VictorJuiz/thesis huggingface dataset:
Identification of Biologically Relevant Binding Targets of SH3 Domains Using a Compensatory Mutation Approach
Keywords: compensatory mutation approach
protein-protein interaction
SH3

Protein-protein interactions are crucial for cellular function. Many protein-protein interactions are mediated by peptide recognition modules (PRMs), small domains in proteins that recognize and interact with short peptide sequences in target proteins. In many systems, the interactions between PRMs and target proteins have been investigated using high-throughput methods. These studies have revealed protein-protein interaction networks where single PRMs apparently interact with dozens of different proteins. Some of these interactions may be false positives, may never occur in vivo, or may not be involved in any biological function. Thus, a key challenge arising from these studies is to identity protein-protein interactions that are “biologically relevant”. In this thesis, I define a biologically relevant interaction as one where specific disruption of the interaction can be definitively shown to cause a detectable phenotype under some genetic background or environmental condition. The goal of my work has been to design and test a generalizable approach, which I call “the compensatory mutation approach”, for determining whether particular protein-protein interactions are biologically relevant. To develop my approach, I studied the function of SH3 domains in budding yeast, Saccharomyces cerevisiae, as a model system. In particular, I focused on the SH3 domains found in two yeast proteins, Abp1p and Nbp2p. Using the compensatory mutation approach, I identified biologically relevant binding targets for both of these domains. Importantly, I showed that the biologically relevant binding targets may change depending on the phenotype tested and the assay conditions. I also demonstrated that the pathway involving Abp1p can be re-wired in such a way as to eliminate the requirement for the Abp1p SH3 domain. This study has provided the first unequivocal identification of biologically relevant targets for the Nbp2p and Abp1p SH3 domains, and established that the compensatory mutation approach can supply unique insights into the functioning of biological pathways.
Transnational Latina Feminism in Toronto, Canada, 1970s–2000s: Treading the New Avenues
Keywords: community agencies
exiles
feminist
Latin American
Toronto
women

Latin American women have a long history of anti-imperialist and anti-capitalist political engagement, expanding into feminist praxis with diverse women’s movements across Latin America. Following connections with the Chicana cannon and establishing connections with other racialized grassroots groups in Canada, a particular Latina feminist subjectivity emerged that complicates Canadian feminism and challenges dominant narratives of Latin American political activities. Canadian feminist historiographies regularly engage in processes of erasure that inscribes Whiteness and imperialism into the Canadian landscape. In this context, Latin American women’s agency, experience, and lives have tended to disappear—along with other racialized women—from Canadian consciousness and literature, relegating them to the perpetual and exclusive role of victims. In this dissertation, four historical junctures have been selected that illustrate the transnational praxis of Latin American women’s feminist subjectivities in the city of Toronto, Ontario, Canada. These key moments showcase complex political debates, including the political crisis in Latin America, the imposition of neo-liberal policies, political repression, and exile. Diasporic Latin American women from the 1970s to the early 2000s developed organizations that responded to the political exigencies of the times, maintaining transnational connections with women’s and feminist movements in Latin America while also creating new spaces in Canada, ultimately evolving a unique diasporic Latina feminist subjectivity. These junctures are: (1) the solidarity movement of the 1970s to the 1990s, focusing on two women’s organizations; (2) the formation of the Latin American Coalition to End Violence against Women and Children (LACEV) in the 1990s; (3) the organization of the First Latin American and Caribbean Women’s Encounter in 1995; and (4) the foundation of MUJER in 2003. These four examples are neither linear nor lacking in controversy. This study is based on a mixed methodology that includes interviews with leading activists, document analysis, and personal experience. Throughout their historical journeys, Latin American women contributed not only to the processes of democratization in their countries of origin but also to Toronto’s feminist political milieu, providing a vital contribution and translating into invaluable organizations that continue to support Latin American women’s agencies and political praxis.
The Role of Tumour Necrosis Factor Receptor Family Members CD137 (4-1BB) and CD30 in Antiviral T Cell Responses
Keywords: 4-1BB
aging
CD30
co-stimulation
influenza
tissue-resident memory

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.
Evolution and Molecular Mechanisms of Photoreceptor Transmutation in Reptiles
Keywords: lizards
molecular evolution
selection
snakes
vision

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.
Controlled Generation of Progenitor T-cells from Hematopoietic Stem Cells and Pluripotent Stem Cells
Keywords: Biomedical engineering
Hydrogels
Immunotherapy
Stem cells
T cell development
T 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.
Methods of Protein Sample Preparation Using Digital Microfluidics and Gas-Phase Techniques with Detection by Mass Spectrometry
Keywords: differential mobility spectrometry
digital microfluidics
mass spectrometry
protein analysis

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.
Barriers and Facilitators to Early Physical Rehabilitation in Mechanically Ventilated Patients
Keywords: Critical care
Knowledge translation
Mechanical ventilation
Theoretical Domains Framework

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.
Characterization of Loss- and Gain- of Function Mechanisms of TDP-43 Pathology in Amyotrophic Lateral Sclerosis and Frontotemoral Lobar Degeneration
Keywords: Amyotrophic lateral sclerosis
Frontotemporal lobar degeneration
TDP-43

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.
Kinetic Requirements for Catalytic Deoxygenation of Alkanals on Solid Brønsted Acid Sites
Keywords: Aldol Condensation
Alkanal (Aldehyde)
Bio-oil
Hydride Transfer
Solid Brønsted Acid
Transfer Hydrogenation

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.