VictorJuiz/thesis huggingface dataset:
Slit2/Robo-1 Signaling in Monocyte and Macrophage Function: A Role in Vascular Inflammation

Atherosclerosis is a maladaptive inflammatory response initiated by the retention of cholesterol-rich low-density lipoproteins. These are rendered pro-inflammatory upon oxidation, leading to activation of endothelial cells and recruitment of monocyte-derived macrophages. While the clearance of oxidized low-density lipoprotein (oxLDL) by macrophages is initially beneficial, excessive accumulation results in cholesterol-laden foam cells that drive disease progression. The uptake of oxLDL is largely mediated by the scavenger receptor CD36 and requires dynamic actin reorganization driven by Rac GTPases. The Slit family of secreted proteins, together with their transmembrane receptor Robo, inhibit the migration of a variety of cell types, in part by inactivating Rac and blocking dynamic actin rearrangement. We show that Slit2 inhibited adhesion of monocytic cells to activated human endothelial cells, and to immobilized ICAM-1 and VCAM-1. Microfluidic live cell imaging showed that Slit2 inhibited the ability of monocytes tethered to endothelial cells to stabilize their actin-associated anchors and to resist detachment in response to increasing shear forces. Transfection of plasmids encoding constitutively-active Rac1 revealed that Slit2 inhibited post-adhesion stabilization of monocytes on endothelial cells by preventing activation of Rac1. Slit2 also inhibited chemotaxis of monocytes towards the chemokines, CXCL12 and CCL2, and administration of Slit2 to atherosclerosis-prone LDL receptor-deficient mice inhibited monocyte recruitment to nascent atherosclerotic lesions. The role of Slit2 in foam cell formation was investigated using a neutral lipid probe, BODIPY 497/503, and quantified by immunofluorescence microscopy and flow cytometry, revealing a decrease in cholesterol ester accumulation in Slit2 treated cells. The binding and uptake of oxLDL was investigated using fluorescently (DiI)-labeled oxLDL, and quantified by microscopy and flow cytometry. Slit2 decreased oxLDL-mediated foam cell formation in human and murine macrophages, by blocking Rac-dependent uptake of oxLDL by CD36. Thus, Slit2 may represent a powerful new tool to inhibit pathologic monocyte recruitment in vascular inflammation, and block foam cell formation in progressive atherosclerosis.

Keywords: adhesion
macrophage
monocyte
oxLDL
Slit2
Advisor: Robinson, Lisa
Role of sFlt1 and Anxa2 in the Glomerular Filtration Barrier Integrity Maintenance

Gene targeting studies have confirmed the role of VEGF-VEGFR system in vasculogenesis and angiogenesis. While VEGFR2 is a mediator of major functions of VEGF, the roles of VEGFR1 (Flt1) and its soluble isoform-sFlt1 are still elusive, they are thought of as decoy receptors for VEGF. However, accumulating studies revealed the physiological roles of sFlt1 in endothelial cells attachment, vascular sprouting and branching, and inhibition of VEGFR2 activation. Podocyte-specific knockout of Flt1 resulted in effacement of foot-processes and proteinuria, and one allele of tyrosine-kinase domain deleted Flt1 rescued this phenotype. Furthermore, exogenous sFlt1 promoted the attachment of podocyte and induced the formation of foot process-like structures. Mass spectrometry identified Anxa2 as a potential binding partner of sFlt1 on the podocyte membrane, and knockdown of Anxa2 in vitro caused drastic cytoskeletal changes in human podocytes. To further investigate the roles of sFlt1 and Anxa2 in the integrity maintenance of glomerular filtration barrier and the interactions between sFlt1 and Anxa2, we generated a transgenic sFlt1 floxed and a transgenic Anxa2 floxed mouse models. We set up the global sFlt1 KO (v1-/-) mouse line, and identified 17 novel sFlt isoforms from v1-/- mouse placentas by 3â -RACE where the major one was sFlt14-1 in place of sFlt1 (sFlt13) in wild type mice. We also confirmed that sinusoidal trophoblast giant cellsâ contribution to generation of these isoforms. Additionally, we showed that hypoxic signaling pathway and cleavage and polyadenylation alternative splicing machinery were also activated in v1-/- mice. We further confirmed that sFlt1 was essential for mouse embryogenesis and development and heart formation in a dose-dependent manner by generating 2 additional mouse models: 1) expressing ONLY the sFlt1 (Flt1TM-TK2); 2) expressing ONLY one-allele of sFlt1 (Flt1del/TM-TK-) on mixed genetic background (CD1 mouse). We generated global and podocyte-specific Anxa2 KO mouse lines, induced diabetes in global Anxa2 KO mice, and found that Anxa2 has an anti-glycation/glycosylation feature. Aged (1.5 years old) podocyte-specific Anxa2 KO mice developed proteinuria. PAS stain identified thick GBM and deposits in the sub-epithelial space. TEM confirmed these findings and demonstrated the intense darkness of the GBM, and further identified similar changes in younger mice (6-month old). The involvement of complement component C3 and other normal GBM components were not confirmed. In summary, these in vivo data provide the first genetic evidence that sFlt1, not full-length Flt1, is essential for mouse embryogenesis and growth and heart development in a dose-dependent manner; and that Flt1 has a large number of soluble isoforms in both human and mouse. In addition, Anxa2 from podocyte is actively involved in the cross-talk between podocyte and GBM.

Keywords: angiogenesis
embryogenesis
podocyte
soluble VEGFR1
Vasculogenesis
VEGF
Advisor: Quaggin, Susan E
Targeting CDK to Prevent and Treat Retinoblastoma and other Cancers

Pharmaceutical investment in cell cycle inhibitors has been immense, yet the efficacy in treating established tumors is poor in most cases. Considering that tumor cells co-opt the cell cycle to grow, it is unclear why most cell cycle inhibitors, like CDK inhibitors, have failed to pass Phase II clinical trials. Since expansion of premalignant cells is critical for tumor establishment, we wondered whether CDK inhibitors would be more effective in prevention than treatment. To examine this, we leveraged pharmaceutical and genetic strategies to demonstrate that initiated retinal cells and nascent retinoblastoma tumors are exquisitely sensitive to CDK inhibition in vivo. To uncover what factors predict CDK inhibitor sensitivity, we generated a transcriptional signature, which reports real-time activity of CDK2 (a potent target of most CDK inhibitors). Surprisingly, we found that CDK2 activity does not predict CDK2 inhibitor sensitivity. Instead, drug sensitivity is predicted by a subset of Protein Processing networks (e.g. translation, mRNA stability, splicing, and protein targeting). Through mining publically available datasets, we unravel a novel co-regulatory/co-dependent relationship between CDK2 activity and Protein Processing networks. Leveraging Protein Processing networks, we define which cancers may be best suited for CDK inhibition in the clinic (e.g. diffuse B-cell Lymphoma). Overall, we offer two strategies to bolster the clinical translation of CDK inhibitors and gain further insight into how cancer cells rewire their cell cycle machinery in order to bypass therapy.

Advisor: Bremner, Rod
Reconstructing the Office Furniture: The Art of the Early Modern Choir Stall

This dissertation examines a range of choir stalls made in northern Europe and Spain between 1450 and 1550 with the aim of reinstating choir stalls as significant objects in our understanding of the arts of the early modern period. This century of prosperity and renewal supported widespread church commissions and resulted in the construction of some of the most complex and inventive sets of stalls ever built. As objects that were central to the ritual life of religious communities and helped define the sacred space of the choir, their form and iconography tell us about the values, beliefs and aspirations of the patrons who commissioned them and the communities who used them. As large, complex microarchitectural structures, they allowed varying and potentially interacting visual programs to be displayed in diverse and innovative ways. Using a variety of approaches, four broad art historical issues are investigated, showing some of the ways that choir stalls contribute to the larger discourse. Historiography elucidates the longstanding emphasis on misericords and the lack of integration of this work with broader art historical research, as well as suggesting ways to move forward. Analysis of a variety of historical documents along with current research on acoustics and design, and engagement with modern theorists of space highlight how the spatial configuration of choir stalls contributed to key aspects of their use and meaning. The examination of a range of documentary sources provides a picture of how a set of stalls might be realized in the early modern period, and the ways in which these practices compare with the construction of other complex objects. Finally, ``thick description'' of early modern choir stalls based on historical data, contemporaneous documents and the stalls themselves combined with assumptions from material culture studies, show how they were used to construct both individual and group identity, which affirms the significance attached to choir stalls in early modern society. Overall, this dissertation demonstrates that choir stalls were an important part of the artistic production of the early modern period and their study is therefore necessary for a fuller appreciation of the arts of this time.

Keywords: choir
church
furnishing
furniture
microarchitecture
stall
Advisor: Kavaler, Ethan Matt
NMR Sensing Strategies Based on Modulation of Chemical Exchange Rate With Small-Molecule Synthetic Sensors

Nuclear magnetic resonance (NMR) spectroscopy is a powerful and versatile tool for monitoring molecular interactions. Dynamic processes, such as conformational changes or binding events, can induce drastic effects on NMR spectra in response to variations in chemical exchange rate. Molecular strategies in which the chemical exchange rate is deliberately controlled can lead to attractive opportunities in NMR sensor design. This thesis explores two main strategies involving structurally similar sensors in which modifications of the chemical exchange rate have been utilized to sense anions or detect changes in pH. The first section of the thesis describes a novel and ultrasensitive approach to detect anions indirectly by NMR and is based on a small synthetic sensor (TUC) displaying conformational rigidity due to intramolecular hydrogen bonding. With an increase in chemical exchange rate, due to anion-induced conformational flexibility, detection and quantification of spherical and tetrahedral anions as low as 120 nM concentrations, which is more than 1000 fold lower than the detection limit of NMR, are possible. The next part describes a slow proton exchange strategy for pH sensing. Monitoring pH change accurately and non-invasively by NMR has important implications in the detection and treatment of pathologies such as cancer, ischemia or cystic fibrosis. Conventional NMR pH detection methods are mostly based on changes in chemical shift due to fast proton exchange between the different protonation states of the sensors. Slowing down proton exchange allows ratiometric pH measurement, which eliminates the errors from pH-independent factors such as artifacts caused by the chemical environment, ionic strength, etc. Therefore, pH can be measured by using the ratio of the different protonation states of the pH sensor. Detection of pH in vitro and in bacterial cultures was successfully demonstrated with the sensor SPE1 using this new method, and preliminary chemical exchange saturation transfer (CEST) experiments were carried out. Optimization of SPE1, through 13C-labeling (13C-SPE1) and new design to decrease the pKa and increase the pH window of the sensor (SPE2), was also studied. Overall, both strategies demonstrate how modulation of chemical exchange rate can provide a platform for the development of new NMR sensors with high sensitivity and accuracy.

Keywords: anion
chemical exchange
hydrogen bonds
NMR
pH
sensing
Advisor: Zhang, Xiao-an
Late Victorian Ballad Translation

This dissertation investigates late Victorian ballad translations that depict non-British people. It focuses on ballads published in the 1890s, at the height of imperialist expansion and at a peak of ballad collecting and editing. In this period, as recent criticism in the field has shown, many ballad scholars, collectors, and publishers treated this verse form like an artifact in a Victorian museum: they framed, bowdlerized, and decontextualized the ballad, as if it were an inanimate object, rendering it easily consumable for the middle classes. By looking at poets who translated the ballad rather than publishers who ‘artifactualized’ it, I show that the ballad remained a vital—even rebellious—politically engaged form, even during this period of intense collecting and cataloguing. My work contributes to the burgeoning field of global poetics through an investigation of four poets: William Morris, Rudyard Kipling, Robert Louis Stevenson, and E. Pauline Johnson. My first chapter, a study of Morris’s ballad translations from Danish and Icelandic, argues that Morris figured the ballad as embodying folk up-rising. My second chapter argues that Kipling’s ballad translations are from “Atkinsese,” a language named after Tommy Atkins, the soldier-speaker the “Barrack-Room Ballads.” Tommy sings of the experiences of empire, but, in the margins of the text, an editor-figure retranslates Tommy’s words in order to undercut the authority that Tommy establishes. In my third chapter, I show that Stevenson’s translations from Tahitian blend Indigenous rhythms with conventions of the classical epic in an attempt at translating ethically. Finally, in my fourth chapter, I argue that Johnson’s The White Wampum includes hybridized balladic monologues that not only translate Indigenous Canadian voices into fluent English, but also translate the concept of the wampum belt to create hybridized “verse-wampum.” This dissertation dwells in the thresholds of Victorian poetry—in the margins of pages, at the borders of nations and of languages—in order to suggest that these zones of interaction and interpretation were broader and more significant than currently conceived.

Keywords: ballads
poetry
translation
Victorian
Advisor: Bolus-Reichert, Christine
Infants and the Child Welfare System: An Exploration of Practice and Policy Responses

The overarching objectives of this three-paper dissertation are to: (1) build upon and extend the minimal knowledge base with respect to infants in a Canadian child welfare context; and, (2) identify opportunities and challenges for promoting the optimal development of infants through an exploration of child welfare practice and policy responses in Ontario. A developmentally-informed framework guided this research. The first two papers utilized data from the 2013 cycle of the Ontario Incidence Study of Reported Child Abuse and Neglect (OIS-2013) to create a clinical portrait of infants and families involved with the child welfare system. The first paper explored the most influential clinical factors associated with the decision to transfer a case to ongoing services (i.e., characteristics of the infant, primary caregiver, household, case and of the short term services provided). The second paper examined the clinical factors predictive of the decision to refer families to services and age-specific differences in the clinical portrait and types of services referred to. The third paper explored how the science of early childhood development could inform and enhance child welfare policies and practices with infants and their families. The findings indicated that the most influential predictor of the provision of ongoing services was having a primary caregiver with few social supports; whereas, being a victim of intimate violence (IPV) and younger caregiver age were the caregiver risk factors that were significantly associated with a referral. There were age-specific differences in both the clinical profiles and the services referred to, suggesting that infants are a distinctly vulnerable subpopulation of children. The findings converge on the importance of developing policies and practices that target the unique needs and experiences of infants and their families. Key findings, themes, implications for social work, and recommendations for future research, policy, practice and advocacy efforts are proposed.

Keywords: child welfare
decision-making
earlychildhood development
infants
service provision
Advisor: Fuller-Thomson, Esme
Feminicidio, Transnational Legal Activism, and State Responsibility in Mexico

This dissertation uses the concept of transnational legal activism to analyze the mobilization of international human rights law as a multi-scalar process that produces and is shaped by gendered political and discursive opportunities. I apply this framework to examine how feminist grassroots activists engaged with supranational human rights institutions, especially the Inter-American Court of Human Rights, to hold the Mexican state responsible for the murders of three young women in Ciudad Juárez, an industrial city that borders the United States, in the case of González and Others “Cotton Field.” The Court declared that Mexico had failed to act with due diligence to prevent, investigate, and punish these crimes. These murders epitomize what activists identified as feminicidio, the systematic killing of women in a context of institutionalized gender discrimination sanctioned by the state; this phenomenon has prevailed in the northern state of Chihuahua where Ciudad Juárez is located since the 1990s. The dissertation also investigates how federal and local state actors responded to grassroots activists’ claims and the judgment of the IACtHR, including the criminalization of feminicidio. Through interviews with Mexican activists and frame analysis of the IACtHR judgment and of federal and local parliamentary debates, I argue that grassroots activists’ involvement in transnational legal activism contributed to expanding and rearticulating the meaning of women’s human rights and state responsibility at the domestic and supranational levels. Throughout, I highlight activists’ agency in this process and in their interactions with transnational organizations specialized in human rights advocacy and supranational litigation. Thus, I challenge assumptions in the literature on human rights and social movements that imply that grassroots actors have a limited access to international law and avenues to participate in transnational advocacy. Last, I suggest that the actions of Mexican grassroots activists extend a Latin American approach to international human rights law.

Keywords: Femicide
Feminicidio
Inter-American Human Rights System
International Human Rights Law
Mexico
Transnational Legal Activism
Advisor: Korteweg, Anna C
Levi, Ron
Unrepresentable Blood: Canadian Blood Donation, “Gay Blood” and the Queerness of Blackness

In this dissertation, I explore the Canadian Blood Services blood donation questionnaire and how the blood stories assembled within this document, and in the larger blood system, intersect with and depict blackness, queer (diaspora) sexualities, and Canadian (homo)nation-making. Narratives on blood produce moments of discipline, regulation, and confinement. Canadian Blood Services argues that its donor questionnaire is designed to effectively screen potential blood donors, with a number of questions focused on preventing an HIV/AIDS outbreak in the general population. The information gathered from these diverse questions constructs a figure of the ideal blood donor, thus creating a distinction between people whose blood gives life and people whose blood brings death. These distinctions result in the ban of particular groups of people, including bisexual and gay men and African people. Through centring a black queer diasporic analytic and reading practice, I am able to interrogate the ontological problem made of blackness. I contend that queerer modalities of thought are necessary to account for the complicated realities of racialized sexuality lived through black queered bodies and by black queer and trans people (and their blood). I analyze a diverse set of archives, including the donor questionnaire; websites of social and political organizations involved in the gay-blood debates; and legal, news, and government documents pertaining to the Canadian blood system. I seek to break the public silence on how blood continues to be used to justify the denigration of the lives of black people, both inside and outside of gay spaces, to push against the narrow, normative Eurocentric structures of gay blood. Thus, this reading acts as a decolonial, diasporic, transgressive project of writing blackness. My intervention into these anti-normative, anti-colonial discussions of blood, queerness, and blackness engages in a form of â epistemic disobedienceâ necessary to think differently about and disrupt both the homonationalist framing of gay blood and, more importantly, how we envision queer communities in our diasporic home-making. It is this that I seek to provoke in this thesis: to bring together the tangible and incoherent realities of our lives in order to articulate and engage in transformative justice.

Keywords: African/Black blood
Black Queer Diaspora
Blood donation
Blood narratives
Gay Blood
Transnational blackness
Advisor: Walcott, Rinaldo W
Characterization of the Calcium Channel Scaffold and the Cav2.2-synaptic Vesicle Tether at the Presynaptic Nerve Terminal

Calcium ion influx through voltage-gated calcium channels (Cav) triggers synaptic vesicle (SV) fusion and neurotransmitter release at presynaptic nerve terminals. A single Cav can trigger transmitter release, suggesting that a molecular tether links SVs to the channel. The spatial organization of Cavs at specialized regions within nerve terminals, known as transmitter release sites, suggests a protein scaffolding mechanism. Presently, the molecular mechanisms involved in channel scaffolding and SV tethering remain unresolved. The objective of the studies presented in this thesis was to examine the role of chick Cav2.2 intracellular region(s) in the abovementioned mechanisms using biochemical assays. First, we explored if Cav2.2 form an inter-channel link through their cytoplasmic regions, an interaction that may contribute to the calcium channel scaffolding mechanism. Pull-down assays were performed with Cav2.2 intracellular region fusion proteins and chick brain lysate. The assays demonstrated that Cav2.2 distal C-terminal region (C3) captures channel from membrane lysate. Furthermore, separate experiments with truncated C3 region fusion proteins and blocking peptides demonstrated that channel-channel association is mediated by the PDZ ligand domain (PDZ-LD) at the Cav2.2 C-terminal tip. Next, we examined the Cav-SV binding site using a novel in vitro SV pull-down assay (SV-PD). Our recent study narrowed down a SV binding site to 49 amino acids upstream of the PDZ-LD within the C3 region. We synthesized five non-overlapping peptides, each ten amino acids long, mimicking the SV binding site. SV-PD assays were carried out with C3 region fusion proteins and mimetic peptides, from which HQARRVPNGY markedly inhibited SV-PD. We demonstrate that a yet unknown PDZ domain protein mediates the channel-channel association, and localized an SV binding site to a 10 amino acid region within the Cav2.2 distal C-terminal. Our study contributes to the understanding of fundamental molecular mechanisms at presynaptic nerve terminals and has important implications in synaptic transmission.

Keywords: Cav2.2
Presynaptic nerve terminal
scaffold
Synaptic vesicle
tether
Advisor: Stanley, Elise F