VictorJuiz/thesis huggingface dataset:
Molecular Control of Collagen Biomineralization in the Periodontium

Hydroxyapatite crystals deposit within and align parallel to the collagen fibrils of bony tissues with important implications for their structural function. Though collagen biomineralization is regulated by cellular processes, biological control over mineralization has a physicochemical origin. This is reflected in the structure and composition of mineralizing and non-mineralizing collagenous tissues. The purpose of this dissertation is to elucidate factors which dictate tissue selectivity and intrafibrillar localization of mineral deposited in dental and periodontal tissues. This was accomplished by characterizing the demineralized structure of natively mineralizing and non-mineralizing collagen fibrils, and by implementing an in vitro remineralization model of these tissues. In tooth attachment, collagen fibrils of the unmineralized periodontal ligament insert into cementum where they are mineralized, presenting an opportunity to investigate structural changes in collagen associated with mineralization. Hydrated collagen fibrils from these tissues are shown to have considerable structural similarity, however cementum fibrils are axially compressed. This is interpreted as lateral disruption of fibrillar structure by intrafibrillar mineralization, consistent with the view that collagen serves a structural role in mineralization. In vitro remineralization of demineralized tissue sections shows that intrafibrillar hydroxyapatite forms preferentially in the natively mineralized tissues cementum and dentin over periodontal ligament, implicating non-collagenous proteins in guiding tissue specificity. However, the remineralization conditions require a polyaspartate additive, otherwise extrafibrillar hydroxyapatite forms quickly on the surface of all tissues. When the remineralization model is extended to osteopontin, a putative protein inhibitor of crystallization, selective intrafibrillar remineralization of cementum and dentin occurs. By contrast, bone sialoprotein, a putative mineral nucleator, does not inhibit extrafibrillar crystallization, but a significant portion of section-associated hydroxyapatite aligns with collagen fibrils, indicating close interaction between hydroxyapatite and collagen, mediated by bone sialoprotein. These results describe an acellular mechanism for intrafibrillar collagen mineralization and tissue-specific mineralization dependent both on collagen-associated promoters of crystallization and soluble inhibitors of crystallization. This demonstrates an important role for mineralizing proteins in inhibiting kinetically favored extrafibrillar hydroxyapatite crystallization to support less favorable intrafibrillar crystallization, with particular significance to synthesis of bone-replacement materials.

Keywords: Biomineralization
Cementum
Collagen
Hydroxyapatite
Transmission Electron Microscopy
Advisor: Sone, Eli D
When the Appellate Court Takes Fresh Evidence in a Civil Case, should the Court Dispose of the Case Immediately or Order a New Trial?

When the appellate court takes fresh evidence, which should the court select, either dispose of a case immediately at the appellate court or order a new trial at the trial court? Because the appellate courtâ s power of taking fresh evidence has been based on inconsistent ideas, historical development of the power does not suggest an answer. In practice, the appellate court tends to order a new trial based on the perception that the trial court is more suitable to evaluate evidence than the appellate court. However this perception is not well-grounded. Because the appellate judges are well capable of finding fact based on evidence; further, psychological studies demonstrate that observing the demeanor of a witness is not useful to evaluate credibility of a witness. To save expenses in cost and time imposed by conducting a new trial, this thesis insists that the appellate court should dispose of a case immediately.

Advisor: Stern, Simon
Exploring Student Entitlement at an Ontario College

The purpose of this study was to explore the concept of student entitlement specifically within the context of an Ontario College, focusing on conceptualizing student entitlement from the faculty perspective, and then determining the extent to which students report entitled attitudes. The study followed a mixed-methods methodology. Faculty were invited to participate in a concept mapping exercise and the resulting concept map proposed a four-factor model for student entitlement; including, “Tuition Equals Outcomes”; “Knowledge is a right that doesn’t require effort”; “I am a customer, serve me”; and, “Problems are due to others, not me”. A Student Entitlement questionnaire was assembled using highly rated items from each factor. From the results of the questionnaire, confirmatory factor analysis and exploratory principal components analysis were conducted, and the analyses suggested that the model for student entitlement at the Ontario college would be best be described by a three-factor model, with the “Knowledge is a right” factor identified by faculty being removed and the items under that factor fitting well within the three remaining factors. Based on the three-factor model, raw and standardized student entitlement scores were assembled. The distribution of entitlement was normal, with approximately 15% of respondents in the High or Very High categories. When controlling for age, over 18% of respondents in the Traditional age category (18-24) were in the High or Very High categories. The impact of entitlement includes harm to the student’s learning, which could ultimately impact society as a whole, particularly in fields where students are working with vulnerable populations, such as in health care or social services. Student entitlement also impacts faculty in terms of lower job satisfaction and a pressure to succumb to students’ demands, particularly when faculty feel a lack of support from the administration. This study made suggestions for practice; including, changing the way we market to students; focusing on a transition/orientation program that would specifically address the transition from customer to student; using students’ customer orientation strategically by improving services for students; and, a review of student feedback questionnaires and the process surrounding their administration.

Keywords: Academic entitlement
College
Entitlement
Ontario College
Student entitlement
Advisor: Dietsche, Peter
The Principles of Fundamental Justice and the "Societal Consensus" Requirement

The Supreme Court of Canada’s test for delineating the principles of fundamental justice requires that a proposed norm enjoy “significant societal consensus that it is fundamental to the way in which the legal system ought to fairly operate”. The author examines the considerations that count in favour of recognizing that a proposed norm satisfies this requirement; they include: the standard employed—be it normative or empirical, the types of sources relied upon and the way a proposed norm is framed or reframed by the Court. The author argues a normative standard is preferable because it allows the Court to retain full competence to deal with those egregiously unfair outcomes that fall under section 7 but are unpredictable ex ante. It is the better standard for a liberal-democratic state which values dialogue as between the Court and Parliament. As for reformulating proposed principles, the author argues procedural safeguards should be introduced.

Keywords: Charter dialogue
Evidence
Fundamental Justice
Popular opinion
Section 7 Test
Standard
Advisor: Stewart, Hamish
High-throughput Analysis and Control of the Pluripotent Stem Cell Microenvironment to Interrogate and Direct Cell Fate

Human pluripotent stem cells (hPSCs) are heralded to transform regenerative medicine and drug discovery and development, as they can potentially supply a scalable source of clinically relevant cell populations. Numerous exogenous and endogenous factors interplay to control stem cell fate; however, these are often unrecognized and unreported, undermining reproducibility and precluding the systematic analysis of hPSC response to molecular and chemical perturbations. We have developed a high-throughput (HTP) platform to screen hPSCs in configurable micro-environments, in which we optimized parameters including colony size and cell density to achieve rapid and robust cell fate responses to cues. We used this platform to perform single-cell protein expression profiling, revealing that Oct4 and Sox2 co-staining discriminate pluripotent, neuroectoderm, primitive streak, and extraembryonic cell fates. We applied this code to analyze dose responses of 27 developmental factors to delineate lineage-specific concentration optima and to quantify cell-line endogenous signaling pathway activation and differentiation bias. We additionally demonstrate that response profiles can classify cell lines by similarity, are stable through passages, predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell-lines reticent to cardiac induction. We next applied this platform to screen 400 small molecule kinase inhibitors, measuring resultant yield and purity of multiple lineages on a per colony basis. Enrichment analysis revealed mTOR inhibitors as strong inducers of mesendoderm cells. This mesendoderm enhancing effect of mTOR inhibitors is blocked by TGFβ or BMP receptor inhibition, and knockdown of Raptor, a mTORC1 component, via siRNA phenocopied the effects of small molecule mTOR inhibitors such as rapamycin. When rapamycin was added to a blood differentiation protocol, CD34+VECAD+ hemogenic endothelium purity and yield were increased 3 fold, with a concomitant enhancement of blood colony forming cells (CFCs). Lastly, to aid translation of these approaches, we validate a protocol of our 96-well hPSC assay in multiple international laboratories via the International Stem Cell Initiative (ISCI) consortium and provide open-source GUI-based software for analysis of spatial context in HTP studies. Ultimately, these technologies and applications enable a more comprehensive elucidation of the regulatory networks linking cell-extrinsic factors, signal transduction, transcription factor regulation, and cell fate decisions.

Keywords: differentiation
drug screening
high-content analysis
human pluripotent stem cell
micro-technology
mTOR
Advisor: Zandstra, Peter W.
Identification and Characterization of the Role of the Circulating Metabolite 3-Carboxy-4-Methyl-5-Propyl-2-Furanpropanoic Acid (CMPF) in Diabetes

Both gestational diabetes (GDM) and type 2 diabetes (T2D) are caused by a failure of the pancreatic beta cell to compensate for increased insulin demand. The underlying cause of this failure, particularly the rapid transition from prediabetes to diabetes, and between GDM and T2D is largely unknown. To identify circulating factors that may play a causal role in beta cell dysfunction we performed global metabolomics profiling on the plasma of GDM and T2D patients compared to normal glucose tolerant controls. We identified the furan fatty acid metabolite 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) as being highly elevated in diabetic plasma. In vivo and in vitro assays demonstrated that CMPF induces glucose intolerance by impairing glucose-stimulated insulin secretion (GSIS), suggesting CMPF may play a causal role in diabetes. Analysis of human patients showed that rapid elevation in CMPF is associated with a significantly increased risk of diabetes development, consistent with elevated CMPF being the “tipping point” in the progression from prediabetes to overt diabetes. The mechanism underlying this dysfunction involves increasing reactive oxygen species (ROS), resulting in altered gene expression, inhibition of beta cell transcription factor activity, as well as uncoupling of glucose metabolism to ATP production, thus reducing insulin biosynthesis and secretion. Additionally, CMPF induces a state of preferential fatty acid utilization, which persists for months following treatment. This suggests that CMPF exposure during GDM may be responsible for the extremely high rate of progression to T2D postpartum. While detrimental for beta cell function, this ability of CMPF to drive fatty acid oxidation has beneficial effects for hepatic insulin sensitivity due to the prevention of steatosis. CMPF induces beta-oxidation, simulating a “fasting-like” state and promoting expression of FGF21, which is required to impair triglyceride synthesis and promote beta-oxidation long-term. Overall, CMPF is significantly elevated in human diabetes and has direct effects on beta cell function and insulin sensitivity in rodent models.

Keywords: Beta Cell
Diabetes
Gestational Diabetes
Insulin
Metabolomics
Steatosis
Advisor: Wheeler, Michael B
Genetic Variation, Adaptability and Gene Expression Pplasticity in heterogeneous Environments

Natural environments change over time and space. How populations respond to different forms of environmental heterogeneity has strong impacts on the health and persistence of the populations. In my thesis, I explored the effects of temporal and spatial heterogeneity on several important aspects of populations—levels of genetic variation, the potential of future adaptation, and gene expression plasticity—using replicated experimental populations of Drosophila melanogaster evolving under alternative regimes: constant regimes maintained in either cadmium- or salt- enriched larvae diets (Cad and Salt, respectively) and two heterogeneous regimes that vary either temporally (Temp) or spatially (Spatial) between the two diets. Whole-genome resequencing of these populations found that for sites likely under differential ecological selection (and those closely linked to them), the pattern of within-population diversity is Spatial > Temp > Salt, Cad. However, for sites that are not affected by differential selection, the pattern is Spatial > Salt, Cad > Temp. Quantitative genetic variance patterns for three fitness related traits are partially consistent with the genomic diversity. Spatial populations tend to harbor more genetic variation than Temp populations or those under a constant environment that is the same as the assay environment. The adaptability of these populations was further assessed by exposing them to high temperature while being maintained in their original larval diet regimes. Both types of heterogeneous populations had a larger adaptive response to high temperature than homogeneous populations for female productivity but not for male siring success. Finally, the transcriptome was measured in both diets for each population to examine gene expression plasticity. Relative to the constant populations, both heterogeneous populations showed increases in adaptive plasticity. Spatial populations are more readily to increase adaptive plasticity than Temp. Whereas for the genes selected for similar expression in both diets, Temp populations more readily evolve to the adaptive state than Spatial.

Keywords: Drosophila melanogaster
environmental heterogeneity
experimental evolution
genomics
quantitative genetics
Advisor: Agrawal, Aneil F
Cardiovascular Effects and Mechanisms of Action of Glucagon-like Peptide-1 Metabolite, GLP-1(28-36)

The incretin hormone, glucagon-like peptide-1 (GLP-1), is a cardioprotective peptide. GLP-1(28-36) is a C-terminal nonapeptide derived from the cleavage of GLP-1 by the endopeptidase NEP 24.11. We investigated the cardioprotective effects of GLP-1(28-36) in mouse models of myocardial ischemic injury and aimed to discover the cellular and molecular mechanisms involved. Chronic 14d pre-treatment with GLP-1(28-36) via osmotic minipumps reduced infarct size following total left anterior descending coronary artery occlusion in mice, as compared to control scrambled peptide. Acute pre-treatment with GLP-1(28-36) prior to ischemia prevented myocardial dysfunctions by improving left ventricular developed pressure (LVDP) at the end of reperfusion, as compared to scrambled peptide, in isolated mouse hearts undergoing ischemia/reperfusion injury (IRI). Isolated hearts from Glp1r-/- mice perfused with GLP-1(28-36) showed similar LVDP recovery after IRI, suggesting GLP-1 receptor (GLP-1R)-independent actions. Pharmacological inhibition of soluble adenylyl cyclase (sAC), an intracellular enzyme responsible for catalyzing cAMP synthesis from ATP, abrogated the LVDP recovery of mouse hearts pre-treated with GLP-1(28-36). At the cellular level, GLP-1(28-36) caused concentration-dependent increases in ATP and cAMP synthesis in mouse coronary artery smooth muscle cells (caSMCs). GLP-1(28-36) underwent cellular uptake, increased cAMP-PKA activity, and prevented cytotoxicity of caSMCs exposed to H202-mediated oxidative injury, as evidenced by decreased release of LDH. These effects were attenuated with KH7, a sAC-inhibitor, or using caSMCs isolated from sAC-/- mice. Using co-immunoprecipitation and subsequent proteomic analysis, we discovered that GLP-1(28-36) interacts with proteins involved in mitochondrial metabolism. Mitochondrial trifunctional protein subunit α (MTPα) was identified as the most significant binder of GLP-1(28-36) in mouse hearts. Altogether, my study reveals GLP-1R-independent cardioprotective actions of GLP-1(28-36), localized to caSMCs. We identify sAC as a novel molecular pathway, although the precise mechanism by which GLP-1(28-36) increases ATP production to upregulate sAC activity remains unclear. Future investigations should focus on the role of GLP-1(28-36) in mitochondrial metabolism.

Keywords: Cardiovascular
Glucagon-like peptide-1
Incretins
Ischemia
Myocardial Infarction
Advisor: Husain, Mansoor
Chiropractic Care and Risk For Acute Lumbar Disc Herniation: A Mixed Methods Approach

Objective: To advance knowledge of the risk for acute lumbar disc herniation (LDH) following chiropractic care. The objectives were to: (1) synthesize current evidence on the incidence and determinants of LDH with radiculopathy in adults; (2) determine clinicians’ beliefs regarding the risk for acute LDH associated with chiropractic spinal manipulation treatment (SMT); and, (3) investigate associations between both chiropractic and primary care physician (PCP) care, and acute LDH with incident early surgery. Methods: (1) Systematic review of the literature. (2) Belief elicitation interviews with 47 chiropractors, family physicians and spine surgeons. Probability distributions for the relative risk (RR) for acute LDH associated with chiropractic SMT were derived. (3) Self- controlled case series study using Ontario administrative health data. Incidence ratios for acute LDH with early surgery in exposed periods after chiropractic visits relative to unexposed periods were estimated within individuals, and compared with incidence ratios following PCP visits. Results: (1) The annual incidence of LDH with radiculopathy ranges between 0.1% and 10%, and varies with the case definition. Risk factors included individual, behavioural and work-related characteristics. (2) In the belief elicitation study, chiropractors expressed the most optimistic belief (median RR, 0.56; IQR, 0.39-1.03); family physicians, a neutral belief (median RR, 0.97; IQR, 0.64-1.21); and spine surgeons, a more pessimistic belief (median RR, 1.07; IQR, 0.95-1.29). (3) The self-controlled case series study found positive associations between both chiropractic and PCP visits, and surgically managed acute LDH. There was no more risk associated with visits to chiropractors than with visits to PCPs. Conclusions: (1) The best evidence synthesis shows the varying quality and heterogeneity of the epidemiologic literature on LDH with radiculopathy. There is a need to develop standardized case definitions that validly classify the clinical spectrum. (2) Clinicians’ beliefs about the risk for acute LDH associated with chiropractic SMT varied systematically across professions. The derived probability distributions can serve as prior probabilities in Bayesian analyses of this exposure-outcome association. (3) Patients with prodromal back pain related to a developing disc herniation may seek healthcare from both chiropractors and PCPs before full clinical expression of acute LDH that is subsequently surgically managed.

Keywords: belief elicitation
chiropractic
epidemiologic methods
lumbar disc herniation
risk
self-controlled case series
Advisor: Cassidy, J. David
Proteomic Identification and Systematic Verification of Biomarkers for Aggressive Prostate Cancer

Despite prostate cancer’s status as the most common cancer to affect men in the Western world, only an estimated 20% of men will die from it. Due to the lack of effective means of prognostication, many men are unnecessarily biopsied and treated. The early and non-invasive identification of lethal disease would have a profound impact on the current practice of prostate cancer management. To this end, the work presented in this thesis describes efforts to characterize proteins in a prostatic fluid known as expressed prostatic secretions (EPS), and to delineate factors in this fluid that demonstrate a high potential as prognostic biomarkers. Using different mass spectrometry-based platforms, I present both a global view of the EPS proteome, as well as targeted, quantitative measures of promising biomarker candidates in this fluid. In order to generate an archive of soluble and secreted factors present in the proteome of EPS, I used shotgun proteomics to analyze direct-EPS and EPS-urine. This led to the identification of over 2000 proteins that comprise the EPS proteome. The data was integrated with other publicly accessible information, such as gene expression profiles and immunohistochemistry images, to characterize this proteome. Furthermore, a collection of prostate-enriched proteins was identified by comparing the proteomes of EPS-urine and urine, demonstrating the unique protein profile of EPS. Next, comparative proteomic profiling of direct-EPS from organ confined and extracapsular tumors produced a long list of potential biomarker candidates. A targeted assay was systematically developed in order to quantitatively assess the performance of the most promising candidates in cohorts of EPS-urines from a heterogeneous population of prostate cancer patients and controls. Utilizing the assay, a number of candidates were verified to be putative biomarkers for prostate cancer diagnosis and prognosis. This information needs to be further validated and holds promise for improved prostate cancer management.

Keywords: Biomarker
Mass spectrometry
Prostate cancer
Proteomics
Translational research
Advisor: Kislinger, Thomas