VictorJuiz/thesis huggingface dataset:
Biophysical Techniques and Assay Design for the Development of Small Molecule Inhibitors

One of the most important pillars in preclinical research involves the biophysical characterization of protein-compound interactions. This characterization, describing the in vitro potency and binding properties of the interaction, is measured through techniques such as fluorescence polarization assays, thermal shift assays, isothermal titration calorimetry experiments, and activity-based assays. A broad biophysical profile of each compound against the target of interest allows for fine-tuning of the molecule through structure-activity relationship-guided design. Biophysical profiling can enable accurate prediction of the interaction within a biological setting and thus provide insight into the mechanism-of-action observed in a cellular setting. This thesis details the recombinant protein expression and purification as well as the biophysical assay development of several attractive therapeutic targets for cancer treatment including STAT3, STAT5B, UBA5, HDAC6, and HDAC8. To aid in STAT inhibitor discovery, exploration of assay development to confirm STAT-phosphorylation inhibition was described. This thesis reports a rapid fluorescence-based screening method to identify SH2 domain binders followed by a phosphorylation-inhibition assay to validate mechanism of action for molecules determined from the high-throughput screening assay. Additionally, the development and optimization of a fluorescence polarization (FP) assay for binding affinity determination of DNA binding domain inhibitors for STAT3 was also described. Similarly, FP assays and thermal shift assays were also validated for drug discovery program geared towards identification of highly selective and potent HDAC6 and HDAC8 inhibitors. Fluorescence-based activity assays and in cellulo nanoBRET assays were also established for HDAC proteins to confirm the results obtained from preliminary biophysical assays. Exploration of high-throughput virtual screening was also employed to identify novel UBA5 inhibitor scaffolds from a diverse natural product and natural product derivative libraries. This experiment yielded a lead candidate with potential for refinement through structure-activity relationship (SAR) to improve drug-like qualities of the molecule. Lastly, the hijacking of the upregulated prenylation pathway in cancer for artificial protein-membrane anchorage was investigated for utility as an alternative protein inhibition mechanism. Collectively, these studies demonstrate the progress in the assay development area for the targeting of multiple proteins to aid the drug discovery process.

Keywords: Assay development
HDAC
High-throughput screening
Prenylation
STAT
UBA5
Advisor: Gunning, Patrick T
Large-scale Network Models of Mild Traumatic Brain Injury and Repetitive Head Trauma

Mild traumatic brain injury (mTBI) may be understood as a multi-scale system deficit where adverse clinical outcomes emerge due to interacting brain changes that span spatial scales. At the micro-scale a neurometabolic cascade affects neurotransmission, while on the macro-scale diffuse axonal injury disrupts long-range connections. Large-scale brain network modeling allows us to make insights across these spatial scales; integrating neuroimaging data with biophysically based models to predict whole-brain dynamics. In this thesis, I used brain network models to study the long-term effects of mTBI and repetitive head trauma. Study 1 found mTBI patients experiencing active post-concussion syndrome symptoms had lower regional inhibitory connection dynamics relative to comparison participants. Study 2 expanded these findings to a sample of mTBI patients recruited from hospital emergency rooms in the semi-acute phase (1-2 weeks) by showing regional inhibitory dynamics were related to chronic TBI outcomes. Finally, in Study 3 I linked lower regional inhibitory connection dynamics with higher concussion exposure, lower cognitive performance, and higher psychosocial complaints in a sample of retired professional hockey players. Together, the work offers converging evidence that lower regional inhibitory dynamics are related to concussion exposure and poorer clinical outcomes following mTBI. The work exemplifies how large-scale network modeling may be used to make cross-scale inferences otherwise inaccessible with conventional neuroimaging.

Keywords: brain network models
mild traumatic brain injury
neuroimaging
sports concussion
Advisor: McIntosh, Anthony R
Addressing Vaccine Hesitancy in Canada: Paediatricians’ Perspectives and Social Work Opportunities

The objectives of this three-paper thesis are to: (1) explore the prevalence and impact of parental vaccine hesitancy on paediatricians in Canada; and, (2) use a social work lens to examine vaccine hesitancy and identify novel approaches involving social work to enhance vaccine uptake for young children in Canada. This research is guided by four theoretical perspectives: The Health Belief Model, The Theory of Planned Behaviour, Ecological Systems Theory and The Transtheoretical Theory of Change Model. The first and second papers are based on survey data collected through the Canadian Paediatric Surveillance Program (CPSP). The first paper examined the frequency with which paediatricians encounter vaccine hesitancy in clinical practice, challenges and impacts of hesitancy on practice and factors associated with vaccine-compliance among parents with concerns about vaccination. The second paper examined factors predicting physician-reported parental intention to vaccinate following the diagnosis of a paediatric vaccine-preventable disease. The third paper explores how social workers could contribute to addressing vaccine hesitancy with respect to early childhood vaccines in Canada. The findings of this dissertation indicate that paediatricians encounter parents with concerns about vaccination frequently in clinical practice. The most significant predictor of vaccine compliance among parents of young children with concerns about vaccination was receiving a strong personal recommendation from their primary care physician. The only significant predictor of physician-reported parental intention to vaccination following the diagnosis of a paediatric vaccine-preventable disease was whether the parent previously refused all vaccines for the child. Vaccine hesitancy is a complex phenomenon which has traditionally remained within the purview of physicians. Social workers have the training and skills to effectively address vaccine hesitancy, particularly among families facing structural barriers to accessing vaccination. This dissertation concludes with a discussion of key findings, implications for social work and healthcare and suggestions for future research, policy and practice efforts.

Keywords: interprofessional practice
paediatricians
paediatrics
social work
vaccination
vaccine hesitancy
Advisor: Craig, Shelley L
Spinoza’s Metaphysics of the Finite: Composition, Essence, Modality

This dissertation is about Spinoza’s metaphysics of ordinary objects and organisms. It focuses on three topics: mereological composition, essence and universals, and the nature of contingency. Chapter one argues that Spinoza’s thinking about parts and wholes provides a powerful alternative to so-called ‘fictionalist’ accounts of mereological composition. I show that even though Spinoza agrees with fictionalists that part-whole reasoning doesn’t aim at literal truth, at the same time he considers it instrumental in elucidating our limited understanding of causal processes. I conclude that Spinoza is an anti-realist about composition because he is a perspectivalist. That is, for Spinoza, expressions such as ‘x is a part of y’ or ‘x is a whole composed of some ys’ convey abstract judgments from a limited perspective. Chapter two is about Spinoza’s account of universal natures, that is, essences shared between members of a certain natural kind. I show that evidence for commitment to universal natures pervade Spinoza’s metaphysics, epistemology, and political philosophy. However, at the same he provides an error theory explaining how extant realist accounts of universals go wrong. On my reading, Spinoza’s seemingly contradictory statements about shared essences in fact constitute a coherent, modest realist account of the genuine qualitative similarity of entities belonging to a certain natural kind or species. Lastly, chapter three makes a novel case for interpreting Spinoza as a necessitarian, which is arguably one of the most important commitments in his metaphysics. I argue that even though Spinoza holds necessitarianism, he allows for contingency, although in a different sense than it is standardly taken to be in contemporary discussions. His definition of contingency is closer in meaning to the familiar use of the word ‘contingent’ according to which something is contingent if it depends on an external cause. On my reading, this means that Spinoza holds modal compatibilism, that is, on his account, something can be both contingent since it depends on an external cause, and necessarily exist in with regard to its causal history.

Keywords: composition
contingent
essence
necessitarian
Spinoza
universal
Advisor: Hübner, Karolina
Protecting Rights in the digital Age: The Application of the Charter in Cyberspace

Since the swift passage of the Anti-Terrorism Act (Bill C-51) in 2015, the Canadian Security Intelligence Service (CSIS) has had the unprecedented and highly controversial authority to take “reasonable and proportionate” measures — often referred to as “threat reduction measures”—to reduce threats to Canadian security. While there are some limits to the types of measures CSIS can employ, the Canadian Security Intelligence Service Act permits the use of measures that would otherwise contravene the laws of Canada or limit a right protected by the Canadian Charter of Rights and Freedoms (Charter) so long as they are judicially authorized by a judge of the Federal Court of Canada. As new threats and threat actors proliferate online, it is anticipated that CSIS will increasingly carry out its threat reduction mandate in the digital world. Yet, in June 2018, CSIS’s former review body, the Security Intelligence Review Committee, reported that CSIS had never sought a threat reduction warrant from the Federal Court. What remains unclear is why. Is it possible that CSIS has never employed a measure that might violate someone’s freedom of association, expression, or mobility rights, or their right to liberty and personal security protected under the Charter? Or has CSIS concluded that the Charter does not govern actions carried out in cyberspace and, as such, that their online conduct falls beyond the reach and oversight of the Federal Court? Unfortunately, Canadian Courts have never been asked, and legal academics have never answered whether the Charter constrains online state action. The Department of Justice and CSIS have also never publicly enunciated their legal position on this subject. To date, the government’s interpretation of the Charter’s application to state cyber operations remains classified. This dissertation dissects these issues for the first time and ultimately concludes that the Charter governs state conduct in cyberspace.

Keywords: Charter of Rights and Freedoms
cyberspace
espionage
extraterritoriality
human rights
national security law
Advisor: Roach, Kent
Community-engaged Theatre Audiences

Community-Engaged Theatre Audiences is an intervention into the reception of politically motivated, community-centered plays. This thesis aims to understand how individuals make meaning or sense out of these complicated sites of performance. It asks “what can scholars and artists learn about community-engaged theatre by asking audience members about their experiences?” Community-engaged theatre is an umbrella term that contains a multitude of theatrical practices, where theatre artists work with members of a community to create performance texts that in some way depict the community, taking up the community’s concerns and grappling with the community’s future. These plays are social justice initiatives, working within dynamic communities to foster social change. This study is predicated on the understanding that communities are neither fixed nor stable, but are rather emerging and constructed. Community-Engaged Theatre Audiences offers empirical audience data to scholars and artists of community-engaged theatre in an effort to understand more fully this performance genre as it is practiced now, both in contemporary southern Ontario specifically, and in Western, English theatre more broadly. This thesis presents three case studies of new Canadian plays, each one employing a contrasting set of dramaturgical practices: verbatim theatre, physical comedy, and site-specific theatre. The plays are Stories from the Generation Gap, by Cameron Crookston,Common Boots Theatre’s The Public Servant by Jennifer Brewin, Haley McGee, Sarah McVie, ii and Amy Rutherford, and finally, A Simple Twist of Faith by Evan and Rafe Malach. Each case study presents an analytical review of audience reflections in order to offer a rich, local understanding of each play, while revealing the fluidity of belonging and the gap between the communities hailed through the performance and the lived realities of the audience members in attendance. This study investigates multiple perspectives from audience members, bringing their language and reflection to the analysis of each play. Community-engaged theatre as a genre is more deeply understood through an analysis of audience member’s responses to specific exemplars. Audiences are so often erased or simply absent from archives and theatre scholarship. This thesis centers their voices in order to understand better community-engaged theatre as an advocacy project.

Keywords: Audience
Community
Community-Engagement
Drama
Theatre
Advisor: Freeman, Barry
Development of Digital Microfluidic Tools for Translational Medicine

Digital microfluidics (DMF) is a liquid handling technology used to manipulate microliter sized droplets on an array of electrodes. The flexibility of DMF makes it particularly useful for a wide range of applications. This thesis describes the development of DMF tools for diagnostic assays and single-cell analysis for genetic testing. Chapter 2 describes the first demonstration of clinical assays using DMF outside the laboratory. A portable DMF control system and low-cost cartridges were developed and used to perform DMF immunoassays for anti-measles and anti-rubella IgG in blood samples collected and analyzed on-site in a refugee camp in Kenya. We achieved clinical sensitivities and specificities of 86/80%, and 81/91% for measles and rubella. Next, recognizing the challenges inherent in uncontrolled environments, we developed tests that could be performed by inexperienced operators. Chapter 3 describes our development of DMF hemagglutination assays for trauma tests. This system was transported to Sunnybrook Hospital, and when run by an inexperienced user, blood typing results were found to be 100% concordant with the gold standard. Next, we switched to a different application, the development of a DMF powered tool for single-cell analysis called digital microfluidic isolation of single cells for -Omics (DISCO). Chapter 4 introduces DISCO, which combines DMF and laser cell lysis, allowing for single cells to be isolated from heterogenous populations in situ. We analyzed the genomes, transcriptomes, and proteomes of single-cells isolated by DISCO, connecting sequencing data to cell phenotype, while also identifying single nucleotide variations. Finally, chapter 5 discusses the application of DISCO for non-invasive prenatal testing (NIPT). Novel strategies were developed for the isolation of viable fetal cells from the cervical mucus of pregnant mothers for genetic testing. We provide preliminary evidence of successful fetal cell isolation, demonstrating potential utility of this method for NIPT. In sum, the tools developed in this thesis demonstrate the utility of DMF for point-of-care diagnostics and single-cell genetic analyses. I propose that the further development of these tools will allow for DMF to be utilized in resource limited settings and for a wide range of assays for trauma monitoring, cancer diagnostics, and beyond.

Advisor: Wheeler, Aaron
The Role of SOX2 in Molecular Circadian Clock Regulation and Suprachiasmatic Nucleus Development

The focal point of my doctoral research, Sox2, is a stem cell gene that has been extensively studied for its function in stem cell maintenance and proliferation, cellular reprogramming, neurogenesis, and tumorigenesis. Sox2 is expressed and maintained in stem cells of different tissues, including the developing central nervous system and adult neurogenic niches. When neural stem/progenitor cells terminally differentiate into neurons, SOX2 protein is usually eliminated and its gene transcription turned off, thus rendering it functionally irrelevant in most mature neurons. However, a hand full of brain regions contain fully differentiated neurons that are spared from Sox2 downregulation and retain strong Sox2 expression. One of such regions is the suprachiasmatic nucleus (SCN), that contains the mammalian central circadian clock. The 20,000 clock neurons in the SCN are responsible for interpreting photic signals and orchestrating oscillations in peripheral tissues. In this study, I demonstrated the “unusual” and “non-canonical” roles of Sox2 in these post-mitotic and differentiated clock neurons. By creating a Sox2 gene knockout specifically in the SCN, I found that Sox2 in SCN neurons is pivotal for high-magnitude expression of clock genes and neuropeptides that are critical for circadian rhythm regulation. In addition, the absence of Sox2 in SCN neurons leads to severe and widespread changes in behavioral rhythms. My study also shows that neuronal Sox2 expression is vital for defining the transcriptional landscape of the SCN and promoting its function as the central circadian pacemaker. These severe consequences of Sox2-deficiency observed in adult mutant animals are, at least in part, due to developmental and differentiation defects of the SCN.

Keywords: behavior
circadian rhythms
RNA sequencing
SOX2
stem cell transcription factor
suprachiasmatic nucleus
Advisor: Cheng, Hai-Ying Mary
Investigating the Underlying Biological Causes of Idiopathic Scoliosis

Idiopathic scoliosis (IS) is a spinal disease characterized by a three-dimensional curvature of the spine that arises in the absence of observable physiological or anatomical defects. Although there has been investigation into the genetic components of IS, the underlying cell biological and molecular mechanisms for disease progression remain unknown. Consequently, treatment options are limited to bracing or surgery, which act more as preventative measures rather than a cure. Our lab has previously described the first genetically defined developmental model of IS, ptk7ahsc9/hsc9 mutant zebrafish. Homozygous ptk7ahsc9/hsc9 mutant fish display hallmark phenotypes associated with human disease, including 3-dimensional rotational spinal curvature and adolescent onset. To identify mechanisms underlying IS, I employed next generation sequencing, transgenic reporters, and pharmaceutical treatment to define the biological origins of spinal curvature in ptk7ahsc9/hsc9 mutant zebrafish. These methods revealed an unexpected up regulation of immune factors and recruitment of immune cells to the site of spinal curvatures. I also demonstrated that administration of anti-inflammatory agents to juvenile ptk7ahsc9/hsc9 fish significantly impacts the onset and progression of scoliosis. To further investigate the mechanisms underlying IS pathogenesis, I also undertook RNA-sequencing on a second zebrafish model of IS, sspodmh4/+. sspodmh4/+ fish display a consistently severe, late-onset, scoliotic phenotype. Investigation of differential gene expression in sspodmh4/+ fish also indicated an neuroinflammatory response driving scoliosis formation. Furthermore, RNA-sequencing of sspodmh4/dmh4 and sspohsc105/hsc105 embryos, which display an embryonic curly-tail down phenotype, indicated that the curly-tail down phenotype may act as a functional scoliosis surrogate model, for use in drug and chemical screens. Together, this data suggests neuroinflammation as a common factor driving scoliosis pathogenesis in multiple models of zebrafish IS, and that modulation of these inflammatory signals could provide a treatment for this disease.

Keywords: Idiopathic scoliosis
ptk7
scospondin
Zebrafish
Advisor: Ciruna, Brian G
Identity Affirmation, Resilience, and Cardiovascular Health Among Sexual Minority People of Colour

Research with sexual minority (SM) people – especially people of Color (PoC) – has been limited in terms of depth and breadth. The existing research involving SM people has historically excluded PoC or neglected to address racialized experience as a factor in stress and wellbeing. Similarly, this body of literature rarely considers the impact of the unique experiences of sexuality (e.g., bisexual vs. monosexual) and gender (e.g., cisgender vs. gender diverse). Because SM-PoC are considered a population with conflicting identity categories, much of the literature focuses on the negative aspects of living with this identity, and rarely employs a strengths-based framework. This dissertation aimed to address these gaps in the literature by expanding on SM-PoC representation and diversifying approaches to examining mental and physical health. Study 1 involved a content analysis which examined the representation of bisexual PoC in SM mental health literature. Overall, this study revealed the underrepresentation of bisexual PoC, the most effective strategies for recruiting this population in research, and the common pathologizing of bisexual PoC in the literature. Findings highlight a great need for intersectional research that includes underserved ethnoracial groups. Study 2 involved a thematic analysis of qualitative survey responses from bisexual PoC. Participants noted novel positive perspectives on identity invisibility, in which they discussed being empowered by their identity and using their ability to pass as cisgender and heterosexual to advocate for others. Study 3 presented the development and initial validation of the Queer People of Color Identity Affirmation Scale (QPIAS), the first scale on positive attitude toward identity for this population. The QPIAS predicts resilience and empowerment beyond existing measures of sexual and ethnoracial identity affirmation. Use of this scale may provide new information on factors contributing to wellbeing in this population and be a beneficial tool in multiculturally competent clinical assessment. Study 4 explored the relationship between the QPIAS and cardiovascular regulation and reactivity in a sample of SM-PoC. QPIAS scores were found to correlate positively with both stress reactivity and self-reported physical health. High QPIAS scores were associated with flexible parasympathetic activity and vagal withdrawal during stress, which may be advantageous to health.

Keywords: Culture
heart rate variability
Identity affirmation
LGBTQ+
Psychometrics
Psychophysiology
Advisor: Andersen, Judith P