3±10.8% CD7+ T-cells. CD7+ progenitor T-cells generated in the engineered niche were capable of thymic engraftment in lymphodeficient SIRPa-/- Rag2-/- gc-/- mice and generated mature functional CD3+ T-cells in vivo. Building on this system, we next immobilized DL4 in a 3D methylcellulose hydrogel (DL4-MC) using thiol-maleimide chemistry. DL4-MC hydrogels exhibited a dose-dependent increase in Notch pathway activation. We demonstrated that production of progenitor T-cells from HSPCs encapsulated in DL4-MC hydrogels could be enhanced when VCAM-MC was incorporated, creating a 3D engineered hydrogel thymus-like niche. Thus, controlled and reproducible differentiation of progenitor T-cells from stem cells can be achieved by engineering a thymus-like niche in vitro using minimum essential cues required for T-cell development. This provides an important step towards systematically engineering the niche for progenitor T-cell development with the ultimate goal of generating histocompatible “off-the-shelf” T-cells from normal or gene-edited stem cells for T-cell immunotherapeutic applications.

Keywords: Biomedical engineering
Hydrogels
Immunotherapy
Stem cells
T cell development
T cells
Advisor: Zandstra, Peter
Methods of Protein Sample Preparation Using Digital Microfluidics and Gas-Phase Techniques with Detection by Mass Spectrometry

Biological samples are complicated, having many different components at a wide variety of concentrations. Some means to simplify these samples while leaving the discrete components unaltered is required. Small structural differences can render a protein unusable role. Mass spectrometry is a valuable tool to characterize proteins but the number of components in these samples present a challenge for accurate detection. Sample preparation and separation methods are effective tools to alleviate this challenge In this dissertation, I present methods for simplifying protein samples for analyze with mass spectrometry using liquid and gas phase methods. In the first chapter, I review some sample preparation options. In Chapter 2, I present a microscale method for high abundance protein depletion using digital microfluidics and functionalized magnetic particles. This is a fast, effective, and multiplexed method for reducing the interference caused from proteins like serum albumin and immunoglobulins resulting in a 4-fold improvement in signal to noise for a low abundant protein. Chapter 3 extends this methodology by employing the same fluid and particle handling technique to immunoprecipitation. This chapter introduces the use of a pre-concentration method called pre-concentration using liquid intake by paper (P-CLIP), a promising ‘world-to-chip’ interface for many digital microfluidic applications. In Chapter 4, gas-phase vapours are used to simplify analysis of peptides by electrospray mass spectrometry. Acetonitrile vapour suppresses in-source fragmentation of fragile ions by clustering with fragile ions resulting in improved limits of detection. Chapter 5 presents a union of liquid chromatography and differential mobility spectrometry for characterization of biopharmaceutical proteins. This new combination, called DMS-SWATH, increases MS/MS sequence coverage for proteins and alleviates problems found in traditional analysis by mass spectrometry and differential mobility spectrometry. Overall this work presents important steps forward in miniaturizing and ameliorating the use of mass spectrometry for the characterization of proteins.

Keywords: differential mobility spectrometry
digital microfluidics
mass spectrometry
protein analysis
Advisor: Wheeler, Aaron R
Barriers and Facilitators to Early Physical Rehabilitation in Mechanically Ventilated Patients

Background: Physical rehabilitation strategies, when initiated in critically ill patients, may improve functional outcomes of survivors and may shorten duration of mechanical ventilation and ICU length of stay, although evidence is conflicting. Despite some conflicting evidence, it has been recommended in clinical practice guidelines, but not been consistently implemented in critical care units. The implementation of complex interventions in healthcare is challenging, but may be improved using theory to understand barriers and facilitators, and ultimately to design interventions. The Theoretical Domains Framework (TDF) is a framework based on health psychology that was designed to improve implementation of evidence based practice. Methods: This dissertation describes a series of projects, based on the TDF, and aimed at understanding barriers and facilitators to early physical rehabilitation in mechanically ventilated patients. First, a systematic review of the literature, using the TDF to synthesize barriers and facilitators was done. The following two research chapters describe a Delphi study of nurses, physical and occupational therapists, respiratory therapists and physicians. The first round of the Delphi consisted of semi-structured interviews. With the TDF, a theoretically driven topic guide was developed and interviews analyzed using a theory-driven content analysis. The following rounds of the Delphi consisted of agree/disagree questions with a Likert scale, based on Round 1 results that were reviewed and prioritized by the research group. Results: The systematic review of the literature found that there was a general lack of theory in assessing barriers and facilitators. Using the TDF to synthesize, we found a significant focus on the domains of Behavioural Regulation, Environmental Context and Resources and Beliefs about Consequences. The Dephi study confirmed these findings, although also found a focus on the domains of Skills, Social and Professional Role, Beliefs about Capabilities, Social Influences, Knowledge and Optimism.

Keywords: Critical care
Knowledge translation
Mechanical ventilation
Theoretical Domains Framework
Advisor: Cuthbertson, Brian H
Characterization of Loss- and Gain- of Function Mechanisms of TDP-43 Pathology in Amyotrophic Lateral Sclerosis and Frontotemoral Lobar Degeneration

Nuclear depletion and formation of cytoplasmic inclusions of the DNA/RNA-binding protein TAR DNA-binding protein of 43 kDa (TDP-43) in disease affected-neurons is a shared neuropathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). This suggests that TDP-43 could contribute to disease pathogenesis through a loss-of-function from the nucleus and/or a gain-of-function in the cytoplasm. Since TDP-43 regulates numerous aspects of RNA metabolism, I determined if abnormalities of TDP-43 would lead to changes in translating mRNAs in motor neurons of a mutant TDP-43 transgenic mouse model by coupling ribosome affinity purification with microarray analysis. Through this I discovered that two RNA-binding proteins, methenyltetrahydrofolate synthetase domain-containing protein (MTHFSD) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58), were differentially expressed in motor neurons of mutant TDP-43 mice and ALS cases. I demonstrated that TDP-43 binds to the MTHFSD and DDX58 RNAs, indicating that TDP-43 could directly regulate their expression. Since TDP-43 autoregulates its own expression and splicing I investigated a C-terminally truncated isoform of TDP-43 (TDP-C-spl) and demonstrated that it is upregulated in ALS spinal cord tissue. TDP-C-spl formed cytoplasmic aggregates in transfected cells, and induced neuronal dysfunction in transgenic TDP-43-C-spl mice, which developed progressive neuropathology, age-dependent decline in cognition and displayed stereotypic behavior, modeling aspects of FTLD. To understand upstream events leading to TDP-43 mislocalization, I investigated its relationship with hexanucleotide repeat expansions in C9orf72, the most common genetic cause of ALS/FTLD using isoform specific antibodies. I further show that the normal localization of C9-S to the nuclear membrane of motor neurons is lost in ALS, and correlates with TDP-43 pathology. In summary, this thesis has uncovered novel functional consequences of TDP-43 pathology and implicated nucleocytoplasmic dysfunction as a possible upstream event causing TDP-43 mislocalization.

Keywords: Amyotrophic lateral sclerosis
Frontotemporal lobar degeneration
TDP-43
Advisor: Robertson, Janice
Kinetic Requirements for Catalytic Deoxygenation of Alkanals on Solid Brønsted Acid Sites

Alkanals undergo catalytic deoxygenation on solid Brønsted acid catalysts (e.g. acidic zeolites and polyoxometalate clusters), producing light alkenes, alkadienes, or heavy aromatic species, under atmospheric pressure and moderate temperature (473-673 K). The details about the reaction mechanisms and kinetics, however, remain ambiguous. In this thesis, C3-C6 n-alkanals were used as model reactants in combined kinetic measurement, chemical titration, and infrared spectroscopic experiments, to investigate the mechanisms and kinetic requirements for alkanal deoxygenation on solid Brønsted acid sites (H+). The reaction network for alkanal (CnH2nO) deoxygenation consists of three kinetically coupled primary reaction pathways: (i) intermolecular C=C bond formation, a bimolecular pathway lengthening the carbon chain via aldol condensation and dehydration and forming larger alkenals (e.g. C2nH4n-2O and C3nH6n-4O), (ii) intramolecular C=C bond formation, an unimolecular pathway evolving alkene (CnH2n) via transfer hydrogenation and dehydration while preserving the carbon backbone, and (iii) isomerization-dehydration, another unimolecular pathway directly ejecting a water molecule from the alkanal and producing alkadiene (CnH2n-2). The secondary cyclization-dehydration following pathway (i) produces aromatic species which provide the H atoms required for the alkanal transfer hydrogenation during pathway (ii); pathway (i) is also kinetically coupled with pathway (iii) as both pathways share a bimolecular surface intermediate and the accessibility of acid-base site pairs dictates the selectivities between these two pathways. In pathway (ii), the kinetic relevant alkanal transfer hydrogenation step proceeds via hydride ion transfer from a hydrocarbon (e.g. aromatic products) as the H-donor to the protonated alkanal as the H-acceptor, upon forming a bimolecular carbocation transition state. The hydride transfer is favored on H+ sites under confinement of molecular dimension because the confined structure solvates and stabilizes the transition state and lowers the activation barrier. The rate of the transfer hydrogenation is dictated by the hydride ion affinity difference between the H-donor-acceptor pair. The chain length of the alkanal reactant determines the stability of the enol tautomer and the hydride ion affinity of the protonated alkanal, as a result, dictating the rates and selectivities of these deoxygenation pathways. This mechanistic knowledge on the multiple catalytic cycles and their kinetic and thermodynamic requirements provides the framework for predicting the rates for larger oxygenates and hydrocarbons during alkanal deoxygenation and could guide the design of catalyst structures to enable tuning of rates and product distributions.

Keywords: Aldol Condensation
Alkanal (Aldehyde)
Bio-oil
Hydride Transfer
Solid Brønsted Acid
Transfer Hydrogenation
Advisor: Chin, Ya-Huei (Cathy)
Quantitative Surface and Sub-Surface Fluorescence Spatial Frequency Domain Imaging for the Enhanced Resection of Glioma

The extent of resection is now considered a significant prognostic factor; yet, safe complete resection is achieved in only 30% glioma patients. The challenge of surgery arises from the diffusive nature of gliomas known to infiltrate normal parenchyma beyond the resection cavity, which are left undetected. To better visualize glioma, intra-operative fluorescence-guided resection (FGR) has been a practical solution, providing real-time tumor contrast. In its most widely used form, FGR is mediated by the preferential overproduction of the fluorophore protoporphyrin IX (PpIX) in malignant tissue after an oral dose of its precursor 5-Aminolevulinic Acid (ALA). ALA-PpIX-FGR has been shown to significantly increase extent of resection. However, the visual assessment and the variable intrinsic optical attenuation of tissue limit this technique to delineating only high-grade tumors that display strong fluorescence. To this end, the work described in this thesis outlines the development of intraoperative quantitative fluorescence imaging (qFI) to quantify tissue fluorescence from superficial and from sub-surface tumors, by exploiting PpIX’s secondary absorption peak at 635 nm. Both developments were facilitated by enabling the quantitative optical-property-mapping and depth-resolving capabilities of a technique called Spatial Frequency Domain Imaging (SFDI). SFDI is a unique form of diffuse optical imaging. Light is spatially modulated at predetermined spatial frequencies and spatially-resolved diffuse reflectance images are collected. Recovering 2D maps of tissue optical properties is a prerequisite for both qFI and depth imaging, thus, a custom-built SFDI system was developed and optimized for both techniques. By determining tissue optical properties at both the absorption and emission wavelengths of PpIX, the raw hyperspectral fluorescence images are converted to 2D maps of PpIX concentration, [PpIX], after applying a quantitative fluorescent model. qFI is now being integrated on a surgical microscope for future clinical trials. While SFD-enabled-qFI proved to be a practical extension to the quantitative probe, both are still blind to sub-surface tumors, due to the limited penetration depth of the excitation light. This limitation prompted the development of quantitative sub-surface fluorescence imaging, where tumor depth and [PpIX] can be recovered to assist the surgeon in making an informed decision on whether to continue resection.

Advisor: Wilson, Brian C
Normalizing Marginality: A Critical Analysis of Blackness and Disability in Higher Education

This dissertation examines the experiences of Black undergraduate and graduate students with disabilities enrolled at the University of Toronto, Canada. The study employs an intersectional framework to explore the experiences of twelve Black students in relation to interpretive categories of Blackness and disability in this university setting. Using interpretive sociology, critical Black and disability studies theories, my study illuminates the ways students navigate the everyday complexities of Blackness and disability in University life. In employing qualitative methodology, this study investigates the University of Toronto’s disability accommodation policies, practices and procedures that organize the lives of Black disabled students. In essence, my study addresses marginalization by mapping the University’s bureaucratic practices influencing students’ academic progress. In relation to this mapping, the dissertation explores how Black disabled students navigate their experiences in accessing the bureaucratic ordering of accommodation and how this influences their academic endeavours. This critical analysis reveals that the University of Toronto is failing Black disabled students through its disability accommodation policies and practices. It also indicates that the marginalization of Black disabled students is normalized through the routine orders of accommodation processes. Ultimately, this study shows how categories of “Blackness” and “disability” act to circumscribe educational opportunities for students with disabilities. These categories are typically informed by anti-Blackness and the bio-medical versions of disability generating “individual lack,” which conceals the complex ways hierarchies of power are enabled by the social construction of normalcy. My aim is to raise a collective awareness of anti-Blackness in negotiating equity in educational opportunities in universities for students with disabilities. The study concludes by discussing how the impact of colonialism and structural inequities within accounts of Blackness and disability continue to produce injustice in university settings.

Keywords: Black Studies
Discursive Intersectionality
Gender Studies
History
Humanities
Social Sciences
Advisor: Titchkosky, Tanya