VictorJuiz/thesis huggingface dataset:
Development and Application of 19F NMR of Proteins
Keywords: protein
NMR spectroscopy

19F NMR studies of proteins provide unique insight into biologically relevant phenomena such as conformational fluctuations, folding and unfolding, binding and catalysis. While there are many advantages to the use of 19F NMR, experimental challenges limit its widespread application. The focus of this thesis has been to address some of these limitations, including resonance assignment and perturbations arising from fluorine probes, and to develop more robust methods of studying protein topology by 19F NMR. 19F NMR experiments designed to measure local hydrophobicity and exposure were developed and evaluated in two systems, Fyn SH3 and calmodulin, labeled with 3-fluorotyrosine. Paramagnetic effects from dissolved oxygen, solvent isotope shifts from deuterium oxide, and 1H-19F NOEs were each sufficient in establishing relative solvent exposure, while the combination of effects from oxygen and deuterium oxide were able to delineate local hydrophobicity and solvent accessibility of 19F probes. Two NMR based resonance assignment protocols were developed using 13C, 15N-enriched 3-fluorotyrosine and 3-fluorophenylalanine, separately biosynthetically incorporated into calmodulin. In the first approach, isotopic enrichment facilitated two-dimensional heteronuclear experiments based on INEPT and COSY magnetization transfer schemes to correlate the fluorine nucleus to sidechain and backbone 1H, 13C, and 15N atoms, providing complete spectral assignment. The assignment of 3-fluorophenylalanine resonances was achieved using 19F-, and 15N-edited homonuclear NOE experiments to connect the fluorine nucleus to intraresidue and neighboring 1H and 15N resonances. While both strategies were successful, the NOE-based method was vulnerable to alternate relaxation mechanisms, including chemical shift anisotropy and chemical exchange. Structural perturbations arising from uniform incorporation of 3-fluorophenylalanine in calmodulin was thoroughly investigated using 19F and 1H-15N NMR spectroscopy, 15N spin relaxation and thermal denaturation via circular dichroism spectroscopy. While stability was unaffected, NMR experiments revealed increased protein plasticity, minor conformers and line broadening. The merit of fractional fluorine labeling in reducing such disruptions was demonstrated, and labeling levels of 60-75% provided an optimal balance between native-likeness and the usual advantages of 19F NMR in our system. The 19F NMR techniques developed here are broadly applicable and will expand the utility of 19F NMR in studies of protein systems.
The Role of the Di-arginine "R553AR555" Motif in Modulating Trafficking and Function of the Major Cystic Fibrosis Causing Mutant (DeltaF508-CFTR)
Keywords: Cystic Fibrosis
Peptide therapeutics
RXR
di-arginine
trafficking
F508
CFTR
correctors

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.
Two-photon Excitation Photodynamic Therapy for Localized Blood Vessel Targeting
Keywords: Photodynamic therapy
vessel targeting

The motivation of this study lies in the necessity for a microfocal therapy to specifically target diseased areas in vascular pathologies such as age-related macular degeneration (AMD). AMD is the most common cause of legal blindness among people over the age of 60 in developed countries. This degenerative condition affects the macula, the central region of the retina, severely impairing detailed vision and hindering everyday activities. Worldwide, 25-30 million people live with some form of AMD. Among them, ~10% suffer from the more advanced and damaging form, wet-AMD, which causes rapid and severe loss of central vision. To date, there is no cure or long-term alternative for this degenerative disease despite intensive research efforts. With recent developments in biophysical tools and experimental procedures, in this study, we demonstrate a highly-localized therapeutic option: two-photon (2-photon) photodynamic therapy (PDT) that could be advantageous for the cure of wet-AMD, either alone or in combination with recently discovered anti-angiogenic therapies. This new approach offers selective targeting of the diseased area, thus minimizing damage to the surrounding sensitive healthy eye tissues, which is a major concern with the clinically-used, standard wide-beam, one-photon (1-photon) PDT. The objective of the research was to test the feasibility of microfocal 1-photon and the inherently localized 2-photon PDT, their optimization and also to evaluate the efficacy of existing 1-photon and novel 2-photon photosensitizers. In this thesis, I illustrated the in vitro (endothelial cell monolayer) and in vivo (window chamber mouse (WCM)) models that can be used to quantitatively compare the 2-photon efficiency of photosensitizers. Using the in vitro model, I compared the 2-photon efficacy of clinically used 1-photon PDT drugs Photofrin and Visudyne, and showed that the Visudyne is an order of magnitude better 2-photon photosensitizer than Photofrin. With the WCM model, I demonstrated a novel designer 2-photon photosensitizer is 20 times more efficient than Visudyne for single vessel occlusion. I also generated the drug and light dose reciprocity curve for localized single-vessel microfocal PDT. This is a necessary step towards applying the method to the relevant ocular models of AMD, which is the next phase for this research.
Understanding Bicultural Identity and its Impact on the Association between Discrimination and Well-being
Keywords: bicultural
discrimination
well-being
identity
culture
ethnicity
self-esteem
depression

How one understands their bicultural identity has implications for how one psychologically reacts to discrimination. The three major goals of this thesis were to explore 1) how young adult, bicultural, ethnic minorities understand their cultural identities; 2) how the individual’s understanding of his or her bicultural identity was associated with reactions to real or imagined experiences of discrimination; and 3) the value of the blended/alternating bicultural distinction in understanding bicultural identity and how that might relate to the association between discrimination and well-being. Young adult undergraduates attending a diverse university campus, mainly of East and South Asian background, were interviewed, asked to complete written questionnaires, or asked to participate in an experimental study concerning cultural identity. Results from the three studies of the thesis suggest common themes in participant descriptions of bicultural identity. Results also demonstrate the direct negative effect of discrimination on mood, and suggest that the distinction between understanding one’s bicultural identity as a stable, cohesive blend of two cultures (Blended Biculturalism) vs. as one that switches between two cultural selves (Alternating Biculturalism), may have implications for differential effects of discrimination on psychological well-being. Alternating biculturals in the discrimination condition reported experiencing more negative mood than alternating biculturals in the non-discrimination condition. Blended biculturals did not show this difference. Alternating biculturals in the discrimination condition also reported experiencing more approach thoughts toward their group than alternating biculturals in the non discrimination condition; blended biculturals did not show this difference either, but rather had high levels of approach thoughts in both conditions. Possible explanations for these results are discussed.
Cricket as a Diasporic Resource for Caribbean-Canadians
Keywords: transnational
Toronto
diaspora
Black masculinity
liming
older men
communitas
sport tourism

The diasporic resources and transnational flows of the Black diaspora have increasingly been of concern to scholars. However, the making of the Black diaspora in Canada has often been overlooked, and the use of sport to connect migrants to the homeland has been virtually ignored. This study uses African, Black and Caribbean diaspora lenses to examine the ways that first generation Caribbean-Canadians use cricket to maintain their association with people, places, spaces, and memories of home. In this multi-sited ethnography I examine a group I call the Mavericks Cricket and Social Club (MCSC), an assembly of first generation migrants from the Anglo-Caribbean. My objective to “follow the people” took me to parties, fundraising dances, banquets, and cricket games throughout the Greater Toronto Area on weekends from early May to late September in 2008 and 2009. I also traveled with approximately 30 MCSC members to observe and participate in tours and tournaments in Barbados, England, and St. Lucia and conducted 29 in-depth, semi-structured interviews with male players and male and female supporters. I found that the Caribbean diaspora is maintained through liming (hanging out) at cricket matches and social events. Speaking in their native Patois language, eating traditional Caribbean foods, and consuming alcohol are significant means of creating spaces in which Caribbean-Canadians can network with other members of the diaspora. Furthermore, diasporas are preserved through return visits, not only to their nations of origin, but to a more broadly defined homeland, found in other Caribbean countries, England, the United States and elsewhere in Canada. This study shows that while diasporas may form a unified communitas they also reinforce class, gender, nation and ethnicity hierarchies and exclusions in diasporic spaces. For example, women and Indo-Caribbeans are mainly absent from or marginalized at the cricket grounds, which celebrates a masculine, Afro-Caribbean culture. Corporeal practices such as sports, and their related social activities, can be deployed as diasporic resources that create a sense of deterritorialized community for first generation Caribbean migrants.
Working in the Creative Economy: The Spatial Dynamics of Employment Risk for Musicians in Toronto
Keywords: Cultural Economy
Music
Labour
Spatial Dynamics

Over the past decade the introduction of MP3’s, file sharing networks and illegal downloading has fundamentally changed the music industry, structurally and spatially. In the wake of this restructuring it is now estimated that 95% of all musicians in Canada operate independently of record companies (Canadian Independent Recording Artist Association or ‘CIRAA’). Digital technologies afford independent musicians greater freedom and control over how and where they live and work. Although economic geographers have been quick to examine the impact of the so called ‘MP3 Crisis’ on record sales and the major record labels, little is known about how changes at the macro-scale affect the working lives of individual musicians in specific locations. As a result, this dissertation focuses on the employment experiences and spatial dynamics of independent musicians in Toronto. Drawing on sixty-five in-depth interviews with musicians and key informants in the music industry, the thesis documents the intersections between technology, work and space. In particular, the analysis highlights the ways in which the new creative and spatial freedoms, associated with independent music production, are accompanied by intensified competition and employment risk, which musicians experience in an increasingly individualized way. Surviving in the current marketplace requires independent musicians to perform a range of new tasks and exhibit a higher degree of professionalism. Accordingly, the research outlines some of the reasons why some musicians are rejecting and reworking traditional bohemian lifestyles, spatial patterns and risk mediation strategies. In particular, there is growing evidence of new forms of networking and of the increased importance of strategic collaborations between musicians and fashion designers. There are also signs that some musicians are relocating from the downtown core to the surrounding suburbs, and that musical talent is becoming redistributed across the city-region. Ultimately, this dissertation demonstrates the need for specialized policies to incubate and retain creative talent in an increasingly global and digital marketplace.
Advanced Reasoning about Dynamical Systems
Keywords: knowledge representation and reasoning
reasoning about action and change
situation calculus
decidable reasoning
description logics
two-variable first-order logic with counting quantifiers
action hierarchies
order-sorted logic
regression
computational advantages

In this thesis, we study advanced reasoning about dynamical systems in a logical framework -- the situation calculus. In particular, we consider promoting the efficiency of reasoning about action in the situation calculus from three different aspects. First, we propose a modified situation calculus based on the two-variable predicate logic with counting quantifiers. We show that solving the projection and executability problems via regression in such language are decidable. We prove that generally these two problems are co-NExpTime-complete in the modified language. We also consider restricting the format of regressable formulas and basic action theories (BATs) further to gain better computational complexity for reasoning about action via regression. We mention possible applications to formalization of Semantic Web services. Then, we propose a hierarchical representation of actions based on the situation calculus to facilitate development, maintenance and elaboration of very large taxonomies of actions. We show that our axioms can be more succinct, while still using an extended regression operator to solve the projection problem. Moreover, such representation has significant computational advantages. For taxonomies of actions that can be represented as finitely branching trees, the regression operator can sometimes work exponentially faster with our theories than it works with the BATs current situation calculus. We also propose a general guideline on how a taxonomy of actions can be constructed from the given set of effect axioms. Finally, we extend the current situation calculus with the order-sorted logic. In the new formalism, we add sort theories to the usual initial theories to describe taxonomies of objects. We then investigate what is the well-sortness for BATs under such framework. We consider extending the current regression operator with well-sortness checking and unification techniques. With the modified regression, we gain computational efficiency by terminating the regression earlier when reasoning tasks are ill-sorted and by reducing the search spaces for well-sorted objects. We also study that the connection between the order-sorted situation calculus and the current situation calculus.
Administrative Law in the Welfare State: Addressing the Accountability Gap in Executive Social Policy-making
Keywords: administrative law
social welfare
public administration

With the rise of the welfare state, democratic, common law governments undertook a new proactive role as social welfare manager; allocating limited health, education, and social services benefits among competing public priorities. In spite of the important impact of this role on the lives of citizens, the various political, managerial, and legal mechanisms designed to hold executive social policy-makers accountable to the legislature have largely broken down. In this dissertation, I argue that our constitutional democracy permits courts to fill this accountability gap by acting as an accountability mechanism of last resort. I propose a new doctrine of administrative review for social policy and programs in which deference is based on evidence of accountability achieved within the policy-making process. Administrative review for accountability would allow courts to evaluate the legitimacy of the process by which social policy is created rather than interfering in the substance of social policy. My dissertation seeks to develop this administrative review doctrine within existing constitutional constraints including the separation of powers.
A Logical Theory of Joint Ability in the Situation Calculus
Keywords: joint ability
multiagent systems
situation calculus
knowledge representation
reasoning about action
artificial intelligence
intelligent agent
iterated elimination of dominated strategies
game theory
computer science

Logic-based formalizations of dynamical systems are central to the field of knowledge representation and reasoning. These formalizations can be used to model agents that act, reason,and perceive in a changing and incompletely known environment. A key aspect of reasoning about agents and their behaviors is the notion of joint ability. A team of agents is jointly able to achieve a goal if despite any incomplete knowledge or even false beliefs about the world or each other, they still know enough to be able to get to a goal state, should they choose to do so. A particularly challenging issue associated with joint ability is how team members can coordinate their actions. Existing approaches often require the agents to communicate to agree on a joint plan. In this thesis, we propose an account of joint ability that supports coordination among agents without requiring communication, and that allows for agents to have incomplete (or even false) beliefs about the world or the beliefs of other agents. We use ideas from game theory to address coordination among agents. We introduce the notion of a strategy for each agent which is basically a plan that the agent knows how to follow. Each agent compares her strategies and iteratively discards those that she believes are not good considering the strategies that the other agents have kept. Our account is developed in the situation calculus, a logical language suitable for representing and reasoning about action and change that is extended to support reasoning about multiple agents. Through several examples involving public, private, and sensing actions, we demonstrate how symbolic proof techniques allow us to reason about team ability despite incomplete specifications about the beliefs of agents.
Recovery of Surface Active Material from Municipal Wastewater Activated Sludge
Keywords: alkaline extraction
biosurfactacts
bio-based adhesives
wastewater activated sludge
ultrafiltration
fractionation

Wastewater activated sludge is produced during the biological treatment of wastewater. After treating the sewage, the sludge is allowed to settle. Part of the settled material is returned to the treatment process as return activated sludge (RAS) and the excess is removed as waste activated sludge (WAS). The handling and disposal of the sludge are energy and capital-intensive treatments, with a significant environmental impact. This work studies the possibility to utilize RAS (an example of wastewater sludge) as a source of surface active agents. The results indicate that higly surface active materials can be extracted from RAS, and that the RAS extract has potential applications as a detergent and wood adhesive. The results also suggest that recovering a suite of products from RAS, a biological heterogenous source, can be technically feasible. An effective alkaline treatment was developed (at pH>12) that can extract up to 75% of the sludge’s organic matter, a yield higher than previously reported. Increasing the extraction pH increased the extract surface activity, which is linked to increasing the amount of higher molecular weight molecules and the presence of phospholipids. Increasing the extraction pH beyond 11 was also related to extensive cell lysis, increasing significantly the amount of recovered material and the surface activity of the extract. The alkaline extract has properties comparable to commercial detergents. Without further purification, the extract has a low surface tension (37 mN/m on average) and performs similarly to synthetic detergents. Further assessment of the RAS extract (insensitivity to pH, surface tension, interfacial tension) suggests that it may be suitable for commercial applications. The RAS extract can also be formulated into wood adhesives using glutaraldehyde as a crosslinker. The extract fraction with 10-50 kDa constituents at pH 9 achieves high adhesive shear strengths (4.5 MPa on average, at 30% relative humidity and 25°C) with 40% of wood failure. The adhesive strength of RAS-based adhesives is strongly correlated to its protein content.
The Gromov Width of Coadjoint Orbits of Compact Lie Groups
Keywords: Symplectic Topology
Lie Theory

The ﬁrst part of this thesis investigates the Gromov width of maximal dimensional coadjoint orbits of compact simple Lie groups. An upper bound for the Gromov width is provided for all compact simple Lie groups but only for those coadjoint orbits that satisfy a certain technical assumption, whereas the lower bound is proved only for groups of type A, but without the technical restriction. The two bounds use very different techniques: the proof of the upper bound uses more analytical tools, while the proof of the lower bound is more geometric. The second part of the thesis is a short report on a joint project with my supervisor, which was concerned with the relationship between two different definitions of orbifolds: one using Lie groupoids and the other involving diﬀeologies. The results are summarized in Chapter 5 of this text.