VictorJuiz/thesis huggingface dataset:
Stem Cells of the Neural and Pancreatic Lineages
Keywords: stem cells
pancreas
neural
diabetes
brain
PMPs

In this thesis, I describe studies identifying and characterizing two putative stem cell populations of the neural and pancreatic lineages. The mechanisms governing the emergence of the earliest mammalian neural cells during development and the ontogeny of neural stem cells remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition, however an instructive process has also been proposed. I utilized mouse ES cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals, and what cellular fate will result. Individual ES cells were found to rapidly transition directly into neural cells by a default mechanism, a process shown to be independent of suggested instructive factors. Further, I provide evidence that the default neural identity is that of a primitive neural stem cell, the earliest identified stem cell of the neural lineage. The exiguous conditions used to reveal the default state were found to present primitive neural stem cells with a survival challenge, which could be mitigated by survival factors or genetic interference with apoptosis. I also report the clonal identification of multipotent precursor cells, PMPs, from the adult mouse and human pancreas. These cells proliferate in vitro to form clonal colonies and display both pancreatic and neural cell multipotentiality. Importantly, the newly generated beta cells demonstrate glucose-dependent calcium responsiveness and regulated insulin release. PMP colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. Moreover, genetic lineage-labeling experiments excluded the neural crest, and established the embryonic pancreatic lineage, as the developmental source of PMPs. The PMP cell was further found to express insulin in vivo, and insulin+ stem cells were shown to contribute to multiple pancreatic and neural cell populations in vivo. These findings demonstrate that the adult mammalian pancreas contains a population of insulin+ multipotent stem cells, capable of contributing to the pancreatic and neural lineages. In the final section of this thesis, I consider the relationships between neural and pancreatic tissues, as well as discussing the relevance of these two novel stem cell populations.
Disruption of Epigenetic Regulatory Elements and Chromosomal Alterations in Patients with Beckwith-Wiedemann Syndrome
Keywords: Genomic Imprinting
Epigenetics
Beckwith-Wiedemann syndrome
DNA Methylation
Cytogenetics
Rhabdomyosarcoma
Monozygotic Twins

Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.
Undoing Wit: A Critical Exploration of Performance and Medical Education in the Knowledge Economy
Keywords: medical education
knowledge economy
performance
ethics

Over the past decade, there has been a turn in applied health research towards the use of performance as a tool for knowledge translation. The turn to performance in applied health sciences has emerged as researchers have struggled to find new and engaging ways to communicate complex research findings regarding the human condition. However, the turn to performance has occurred within the political landscape of the knowledge economy, and thus conforms to contemporary practices of knowledge production and evaluation. Recent studies about health-based performances exhibit two hallmarks of economized modes of knowledge production. First, these studies focus their attention on the transmission of knowledge to health care professionals through an exposure to performance. Knowledgeable, and thus more useful or efficient, health care providers are the end-product of this transaction. Second, many of these productions are created in the context of application, and thus are driven by an accountability and goals-oriented approach to knowledge acquisition. This thesis argues that economized and rationalized modes of knowledge production do great harm to performance’s pedagogical and ethical potential. By utilizing scientific evaluative methodologies to monitor performance’s ‘success’ as an evaluable, predictable and ends-oriented practice obscures performance’s libratory value, and thus misses performance’s potentially most potent and critical contributions. To mount this argument, I present a case study of Margaret Edson’s play Wit, which has been used widely in medical education. Drawing from the philosophy of Emmanuel Levinas, I critically explore the impact of the knowledge economy on arts-based pedagogical models within health research and education. Further, I seek to redress potential harms inflicted by the knowledge economy by developing the notion of ethical “response-ability.” Through this concept I argue that performance challenges normative conceptions of reason, rationality and scientific evaluation, making the use of theatre in contemporary educational settings at once troublesome and vital.
Priority Setting for Expensive Biopharmaceuticals: An Analysis of Six Drug Case Studies
Keywords: Priority Setting
Decision making
Accountability for reasonableness
Case studies

Priority setting for expensive biopharmaceuticals is one of the most important challenges for publicly funded health systems. One of the drivers of rising healthcare expenditures is pharmaceuticals (i.e., drugs). Moreover, people are living longer and their expectation of, and demand for, health care, drugs, and services are continually increasing. The overall aim of this research was to describe and evaluate reimbursement decisions for six expensive biopharmaceuticals across five countries in order to ascertain if the processes were legitimate and fair. I conducted qualitative case studies of six expensive biopharmaceuticals in order to describe and evaluate the priority setting activities of eight committees across five countries, including Canada, England and Wales, Australia, Israel and the United States. Data sources included: 1) 32 documents and 2) 56 interviews with informants. The recommendations process of each committee partially met the four conditions of ‘accountability for reasonableness’. My main finding is that, while a number of values were considered by committees when making reimbursement decisions, committees tended to focus on values of evidence, effectiveness and efficiency, but not the full range of relevant values. Thus, these contexts did not fully meet the conditions of legitimacy and fairness. I have provided an in-depth description of the eight committees’ priority setting activities regarding the study drugs, as well as committee members’, patients’ and industry representatives’ views regarding the process. I developed practical guidance for leaders for improving reimbursement decisions for expensive biopharmaceuticals, the implementation of which would enhance the fairness and legitimacy of priority setting. This study has demonstrated that in order to create a fair and legitimate drug reimbursement process, we need to ensure the incorporation of a wide range of values, and the involvement of multiple stakeholder groups within the deliberative and appeals/revisions processes.
Development and Refinement of New Products from Multi-angle Remote Sensing to Improve Leaf Area Index Retrieval
Keywords: LAI
multi-angle remote sensing

Remote sensing provides methods to infer vegetation information over large areas at a variety of spatial and temporal resolutions that is of great use for terrestrial carbon cycle modeling. Understory vegetation and foliage clumping in forests present a challenge for accurate estimates of vegetation structural information. Multi-angle remote sensing was used to derive and refine new information about the vegetation structure for the purpose of improving global leaf area index mapping. A field experiment with multi-angle, high resolution airborne observations over modified and natural backgrounds (understory, moss, litter, soil) was conducted in 2007 near Sudbury, Ontario to test a methodology for the background reflectivity retrieval. The experiment showed that it is feasible to retrieve the background information, especially over the crucial low to intermediate canopy density range where the effect of the understory vegetation is the largest. The tested methodology was then applied to background reflectivity mapping over conterminous United States, Canada, Mexico, and Caribbean land mass using space-borne Multi-angle Imaging SpectroRadiometer (MISR) data. Important seasonal development of the forest background vegetation was observed across a wide longitudinal and latitudinal span of the study area. The previous first ever global mapping of the vegetation clumping index with a limited eight-month multi-angular POLDER 1 dataset was expanded by integrating new, complete year-round observations from POLDER 3. A simple topographic compensation function was devised to correct negative bias in the data set cause by topographic effects. The clumping index reductions can reach up to 30% from the topographically non-compensated values, depending on terrain complexity and land cover type. The new global clumping index map is compared with an assembled set of field measurements, covering four continents and diverse biomes. Finally, inclusion of the new vegetation structural information, including background reflectivity and clumping index, gained from the multi-angle remote sensing was then shown to improve the performance of LAI retrieval algorithms over forests.
Molecular Mechanisms Regulating Embryonic Cerebral Cortex Development
Keywords: cerebral cortex development
cellular differentiation
in utero electroporation
cortical precursors

Cerebral cortex development is a complex process that integrates both extrinsic and intrinsic mechanisms. The surrounding cellular environment triggers receptor activation, which in turn initiates components of different signalling cascades and subsequently gene transcription, influencing cell survival, proliferation, and differentiation. Genetic mutations causing a loss-of-function or gain-of-function of signalling pathways elements can lead to cortical abnormalities and result in cognitive dysfunctions. In this thesis, I examined the receptor tyrosine kinase (RTK) TrkB and TrkC, the small GTPase Ras, and the C/EBP family of transcription factors, investigating their roles during cerebral cortex development. First, I looked at the role of C/EBPs during cortical cell fate determination. I determined that inhibition of C/EBPs decrease neurogenesis, keeping precursors in an undifferentiated state and later promoting their differentiation into astrocytes, while expression of an activated form of C/EBP promoted neurogenesis and reduced astrogenesis. Moreover, the inhibition of MEK, a mediator of C/EBPβ phosphorylation, also caused a decrease in neurogenesis. Thus, activation of the MEK-C/EBP pathway biases precursor cells to become neurons rather than astrocytes, thereby acting as a differentiation switch. Second, I examined the involvement of Trk signalling during cortical development. I showed that genetic knockdown using shRNA, or inhibition using dominant negative of TrkB and TrkC lead to a decrease in proliferation and later to postnatal precursor cells depletion. Moreover, it caused a reduction in number of neurons combined with mislocalization of the generated neurons to the different cortical layers. Thus, Trk signalling plays an essential role in the regulation of cortical precursor cell proliferation and differentiation during embryonic development. Third, I elucidated the effect of Costello syndrome H-Ras mutations during cerebral cortex formation. I determined that these mutations promoted cell proliferation and astrogenesis, while reducing neurogenesis. Together, these data support a model where proper Trks/Ras/MEK/C/EBP signalling is essential for normal genesis of neurons and astrocytes and show that cortical development perturbations can ultimately lead to cognitive dysfunction as seen in Costello syndrome patients.
Molecular Mechanisms Involved in Insulin and Palmitate Actions on Clonal, Hypothalamic Cell Lines Expressing Neuropeptide Y and Agouti-related Peptide
Keywords: Hypothalamus
Insulin resistance
Hyperinsulinemia
Type 2 diabetes mellitus
Free fatty acids
Palmitate
Neuropeptide Y
Agouti-related peptide
Cell biology
Molecular biology

Type 2 diabetes mellitus (T2DM) ensues from diminished insulin sensitivity and abated compensatory insulin secretion. While diminished insulin secretion has a strong genetic origin, environmental factors are central in the development of insulin resistance; these include hyperinsulinemia and lipotoxicity. Insulin resistance results in the dysregulation of hypothalamic neurons that mediate its central actions: a key intermediary neuron is the neuropeptide Y/agouti-related peptide (NPY/AgRP) neuron. The hypothesis therefore was generated that insulin directly regulates NPY/AgRP neurons, and that prolonged insulin or palmitate, a prevalent free fatty acid (FFA), exposure inhibits neuronal insulin signaling. Using well characterized hypothalamic cell lines, mHypoE-46 or mHypoE-44, which express NPY, AgRP and insulin receptor signaling machinery, this hypothesis was examined in three studies. Correspondingly, insulin decreased NPY and AgRP mRNA expression in the mHypoE-46 cells, through an extracellular signal-regulated kinase (ERK) dependent mechanism; whereas prolonged exposure of NPY/AgRP cells to insulin or palmitate attenuated insulin signaling, determined by analysis of phosphorylated Akt. Insulin induced insulin receptor substrate-1 (IRS-1) serine 1101 phosphorylation in mHypoE-46 cells, utilizing the mTOR-S6K1 pathway, as the mTOR inhibitor rapamycin prevented IRS-1 serine phosphorylation. Insulin also decreased insulin receptor and IRS-1 protein levels; this was prevented by lysosomal and proteasomal pathway inhibitors, 3-methyladenine and epoxomicin, respectively. Importantly, rapamycin, epoxomicin or 3-methyladenine pre-treatment decreased the attenuation of insulin signaling during long-term insulin exposure. On the other hand, palmitate activated c-Jun N-terminal kinase (JNK), the apoptosis effector caspase 3, and induced endoplasmic reticulum (ER) stress in mHypoE-44 cells: JNK inhibition prevented ER stress. In an attempt to avert the deleterious effects of palmitate, the neuronal cells were treated with the 5`AMP-activated protein kinase (AMPK) activator AICAR, a possible insulin sensitizer. Interestingly, AICAR attenuated JNK and caspase 3 activation, and restored insulin signaling. These findings demonstrate that insulin directly regulates NPY/AgRP neuronal cells, and that insulin and palmitate provoke neuronal insulin resistance through different mechanisms. These findings substantiate the idea that environmental factors known to trigger peripheral insulin resistance may have consequences at the level of the individual hypothalamic neuron, which may ultimately contribute to the resulting pathophysiological states of obesity and T2DM.
The Ecological Consequences and Adaptive Function of Nectar Secondary Metabolites
Keywords: plant-animal interactions
chemical ecology
pollination biology
toxic nectar
Bombus impatiens
Gelsemium sempervirens
Asclepias
Crithidia bombi
behavioural ecology

Plants are under selection to simultaneously attract pollinators while deterring herbivores. This dilemma can lead to tradeoffs in floral traits, which are traditionally thought to be optimized for pollinators. My dissertation addresses the ecological costs and putative functional significance of nectar secondary metabolites, a paradoxical but widespread phenomenon in the angiosperms. I investigate this issue from the pollinator’s perspective using a series of controlled laboratory investigations focused primarily on the bumble bee Bombus impatiens and the nectar alkaloid gelsemine, from Gelsemium sempervirens. I begin by demonstrating that nectar enriched with the alkaloid gelsemine significantly deters visits from bumble bees at a range of natural alkaloid concentrations. However, this aversion can be mitigated by increasing the sucrose concentration such that the alkaloid-rich nectar is more rewarding than its alkaloid-free counterpart. I then demonstrate that the consumption of gelsemine-rich nectar can inhibit oocyte development and protein utilization in bees, but that this effect is limited to bees of suboptimal condition. Continuous consumption of the nectar alkaloid gelsemine also leads to a reduction in the pathogen load of bumble bees infected with Crithidia bombi, but direct interactions between the pathogen and the alkaloid have no impact on infection intensity. Gelsemine also fails to inhibit floral yeast growth, suggesting that nectar alkaloids may not be universally antimicrobial. Finally, I demonstrate that gross nectar cardenolides from the genus Asclepias are strongly correlated with gross leaf cardenolides and that the majority of individual cardenolides found in nectar are a subset of those identified in leaves. This pattern suggests that nectar cardenolides are a consequence of defense for Asclepias; however, they may not be a costly corollary because bumble bees show an overall preference for nectar cardenolides at mean concentrations. Altogether, my dissertation provides a new perspective on the role of chemical defenses against herbivores in plant-pollinator interactions.
Involvement of Reactive Metabolites in Idiosyncratic Drug Reactions
Keywords: drug metabolism
mass spectrometry
adverse drug reactions
drug safety
LC/MS

Idiosyncratic drug reactions (IDRs) represent a significant medical problem and pose a great challenge to drug development. Circumstantial evidence suggests that, in most cases, reactive metabolites of the drug are responsible. The major focus of this thesis is the identification of reactive metabolites and the synthesis of analogs required to test several hypotheses related to involvement of metabolism and covalent binding in the mechanisms of IDRs. Minocycline is unique among tetracyclines in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. In this study, we demonstrated that minocycline is oxidized to reactive intermediates by myeloperoxidase/H2O2/Cl-, HOCl, horseradish peroxidase/H2O2, or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N, N-dimethylamino group, implying that the reactive intermediate was a quinone iminium ion. The other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings, suggesting that the HOCl added across the double bond of the B ring leading to a reactive molecule, and then NAC displaced the chloride ion. Nevirapine, an anti-HIV drug, is associated with idiosyncratic skin rashes in humans. The goal of this project was to investigate whether the 12-hydroxylation pathway is responsible for the skin rash. To test a part of this hypothesis, 12-trideuteronevirapine, 12-OH-NVP sulfate, and several other analogs of nevirapine were synthesized. D-penicillamine is known to cause idiosyncratic autoimmune reactions in humans. The goal of this project was to test whether D-penicillamine covalently binds to macrophages and triggers downstream events leading to autoimmunity. To test a part of this hypothesis, D-penicillamine conjugated to biotin was synthesized. In summary, reactive metabolites of minocycline were found that likely explain why minocycline has an IDR profile unique among the tetracyclines. In addition, analogs of nevirapine and D-penicillamine required for mechanistic studies of nevirapine and D-penicillamine-induced IDRs were synthesized. These studies provide additional support for the involvement of reactive metabolites in the mechanisms of IDRs.
Investigation of Unilamellar Phospholipid Vesicle Interactions with PNIPAM Based Hydrogel Beads
Keywords: Vesicle
PNIPAM
Microgel
NMR

Phospholipid liposome binding to hydrogel beads based on poly(N-isopropylacrylamide) (PNIPAM) is accomplished employing either avidin/biotin conjugation or hydrophobic modification of the microgels, and the ability to form single supported lipid bilayers is explored. The co-monomer acrylic acid (AA), evenly distributed or localized to the shell of the microgel, is included to facilitate post-polymerization chemical modification of the hydrogel beads. The degree of chemical modification of the microgels as well as the thermal behavior is monitored via 1H and 13C nuclear magnetic resonance (NMR). Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phoshocholine (POPC) and a small amount of commercially available biotinylated-lipid are shown to bind as intact entities while sequestering internal contents to biotinylated hydrogel beads, utilizing avidin as the coupling agent. Under fusogenic conditions, these bound liposomes remain as individual vesicles. Alternatively, POPC liposomes are shown to bind to microgels modified to display single chain alkyl groups or cholesteryl moieties, and remain as intact vesicles. It is demonstrated that these liposomes become permeable at high hydrophobe content. Bound liposomes will fuse into larger structures under high hydrophobe content conditions, but remain permeable. The volume phase transition (VPT) characteristic of PNIPAM microgels is shown to influence the permeability of hydrophobically bound (low hydrophobe content), but not avidin/biotin conjugated, liposomes. The degree of liposome binding, as well as their resulting structures and permeability are investigated utilizing 31P NMR, fluorescence spectroscopy and microscopy. The microgel-bound liposome and microgel-supported lipid bilayer hybrid systems would be ideally suited to drug delivery and tissue engineering applications. The microgel-supported single lipid bilayer system would, in addition, potentially act as a cell model system for membrane dynamics and embedded amphiphile NMR studies.