VictorJuiz/thesis huggingface dataset:
Solid-phase On-chip Nucleic Acid Hybridization Assays using Spatial Profiles from Fluorescence Resonance Energy Transfer by Immobilized Quantum Dots
Keywords: Biosensor
Fluorescence Resonance Energy Transfer
Hybridization
Microfluidics
Nucleic Acids
Quantum Dots

The research that is presented herein explores the development of a solid-phase DNA hybridization assay in an electrokinetically controlled biochip. Transduction of nucleic acid hybridization is accomplished by fluorescence resonance energy transfer (FRET) from a layer of immobilized quantum dots (QDs) in microfluidic channels. The chip assay platform was assembled as a composite of glass and polydimethylsiloxane (PDMS), where the glass surface was functionalized with immobilization chemistry to support the spontaneous assembly of QDs. Probe oligonucleotides were subsequently conjugated to the immobilized QDs and hybridization served as the selective interaction for target binding. Since the entire microchannel was derivatized with the transduction element, hybridization of dye labeled oligonucleotides along the channel length created well defined spatial profiles of FRET sensitized acceptor emission. The length of the FRET spatial profiles was related to the quantity of nucleic acid delivered and enabled quantitative transduction of tens of fmol amounts of target within minutes. This chip based assay offered multiplexed analysis where the concurrent detection of two targets was possible with a dynamic range spanning more than an order of magnitude. The robustness of the assay was demonstrated by transduction of nucleic acid targets in a variety of complex matrices including sample solutions that contained an excess of genomic DNA and also serum proteins. Furthermore, the assay offered excellent selectivity toward determination of the presence of a single nucleotide polymorphism (SNP), with contrast ratios exceeding 100:1. A non-traditional approach to SNP transduction was explored, where the size of the QD was found to impact the stringency of interfacial hybridization. Detection of unlabeled target oligonucleotide using a sandwich assay approach enabled transduction of oligonucleotides up to 40 nucleotides in length. The research described herein advances the development of a selective interfacial transduction strategy for the detection of nucleic acid markers such as might be characteristic of disease or pathogens. The on-chip assay format is amenable to point-of-care analysis and can be combined with nucleic acid amplification technologies for highly sensitive on demand DNA analysis.
New Mutations: Condition-dependent Rates and Sex-specific Effects
Keywords: Drosophila melanogaster
Genetic variation
Genome
Mutation load
Sexual selection
Spontaneous mutations

Mutations arise spontaneously in all populations, and can severely diminish fitness. This mutation load is a key issue in evolutionary biology, affecting population persistence, the maintenance of genetic variation, and mating system evolution. The rate at which new mutations arise and their effects on fitness are generally treated as constant within a population, but theory suggests that variation in these parameters can have profound impacts. I explored the sex-specific effects of new mutations, and the role of condition in determining the mutation rate, using genotypes of Drosophila melanogaster loaded with spontaneous mutations. I found that the effect of mutations was more severe on male than on female fitness, pointing to a role for sexual selection in purging deleterious genetic variation. The expected consequence is that deleterious mutations will be eliminated at the expense of males, reducing the mutation load of sexual females, and thereby reducing the cost of sexual reproduction. I also found evidence that infection with a bacterial pathogen can heighten the degree of sex-specific selection. However, sex differences in selection could also reduce mean fitness if many alleles have sexually antagonistic effects. By comparing mutational and standing genetic variation, I tested for non-mutational variation in sexual fitness, which would be a signature of sexual antagonism. While there was some suggestion of sexual antagonism, deleterious mutations adequately explained levels of standing variation. Mutation load can also be affected if mutation rates depend on condition. I compared mutation rates across genotypes that varied in condition due to differences in genetic quality, and found that low condition caused elevated rates of mutational fitness decline. A genomic analysis revealed condition-dependent DNA repair pathway usage as the likely reason for this difference. Condition-dependent mutation is expected to reduce mean fitness in sexual populations, but may also increase the rate of extinction in asexuals.
Pancreatic Dysfunction in Shwachman-Diamond Syndrome
Keywords: Acinar cells
Exocrine pancreatic dysfunction
Ribosomopathy
Senescence
Shwachman-Diamond syndrome
Translation

Shwachman-Diamond syndrome (SDS) is an autosomal recessive ribosomopathy caused by loss of the ancillary translation factor Shwachman-Bodian-Diamond syndrome (SBDS) protein, which functions in ribosome subunit joining and protein synthesis. SDS is typified by exocrine pancreatic dysfunction, skeletal abnormalities, and bone marrow failure with leukemia susceptibility. My objective was to determine the
Shareholder Activism and Canada's 'New Corporate Norm'
Keywords: Shareholder Activism

The purpose of this project is to attribute shareholder activism's dramatic growth in Canada over the past decade to the cumulative effect of three structural factors. These components include: (1) the rise of institutional investing, which has resulted in an array of strategic enhancements developed by activist hedge funds, strengthening minority shareholder influence; (2) macro-economic volatility, which has produced the situational urgency for short-run share price improvement in traditionally strong Canadian sectors; and lastly, (3) the development of a corporate and securities law regime in Canada that has given rise to a localized activist-haven, providing procedural windfalls that have allowed for minority shareholders to exert unprecedented levels of control in corporate governance matters. The thesis argues that each of these three components have given rise to a surge in shareholder activism, whilst contemporaneously influencing modern Canadian investment practices so significantly, that a new corporate norm (NCN) now exists in Canada.
Availability of TGF-1 in the Myofibroblast Matrix: A Matter of Strain and Fibronectin
Keywords: ECM
Myofibroblast
TGF-1

Fibrosis is a detrimental disease that causes organ failure with no effective therapy available to date. Activated fibroblasts -myofibroblasts- are responsible for the excessive secretion of extracellular matrix (ECM) and contraction into the stiff collagenous scar tissue that characterizes fibrosis. Transforming growth factor 1 (TGF-1) is the main cytokine that activates myofibroblasts from a variety of different precursor cells and is central regulator of all fibrotic processes. Because TGF-1 also positively controls vascular, inflammatory, and epithelial cell homeostasis, global TGF-1 inhibition strategies failed to treat fibrosis due to severe side-effects on other cell types. My thesis aims to identify myofibroblastic-specific mechanisms of TGF-1 activation as potential therapeutic targets to treat fibrosis. I am revealing how the mechanical and compositional properties of the ECM contribute to the extracellular storage and activation of latent TGF-1 by myofibroblast contraction. We are the first to establish a direct link between the ECM remodeling activity of myofibroblasts and TGF-1 bio-availability. Analogous to Toronto squirrels storing chestnuts for the winter, myofibroblasts establish stores of latent TGF-1 in the ECM for later activation to gain the contractile strength required to remodel scar tissue. My results demonstrate that: (1) Direct application of mechanical force to latent TGF-1 through cell surface receptor integrins induces a conformational change in the TGF-1-associated pro-peptide and releases the active growth factor. (2) Akin to loading a mechanical spring, myofibroblasts gradually strain the ECM that stores TGF-1 and thereby mechanically prime the latent complex for more efficient release of active TGF-1. (3) The presence of extracellular domain-A (ED-A), being characteristically spliced into cellular fibronectin in conditions of tissue remodeling, enhances binding of latent TGF-1 to the myofibroblast-specific ECM. In the course of these studies, I further show that inhibiting myofibroblast force production and integrin force transmission, blocking the binding of latent TGF-1 to ED-A fibronectin, and releasing the myofibroblast ECM from strain all result in reduced amounts of active TGF-1 being available to bind receptors in the cell membrane. Consequently, these interventions interrupt the feed-forward loop of myofibroblast activation by TGF-1, enhanced contraction, and enhanced auto-activation of TGF-1. Eventually, these findings are poised to lead to novel strategies to attenuate fibrosis progression in vivo using the myofibroblast and its ECM as specific target.
Role of the Glucagon and Glucagon-like Peptides in Pancreatic Beta Cell and Cardiovascular Function
Keywords: Cardiovascular disease
Diabetes
Glucagon
Incretin
Insulin
Obesity

Glucagon is inappropriately elevated in diabetes, and inhibition of glucagon receptor (Gcgr) signaling is beneficial for glycemic control. However, the mechanism through which inhibition of Gcgr signaling leads to improved glycemic control is unknown. Additionally, Gcgr is expressed in the cardiovascular system; however the role of Gcgr signaling in the cardiovascular system is unexplored. In order to develop therapies targeting Gcgr signaling for the treatment of type 2 diabetes (T2D) it is vital to understand Gcgr' s role in regulation of glucose homeostasis and cardiovascular function. In mice, genetic deletion of the glucagon receptor results in increased levels of the insulinotropic hormone, glucagon-like peptide-1 (GLP-1). I hypothesized Glp1r signaling contributed substantially to the improved glucose tolerance observed in Gcgr-/- mice. I have generated and characterized the Gcgr and Glp-1 receptor double knockout (Gcgr-/-:Glp1r-/-) mice. My studies demonstrate that Glp1r substantially contributes to the delayed gastric emptying and improved intraperitoneal glucose tolerance in Gcgr-/- mice, but it did not contribute to the improved oral glucose tolerance seen in the Gcgr-/- mice. Interestingly, expression of non-classical incretin receptors and sensitivity to their exogenous agonists were increased in Gcgr-/-:Glp1r-/- mice, suggesting that in the absence of the classical incretin receptors the non-classical incretin receptors compensate to maintain the enteroinsular axis. I explored the role of Gcgr signaling in the cardiovascular system under normal and ischemic conditions. My studies showed that exogenous glucagon increased mortality from myocardial infarction in WT mice in a p38 MAPK-dependent manner. Conversely, Gcgr+/- and cardiac specific Gcgr-/- mice (GcgrCM-/-) had improved survival following myocardial infarction. Gene expression profiling of hearts from GcgrCM-/- mice showed reduced expression of fatty acid oxidation gene, consistent with a reduction in long-chain acylcarnitines observed upon metabolic profiling of GcgrCM-/- hearts. Therefore, partial or cardiomyocyte specific loss of Gcgr signaling enhanced protection to ischemic injury by regulating fatty acid oxidation. In conclusion, my studies suggest Gcgr signaling is essential for beta cell nutrient sensing and fuel metabolism in cardiomyocytes and therapies aiming at modulating Gcgr signaling for the treatment of T2D require careful assessment of cardiovascular outcomes.
Hedgehog Signaling Modulates Cholesterol Homeostasis in Chondrocytes and in Osteoarthritis
Keywords: cartilage
cholesterol
Hedgehog signaling
microarray
osteoarthritis
statin

Osteoarthritis (OA) is a common degenerative disease of the joint that is characterized by degradation and calcification of articular cartilage, and subchondral bone changes. Hedgehog (Hh) signaling is known to be activated in human and murine OA. Since Hh signaling regulates Gli-mediated gene expression, we identified Hh target genes that are expressed in chondrocytes. Microarray analyses were performed to detect changes in gene expression when the Hh pathway was modulated in human OA cartilage samples. Results from the Affymetrix Human Gene 1.0 ST microarray were analyzed for differentially expressed genes from three patient samples. Using Ingenuity® Pathway analysis, several genes known to be involved in sterol homeostasis were found to be modulated with Hh inhibition. We hypothesized that Hh signaling regulates cholesterol biosynthesis in chondrocytes, and that modulating cholesterol homeostasis impacts the severity of OA. To investigate the function of cholesterol in the cartilage, mice with chondrocyte-specific cholesterol accumulation were generated. This was achieved by excising Insig1 and Insig2, major negative regulators of cholesterol homeostasis. Over time, mice with chondrocyte-specific cholesterol accumulation exhibited impaired growth of the long bones. With aging or surgically induced joint instability, these mice developed more severe OA than control littermates. They expressed typical OA markers, such as type X collagen, indicating chondrocyte hypertrophy in the articular cartilage. Genetic manipulation of Hh signaling in these mice suggests that Hh signaling is modulating the phenotype by regulating sterol homeostasis. Hh reduction and statin treatment both lowered cholesterol production and rescued the phenotype, reducing the severity of OA. Here we identified novel Hh target genes in chondrocytes which regulate intracellular cholesterol levels, and found that cholesterol dysregulation in the chondrocytes predisposes to OA. These data suggest that pharmacologic correction of intra-articular sterol imbalance can be used as a treatment for osteoarthritis.
The Fundamental Roles of the Tissue Inhibitor of Metalloproteinase Family in Mammalian Biology
Keywords: chondrocyte
CXCL12
IHH
metalloproteinase
skeleton
TIMP

The metalloproteinases are a family of extracellular proteases responsible for degrading components of the extracellular matrix as well as shedding growth factors, cytokines and their receptors from cell surfaces, impacting signaling, tissue remodeling and homeostasis. The four mammalian tissue inhibitors of metalloproteinases (TIMP1-4) regulate metalloproteinase activity in the tissues. While the TIMPs exhibit some target specificity, functional redundancy exists due to overlapping expression patterns and shared proteinase targets. It is known that the TIMP-metalloproteinase network influences a vast spectrum of biological process and is implicated in most human diseases. Despite this, the fundamental roles of the TIMP family in mammalian development remained unidentified. This thesis describes the generation and characterization of quadruple knockout TIMPless mice deficient for
Polarized Light Imaging for Assessment of Anisotropy in Turbid Media: Instrumentation and Application in Urology
Keywords: anisotropy
bladder
imaging
polarization
turbid media

Assessment of anisotropy has many applications in tissue engineering and early detection of disease such as cancer or stroke. One of the emerging optical technologies for quantifying anisotropy in biological tissues is polarized light imaging. In this technique, the full Mueller matrix of the tissue is measured and then decomposed to yield optical retardance, which is proportional to the tissue anisotropy. The theory of polarized light imaging, its biomedical application and instrumentation are the subject of this thesis. To retrieve more information from the Mueller matrix, a quantitative analysis of generic turbid media's Mueller matrices beyond their decompositions was proposed. Two new metrics based on Mueller matrix were established in this thesis: 1) Tissue depolarization, calculated from the Mueller matrix, can be used to estimate its optical properties, and 2) Symmetry of the off-diagonal elements of a turbid medium's Mueller matrix can be used to detect the axial heterogeneity of anisotropy. Polarized light imaging, followed by Mueller matrix decomposition, was then successfully applied to locate the structural disorders induced by bladder outlet obstruction disease, in ex vivo functioning rat bladders. Motivated by the result of this study and to enable in vivo polarized light measurements of the bladder, a novel thin fiber based polarimetric probe which can measure the full Mueller matrix of the turbid media was suggested. Finally, to enhance the speed of the ex vivo examinations of biological tissues and avoid artifacts, a new rapid benchtop polarized light imaging system based on photoelastic modulators (PEM) and a charge-coupled device (CCD) was proposed and implemented. The demonstrated scheme does not involve using mechanically moving components and thus reduces systematic errors. This imaging system proved to be the fastest high resolution polarimetric characterization tool for imaging turbid media to date.
The Role of Transcription Factor 7-Like 2 in Hepatic Glucose Metabolism
Keywords: Gluconeogenesis
Glucose production
Liver
TCF7L2
Wnt signaling

Transcription factor 7-like 2 (TCF7L2) is a principal downstream transcription factor that mediates the function of the developmental canonical Wnt signaling pathway. When partnered with -catenin, TCF7L2 stimulates Wnt target gene expression and mediates certain effects of other signaling cascades such as cAMP and insulin in cell type-specific manners. Recent genome-wide association studies have reproducibly revealed that common single nucleotide polymorphisms in TCF7L2 have the strongest genetic association with type 2 diabetes risk in a multitude of ethnic backgrounds. Following this hallmark discovery in 2006, the role of TCF7L2 in regulating metabolism and glucose homeostasis has been intensely investigated. Recent studies have shown that TCF7L2 regulates pancreatic -cell function and survival as well as the production and function of the incretin hormone glucagon-like peptide-1 (GLP-1). As human carriers of TCF7L2 diabetes risk polymorphisms also exhibited increased hepatic glucose production, we aimed to determine the role of TCF7L2 in hepatic glucose metabolism. We revealed that hepatic TCF7L2 expression is regulated by nutrient availability as feeding increased TCF7L2 levels in mouse livers while insulin treatment stimulated TCF7L2 expression in hepatic cell lines. Treatment of hepatocytes with the Wnt ligand Wnt-3a decreased, while TCF7L2 knockdown increased gluconeogenic gene expression and glucose production, suggesting that TCF7L2 and Wnt signaling repress gluconeogenesis. To further examine the
Characterization of Protein Tyrosine Kinase 7 (Ptk7) in Development and Disease
Keywords: disease modelling
embryogenesis
scoliosis
zebrafish

Protein Tyrosine Kinase 7 (Ptk7) is a transmembrane pseudokinase implicated in Wnt signal transduction during embryonic development and in cancer. Ptk7 is a known regulator of mammalian neural tube closure and Xenopus convergence extension (C) movements; however, conflicting reports have indicated both positive and negative roles for Ptk7 in both non-canonical Wnt/PCP and canonical Wnt/beta-catenin signaling pathways. To clarify the function of Ptk7 in vertebrate embryonic patterning and morphogenesis, I generated maternal-zygotic (MZ) ptk7 mutant zebrafish and found that early loss of zebrafish Ptk7 leads to defects in axial C, neural tube morphogenesis, and planar cell polarity (PCP). MZptk7 mutant embryos establish normal Wnt-dependent dorsoventral patterning at the onset of gastrulation; however, post-gastrulation MZptk7 mutant embryos display significant up-regulation of beta-catenin target gene expression. MZptk7 mutants show expansion of the mesodermal compartment within the tail bud, suggesting an important role for Ptk7 in regulating Wnt/beta-catenin-dependent cell fate specification. Furthermore, a plasma membrane-tethered Ptk7 extracellular fragment is sufficient to rescue both PCP morphogenesis and Wnt/beta-catenin patterning defects in MZptk7 mutant embryos, indicating that the extracellular domain of Ptk7 acts as an important regulator of both non-canonical Wnt/PCP and canonical Wnt/beta-catenin signaling in multiple developmental contexts. Zygotic ptk7 mutant zebrafish display no obvious embryological defects and develop to adulthood; however, as juveniles these fish develop severe spinal curvatures analogous to human idiopathic scoliosis (IS). Scoliosis is a complex genetic disorder, characterized by three-dimensional rotations of the spine associated with underlying vertebral malformations (congenital scoliosis, CS) or with no identifiable underlying abnormalities (idiopathic scoliosis, IS). Vertebral abnormalities are not associated with spinal curvature in zygotic ptk7 mutant zebrafish; however, MZptk7 mutants display vertebral malformations analogous to those found in humans with congenital scoliosis (CS). These defects are linked to abnormalities in embryonic segmentation, suggesting an important role for maternal ptk7 in axial patterning. Therefore, MZptk7 and zygotic ptk7 mutant zebrafish represent novel models of CS and IS (respectively) and suggest that both types of deformity share genetic mechanisms that relate to altered Wnt signal transduction.
TIMPs in Mammary Stem Cell Homeostasis and Breast Cancer Progression
Keywords: aging
breast cancer
mammary gland
mammary stem cell
stem cell
TIMP

The mammary gland repeatedly undergoes reorganization and cellular turnover in the adult female. Ductal invasion and branching morphogenesis during puberty, cyclical expansion during reproductive cycles, functional differentiation following pregnancy and long-term maintenance of the ductal tree all require mammary stem cell activity. Through the shedding of signaling molecules and turnover of structural matrix proteins many of these remodeling processes are regulated by metalloproteinases and their inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). Here I report on the role of TIMPs during mature mammary gland homeostasis and breast cancer progression. Across individual and compound
Understanding the Unwritten Rules: Examining the use of Ontario Parliamentary Convention to Guide the Conduct of Elected Officials in Ontario
Keywords: Accountability
Conflict of Interest
Ethics
Government
Integrity
Transparency

Government ethics legislation in Canada guides and restricts the conduct of parliamentarians. Ontario established an independent officer of the legislature in 1988 who is responsible for administering its government ethics legislation. In 1994, an undefined behaviour-guiding standard called “Ontario parliamentary convention” was added to the legislation. This paper explores the history of Ontario’s government ethics regime and examines its response to specific scandals, in order to describe the emergence of Ontario parliamentary convention as a standard of conduct. It then provides a full discussion and complementary summary of the specific parliamentary conventions that Ontario’s legislative ethics officers have determined must guide the conduct of Ontario’s parliamentarians. This discussion and summary illuminate the problematic and inconsistent use of this standard by Ontario’s legislative ethics officers. Two sets of recommendations are then offered to help modernize and strengthen the legislation for the benefit of Ontario’s parliamentarians.