VictorJuiz/thesis huggingface dataset:
Cell Engineering to Overcome Bottlenecks in Human Pluripotent Stem Cell Based Therapeutic Manufacturing

Cell-based therapies have the potential to cure currently incurable diseases by replacing missing or damaged tissues within the body or by generating cells with a unique biological function. As cell-based therapies begin clinical translation, pluripotent stem cells (PSC) have emerged as a robust, reliable source of cells for these therapies since they can propagate indefinitely in vitro and differentiate into clinically relevant cell types. However, efforts to translate laboratory-scale human (h) PSC culture to industrially-relevant suspension bioreactors necessary to manufacture clinical quantities of hPSC have resulted in low-yield, highly variable processes, in direct contrast to proof-of-principle high-yield, robust mouse PSC bioprocesses. We hypothesize that by engineering the cell state we can convert hPSC to a high suspension yield, homogeneously aggregating state. Guided by protocols to convert hPSC to an earlier, "naĂŻve" developmental state resembling that of high yield mouse PSC, chemical modulation of hPSC enabled high yield suspension manufacturing. Enhanced single cell seeding efficiency, growth rate, aggregation, and shear tolerance contribute to the mechanism of action of "4i-hPSC" leading to 6.7Âą0.2 times higher suspension yields than conventional hPSC cultures. 4i-hPSC can be efficiently directed to differentiate towards pancreatic progenitors, and could be used in future cell therapy manufacturing. Development of the 4i-hPSC system demonstrated the criticality of controlling cell aggregation in hPSC suspension expansion. Heterogeneity in aggregate expansion of hPSC hinders the translation of hPSC-derived cell therapies to industrial manufacturing and clinical use. We characterized a class of polymers which promote repeatable, controlled, and tunable suspension aggregate formation of hPSC, both in orbital shaker as well as stirred tank bioreactor systems. Converting hPSC to a state of homogeneous, small aggregate formation will increase the robustness of suspension hPSC bioprocessing and promote the use of quality manufacturing frameworks such as Quality by Design in PSC manufacturing. Combining these two strategies could enable efficient bioprocessing of cell lines that are typically refractory to suspension processing as well as sustained single cell hPSC expansion. Cell engineering to create alternative hPSC states with enhanced and controlled suspension expansion capabilities will enable increased patient access to curative cell therapies through improved manufacturing methods.

Advisor: Zandstra, Peter W
(Re)-Constitutionalizing Duress and Necessity

The Supreme Court of Canada’s decision in Ruzic established that the principle of moral involuntariness forms both the conceptual and constitutional basis for the duress and necessity defences. Scholars have contended that the moral involuntariness principle is not only inconsistent with the legal requirements the Court has developed for the duress and necessity defences, but also serves to veil the underlying values which might otherwise form the bases of these defences. The practical effect of ignoring the moral distinctions underlying an accused’s act is to unjustly deny some accused a criminal defence, contrary to s. 7 of the Charter. By re-constitutionalizing the defences along a continuum of principles—moral involuntariness, moral permissibility, and moral correctness—I argue that the law will be able to develop in a manner which permits the legal requirements for duress and necessity to be commensurate with the moral qualities of an accused’s act.

Keywords: Duress
Necessity
Principles of Fundamental Justice
Advisor: Roach, Kent
Functional Characteristics of Neural Networks in Human Associative Learning

Learning and memory are highly complex and adaptive cognitive processes of the human brain. The present body of work examined functional characteristics of neural networks that supported associative learning in multiple dimensions (e.g. space, time, frequency) and related them to cognitive markers and behavioral performance. Research was conducted with magnetoencephalography (MEG). Given the multidimensional nature of MEG data, several methodological tools were developed concurrently to enable better identification of learning-induced changes in the brain. Study 1 systematically examined the impact of different artifacts on scalp signals. Combined with simulations, it was established that detection of deep/weak sources (e.g. medial temporal lobes, striatum etc.) was substantially improved by removing noise from artifacts. This work culminated in the development of an artifact correction tool that is freely available for reseach use. Study 2 used artifact-free data from Study 1 to examine relationships between time-dependent changes in functional network organization, cognitive skills and behavioral performance. Results showed that individual variations in learning were supported by differences in cognitive ability and time-sensitive connectivity in functional networks. This research has translational scope for customizing rehabilitative practices based on an individualâ s learner profile. Study 3 examined frequency-specific changes in functional network interactions and their implications for adaptive control of behavior during learning. First, an automated data-driven pipeline was developed to obtain distributed brain sources. Phase synchronization was used to measure changes in network interactions. Reorganization of functional networks was related to distinct behavior types â eye movements and error rate. Results highlighted a shift in theta frequencies from early to late periods of learning. Both behavioral measures were shaped by similar network configurations early in learning and dissociable functional networks late in learning. The direct comparison made between implicit and explicit behaviors in this study has practical implications for research in special populations (e.g. development, disease) whereby implicit measures may be the only option for evaluating integrity of brain function.

Keywords: brain-behavior correlations
eye movements
large-scale neural networks
learner profiles
magnetoencephalography (MEG)
multivariate methods (ICA
PCA
PLS)
Advisor: McIntosh, Anthony R
Motivational Interviewing as a Lifestyle Management Strategy for Dyslipidemic Overweight and Obese Adolescents

Background: Dyslipidemia, overweight and obesity (OW/OB) in the pediatric population contributes to metabolic complications and future cardiovascular disease. The prevalence varies but the problem is a global issue. Adolescence is a challenging developmental stage with many factors influencing adolescent health. Lifestyle management is the preferred treatment. Motivational interviewing is a promising counselling approach; however, evidence is limited for this population. Objectives and Methods: The substantive focus involved exploring MI for dyslipidemic adolescents. Study A was a retrospective cohort comparison aimed to assess a collaborative educational approach with MI elements for pediatric patients (n=38) newly referred to a specialized lipid clinic, versus historical patients who had received a more didactic educational approach (n=31). Study B involved theoretical research to build and graphically represent a conceptual framework for MI, including special applications to adolescents. Study C was a mixed-methods, randomized controlled trial (N=32) to evaluate the outcomes of MI counselling, comparing adolescents together with a parent versus adolescents alone. A qualitative component occurred following the quantitative data collection to explore the perceptions of participants. Findings: Despite some limitations, sufficient promise was observed in the Study A findings to move forward with a detailed plan to rigorously explore MI. Significantly improved lifestyle and anthropometry outcomes were observed favouring the collaborative education approach with MI. The theoretical work in Study B defined four key elements for a MI framework: the Person, the Problem, the Process, and the MI Counsellor. The between group results for Study C were equivocal; however, with both groups combined in a secondary analysis, there were significant improvements in nearly all outcomes. The interviews enriched an understanding of the participantsâ lives, lifestyle change, and the counselling process. Conclusion: Motivational interviewing is an effective and desired counselling strategy for adolescents with dyslipidemia and overweight/obesity. Further research with a longer period of observation is needed to determine whether achieved lifestyle improvements can be sustained in the long-term.

Keywords: Adolescents
Dyslipidemia
Motivational Interviewing
Overweight/Obesity
Advisor: McCrindle, Brian W
Extraterritorial Heat Hyperalgesia in Mice Following Infraorbital Nerve Transection

Extraterritorial Heat Hyperalgesia in Mice Following Infraorbital Nerve Transection Mariam Mashregi Doctor of Philosophy Faculty of Dentistry University of Toronto 2017 Abstract Orofacial nerve injuries often lead to long-lasting pain which, is frequently perceived outside the field of injury (extraterritorial pain) and continues long after healing is complete. It has been suggested that sex and genetic factors may contribute to the development of neuropathic pain. These aspects were addressed in the present dissertation by first assessing nociceptive responses to noxious radiant heat stimuli in healthy male and female mice of the AXB-BXA recombinant inbred panel (A/J and C57BL/6J parental strains and their 23 descendant strains), and then comparing baseline responses to nociceptive responses following unilateral infraorbital nerve transection (IONX) or sham surgery, in the ears, ventral and dorsal tail, and hind-paws. These body sites were extraterritorial to the field innervated by the infraorbital nerve. Strain differences in heat hyperalgesia were utilised as phenotypes for genetic linkage analysis to identify chromosomal regions harbouring candidate genes. Since heat absorption depends on skin pigmentation, and the 25 strains were comprised of black, agouti (light-brown/taupe), and white coat colours, a standard heat intensity that elicited a threshold withdrawal at each body site was determined for each coat colour group and used for subsequent studies. Significant body-site-specific and strain-specific differences in baseline heat nociception were found, however, no sex differences were noted except in the ventral tail (male > female). Sensitisation to repeated noxious stimulation within the same test session was also noted in all body sites tested. Following surgery (IONX and sham), significant heat hyperalgesia was seen in many strains in all tested body sites. Sex differences were seen only in the ventral tail (female > male). The same post-operative increases were seen in both the sham and IONX groups, which might be explained by surgery-induced neuritis. Narrow-sense heritability estimates for baseline heat nociception and post-operative extraterritorial heat hyperalgesia ranged from 0.09 to 0.49 (in all body sites). Suggestive (but not significant) quantitative trait loci were mapped for baseline heat nociception in chromosomes 3,7,9, and 13 and for post-operative heat hyperalgesia in chromosomes 4, 11, and X. Several candidate genes were identified in these suggestive regions thus, necessitating future research to identify the causative genes.

Keywords: Animal models
Genetics
Neuropathic pain
Neuroscience
Pain
Sensory behavioural
Advisor: Seltzer, Ze'ev
The RSC Complex Maintains Ploidy by Promoting Spindle Pole Body Duplication in Saccharomyces cerevisiae

Ploidy, the number of complete sets of chromosomes an organism has, is tightly regulated in eukaryotic cells, and is critical for cell function and survival. Cells must coordinate multiple pathways to ensure replicated DNA is segregated accurately and in a timely fashion to prevent changes in chromosome number. In this study, I characterize an unanticipated role for the Saccharomyces cerevisiae Remodels the Structure of Chromatin (RSC) complex in ploidy maintenance. First, I demonstrate that deletion of any one of six non-essential RSC genes causes a rapid transition from haploid to diploid DNA content following germination of haploid mutant spores. I show that this increase-in-ploidy phenotype is caused by a non-disjunction event rather than mating type switching or endoreplication. Furthermore, I find that diploidization gives RSC mutant cells a fitness advantage relative to the haploid state, but that tetraploidization causes decreased viability. Interestingly, diploidized rscâ mutants were morphologically distinct from wild-type diploids suggesting a relationship between spontaneous diploidization and cell morphology. Microscopic analysis revealed that rscâ mutants have defects in spindle pole body (SPB) duplication and maturation, which causes chromosome missegregation. Surprisingly, RNA sequencing indicated that these SPB defects were not due to changes in transcript levels, and I was unable to detect physical interactions between the RSC complex and the SPB. Instead, genetic interaction data revealed that RSC is important for the function of the SPB protein Nbp1 at the SPB. Since the RSC complex is important for nuclear pore complex stability and function, I propose a model where the RSC complex stabilizes the nuclear pore complex to allow appropriate nuclear transport of Nbp1, a critical step for inserting the newly duplicated SPB into the nuclear envelope during the SPB duplication cycle. Thus, I define an unanticipated function of the RSC complex in maintaining ploidy in S. cerevisiae.

Keywords: Diploidization
Nuclear Pore Complex
Ploidy Maintenance
RSC Complex
Saccharomyces cerevisiae
Spindle Pole Body
Advisor: Brown, Grant W
Characterizing the Role of DJ-1 in Experimental Models of Sepsis

DJ-1 is an ubiquitously expressed antioxidant associated with a variety of cellular functions. The role of DJ-1 in sepsis is previously uninvestigated. In a murine model of lipopolysaccharide (LPS)-induced lung injury, DJ-1 expression reduced oxidative stress, inflammation and parenchymal cell death. Mice lacking DJ-1 were susceptible to LPS-induced morbidity and mortality. Interestingly, despite enhanced inflammation and oxidative stress, DJ-1 knockout mice were resistant to cecum ligation and puncture (CLP)-induced polymicrobial sepsis. Loss of DJ-1 enhanced bacterial clearance in vivo and bacterial phagocytosis and killing in vitro. Binding of DJ-1 to NADPH oxidase complex caused dissociation and ubiquitination of the complex, impairing ROS production. Transfection of septic human neutrophils with a small interfering RNA against DJ-1 enhanced oxidative burst. In septic patients, higher circulating DJ-1 was associated with increased morbidity and mortality. The striking difference in outcomes between endotoxin versus live bacterial stimulation was similarly confirmed in other models. In the presence of bacteria, DJ-1 deficiency triggered LC3-associated phagocytosis (LAP) while in the absence of bacteria, enhanced inflammation resulted in incomplete autophagy. DJ-1 negatively regulates Rubicon, a molecular switch, to inhibit LAP following bacterial infection while promoting autophagy after endotoxin stimulation. Bone marrow derived mononuclear cells and adoptive bone marrow transplantation of DJ-1 deficient circulating cells confirmed enhanced survival following polymicrobial sepsis is conferred by absence of DJ-1 in immune cells. In non-immune cells, loss of DJ-1 confers early resistance to CLP-induced myocardial depression, possibly through upregulation of compensatory Nrf2 and bioenergy pathway genes. Collectively, these findings reveal unappreciated and complex stimuli and cell specific roles of DJ-1 in regulating oxidative stress, inflammation and innate immune responses in sepsis. These studies add to the current shift in the sepsis paradigm to distinguish infection-induced organ dysfunction and reiterate the concept that optimal innate immune function is essential in the resolution of infection.

Keywords: bacteria killing
cecal ligation and puncture model
DJ-1/PARK7
lipopolysaccharide
ROS production
sepsis
Advisor: dos Santos, Claudia C
Zhang, Haibo
From Greenspace to Greenbelt: The Role of Civil Society in Landscape Protection in the Toronto Region

Greenspace has been increasingly identified as an important element of environmental, social and economic health, yet proponents of greenspace protection from within civil society often find themselves with limited resources in political battles with powerful pro-growth coalitions. This was the case with the three citizen-led campaigns that managed to protect progressively larger landscapes in the Toronto region, culminating with the creation of a 1.8-million-acre greenbelt. How did they do this? This study uses qualitative methods and draws on institutionalism, participation theory, and social capital approaches to examine the evolution of environmental civil society groups and how they learned to exploit governance processes to persuade state actors to break with the status quo. The study looks at how cultural understandings, historical legacies, legal institutions, planning rules and public preferences shaped the landscape of the Toronto region. The research finds that citizen environmentalists were able to use their social capital resources as civil society groups to adapt governance structures – particularly public participation processes – to build a durable constituency for greenspace protection with payoffs for political actors. Particularly influential were: historical legacies of deploying natural solutions to prevent natural disasters, public participation exercises that were open to manipulation, bridging social capital that helped build large coalitions, and bonding social capital that helped coalition members unite and carry out strategies that included keeping the vision alive for decades as election cycles came and went, and, finally, protecting new state partners from criticism. However, state actors under the influence of the still-strong growth coalition remain reluctant stewards of the environment and have chosen implementation strategies that could undermine greenspace protection over time. The study adopts an inclusive definition of civil society and finds that a new typology based on two measures of social capital – inclusiveness and (private vs. public) interests – shows that certain groups are more useful in coalitions campaigning to expand public goods. The study also finds that difficulties in developing the social capital needed to develop working relationships in short-term stakeholder groups were overcome by resorting to selective membership and secret meetings.

Keywords: Civil society
Environmental politics
Greenbelts
Greenspace protection
Public participation
Social capital
Advisor: Sorensen, Andre
Factors Influencing the Input Side of Folate Nutrition

Mandatory folic acid fortification of the food supply in Canada has led to a 50% reduction in neural tube defects. However, fortification in addition to folic acid supplement use has led to high blood values in the non-target population. To generate evidence to inform future fortification and supplementation policy, the aim of my thesis was to establish a better understanding of the ‘‘input side’’ of folate nutrition. I investigated the impact of extending folic acid fortification to whole wheat flour and the contribution of colonic bacterial synthesis of folate. Using data from the 2004 Canadian Community Health Survey 2.2 (n = 35,107), a modelling exercise predicted that extending folic acid fortification to whole wheat flour would decrease the prevalence of folate inadequacy in whole wheat consumers while not putting the entire population at additional risks. In the second study, I analyzed colonic evacuant collected from rural Africans (n=20) and African Americans (n=20) consuming their regular diets and after a two-week reciprocal dietary switch. Mean colonic folate content was increased by 40% among African Americans consuming a high fibre diet patterned after a typical rural African diet (43 ± 5g/d of fibre), highlighting folate producing capacity of gut microbiota could be manipulated using dietary approaches. In the third study, I established an approach to detect stable isotopic folate in urine and confirmed pre-formed folate can be absorbed across the human iii colon using urinary excretion. While urinary enrichment was detected after administration of single oral and IV dose of 13C5-5-formyltetrahydrofolate (n=9), it approached the limit of detection and hence was variable. In future studies, inclusion of a pre-study protocol whereby subjects consume a daily folate supplement to saturate tissue stores may be worthwhile. A better understanding of the input side of folate nutrition will help develop dietary strategies that achieve optimal health.

Keywords: fibre
folate
folate bioavailability
fortification
gut microbiota
stable isotope
Advisor: O'Connor, Deborah L
We Teach Life: Exile, Hip Hop and the Radical Tradition of Palestinian Cultural Resistance

This dissertation argues that cultural resistance is an important site of struggle for Palestinians fighting against (settler) colonial, imperialist, racist regimes, in search of their national liberation. Cultural resistance is significant because material destruction of Palestinian culture and erasure of its history are at the core of the formation of the Israeli settler-colonial state. Through oral-history interviews with Palestinian cultural producers in exile and analysis of their cultural work and political activism, my research finds they resist dominant geographies by emplacing their people back into the geography and history they have been evicted from through words. While racist knowledge circulates about Palestinians in the global sphere, my research finds that Palestinians have their own anticolonial epistemologyâ transmitted inter-generationally through orality and cultural texts that teach Palestinian history and identity. This shapes the political consciousness of Palestinians and, thus, the commitments of many to seek change through activism and cultural production. The dissertation finds that third-generation cultural producers continue in the footsteps of their ancestors, who developed a radical tradition of Palestinian resistance culture. By mobilizing both Indigenous (dabke/folklore) and non-Indigenous (hip hop) cultural forms to Palestine, the participants choose to speak out through the realm of culture. The study finds that cultural producers, thus, challenge racist, Orientalist tropes imposed upon the Palestinian body and nation by colonial, imperialist forces. They use culture as an arena for struggle to preserve a national Palestinian identity across time and space in the absence of a state; they also produce anticolonial knowledge that centres the experiences and history of the colonized through their perspectives. Additionally, they raise critical consciousness on the question of Palestine across geographiesâ specifically in other settler-colonial states complicit in the Zionist settler projectâ to inspire political action; they thus forge global political solidarities with various struggles, to build a revolutionary and liberated future(s).

Keywords: Cultural Production
Exile
Hip Hop
Oral History
Palestine
Resistance
Advisor: Gaztambide-Fernandez, Ruben A.