VictorJuiz/thesis huggingface dataset:
Identification and Verification of Candidate Biomarkers for Down Syndrome and Discovery of Dysregulated Molecular Pathways in Amniocytes by Proteomics Approaches
Keywords: Proteomics
Down syndrome

Down syndrome (DS), caused by an extra chromosome 21, affects 1 in 750 live births, and is characterized by cognitive impairment as well as several congenital defects. Currently, little is known about the molecular pathogenesis of DS and no direct genotype-phenotype relationship has yet been confirmed. The current screening test for DS subjects many women to undergo invasive procedures such as amniocentesis due to suboptimal sensitivity and specificity. Therefore, this study aimed to discover novel biomarkers to improve screening tests, and to discovery dysregulated molecular pathways in DS-affected fetus to better understand pathogenesis. To achieve this objective, proteomic analyses of amniotic fluid (AF) and amniotic fluid cells (amniocytes) were performed using mass spectrometry (MS), which allows discovery of a large number of proteins in complex biological samples. Since AF contains the most information of the developing fetus, we first generated the most comprehensive list of proteins present in AF by using high resolution MS. We then performed quantitative analyses of proteins from AF as well as amniocytes to reveal novel biomarkers and clues to altered molecular mechanisms of DS. Comparison between the proteome of AF from unaffected and DS-affected pregnancies allowed selection of 60 candidate biomarkers based on spectral counting. Two candidates, APP and TNC-C, were verified by immunoassays to show two-fold increase in AF from DS-pregnancies. Additionally, CPA4, MUC13, CEL, DPP4 and MMP2 were verified to be differentially expressed in trisomy 21-AF via selected reaction monitoring assays using triple-quadruple mass spectrometer. Amniocytes from DS-affected and unaffected fetuses were also quantitatively analyzed by using Stable Isotope Labelling of Amino acids in Cell culture technique. Over 4900 proteins were identified from amniocyte lysate and supernatant by LTQ-Orbitrap mass spectrometer, and 85% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labelled amino acids. Proteins that consistently showed aberrant expression from affected amniocytes have been selected for further verification and molecular network analyses since they may play a role in DS pathogenesis.
Generation of Mouse Models of Human Hematopoietic Disease and their Use to Analyze Hematopoietic Development and Function
Keywords: n-ethyl-n-nitrosurea
Forward Genetic Screen
5-fluorouracil
Hereditary Spherocytosis
Hematopoiesis
Myh9 Related Disease

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.
Synthesis of Fused Nitrogen Containing Heterocycles via Cascade Reactions
Keywords: Cascade
Domino
Cyclobutane
Pericyclic
Sigmatropic
Electrocyclic
Heterocycle
Nitrogen

The synthesis of fused nitrogen heterocycles has been investigated. Chapter 1 describes the synthesis of quinolones through a copper catalyzed amide coupling to ortho acetyl aryl halide followed by an aldol condensation that produces the quinolone in modest to good yields. The reaction works well with lactam partners and leads to tricyclic ring systems. Chapter 2 describes the synthesis of 8 membered enamide rings through a [3,3] sigmatropic rearrangement ring expansion of N-vinyl β-lactams. The N-vinyl β-lactams also undergo a 6π electrocyclization following the [3,3] sigmatropic rearrangement to furnish amino cyclobutanes. Previously reported amino cyclobutanes have been made using [2+2] photocycloaddition and resulted in a mixtures of diastereoisomers. Our method produces the amino cyclobutanes in good to high yields and with excellent levels of diastereoselectivity. Chapter 3 describes the synthesis and applications of reduced hydantoins from aziridine aldehydes and isocyanates. The resulting heterocycles contain an aziridine ring fused to a 5 membered urea. We also demonstrate that the reduced portion of the hydantoin, namely the hydroxyl group, can undergo further displacement with β-ketoesters similar to iminium ion chemistry.
Identification and Characterization of Novel Interactors of Human Protein Phosphatase 4 using Mass Spectrometry Technology
Keywords: Molecular

Within the phosphatase field, identification of regulatory subunits and associated proteins has proven successful in determining the cellular role and potential substrates of phosphatases. This has been especially valuable for the PPP family of phosphatases due to its complex association with myriad of regulatory subunits, which dictate the activity, localization and substrate specificity of the phosphatases. To identify interactors of protein phosphatase 4 (PP4), I established a sensitive affinity purification coupled with mass spectrometry (AP-MS) approach to generate a high-density PP4 interaction network from the soluble fraction of human cell extracts. Our proteomic approach uncovered previously identified as well as new interactors; some may function as auxiliary regulators and others may serve as potential substrates. One of the interactor identified is a novel cytosolic PP4 regulatory subunit, which we termed PP4R4. PP4R4 displays weak homology to the PP2A A subunit of PP2Ac, but interacts specifically with PP4c and does not function as a scaffolding subunit to bridge other known regulatory subunits of PP4c. Remarkably, the PP4 interaction network revealed significant enrichment for proteins involved in transcription elongation and RNA processing. These interactors associate exclusively with PP4R3-PP4R2-PP4c holoenzyme. Consistent with this finding, PP4R3A possesses characteristics resembling that of splicing and transcription factors. We provided evidence suggesting that depletion of PP4c significantly reduced the transcription-elongation regulated genes, JUN and FOS, and altered the exon inclusion of selective genes. Our results define a high-density interaction network for the mammalian PP4 and uncover a potential role PP4 play in regulating the process of transcription elongation and alternative splicing.
The Differential Regulation of Subtypes of N-methyl-D-aspartate Receptors in CA1 Hippocampal Neurons by G Protein Coupled Receptors
Keywords: NMDA receptor
G protein coupled receptor

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.
Trunk Stability during Postural Control: Tool Development and Analysis
Keywords: biomechanics
motor control
trunk
posture
mathematical modeling
neuroprosthesis

Trunk instability is a major problem for people with spinal cord injury (SCI); it not only limits their independence, but also leads to secondary health complications such as kyphosis, pressure sores, and respiratory dysfunction. In exploring mechanisms that may facilitate or compromise postural stability, dynamic models are very useful because the spine dynamics are difficult to study in vivo compared to other structures of the body. Therefore, one objective of this work was to develop a detailed three-dimensional dynamic model of the human trunk as a tool for investigating the neural-mechanical control strategy that healthy people apply to maintain trunk stability during various tasks. Since trunk control is fairly complex, however, another objective of this work was to provide insights into the balance control strategy of a simpler neuro-musculo-skeletal system that may facilitate future studies on trunk control. For this purpose, the control of the ankle joint complex during quiet standing (anterior-posterior degree of freedom) was studied in place of the trunk. The obtained results reveal that a neural-mechanical control scheme using a proportional-derivative controller as the neural control strategy can overcome a large sensory-motor (feedback) time delay and stabilize the ankle joint during quiet standing. Moreover, a detailed dynamic model of the trunk has been developed that is: (1) based on highly accurate geometric models; and (2) universally applicable. Thus, this work also responds to the postulation that structurally more complex models are needed to better characterize the biomechanics of multifaceted systems. Combining the developed biomechanical tools for the trunk with the postural control insights for the ankle joint during standing will be beneficial for: (1) understanding the neural-mechanical control strategy that facilitates trunk stability in healthy people; and for (2) developing neuroprostheses for trunk stability after SCI and other neurological disorders.
Research during an Emergency: A Series of Inquiries Concerning the Outbreak of Severe Acute Respiratory Syndrome (SARS) in Toronto
Keywords: Bioethics
Research Ethics

Background: Researchers and research ethics boards (REBs) in Toronto were unprepared for the SARS outbreak. There is a paucity of literature about how to review emergency-related protocols during a public emergency and so REBs had no guidance about how to review SARS-related protocols. Research questions: The thesis presents four related research inquiries based on the following four objectives: 1) to conduct a comprehensive evaluation of the one-year outcomes in SARS survivors; 2) to explore the ethical issues that emerged during the conduct of the SARS outcomes study; 3) to understand the impact of the SARS outbreak on research ethics review (RER) of SARS-related protocols; and 4) to propose a new framework of RER for use during public emergencies. Methods: Included in this thesis are an observational study, an analytic reflection, a grounded theory study, and a translation of the knowledge gained in the first three parts of the thesis into a framework of RER that is meaningful and actionable. Results: Part I describes the recovery made by SARS survivors from their acute illness. In part II, I explore ethical issues that arose during the conduct of the study including: social and scientific value and scientific validity of emergency research, and respect for privacy and confidentially. Part III presents a theory about how researchers, REBs and public health interacted during the outbreak and in part IV I propose ‘emergency review’ a framework for RER for use during a publicly declared emergency. Conclusions: The natural experiment that was the SARS outbreak in Toronto revealed the vulnerabilities in the structure of REBs. I highlight three conclusions which are the highest priority to provide further development in this field. These are: 1) when REBs, researchers and public health are not effectively communicating during a public emergency, the work of each group is disrupted; 2) institutional conflict of interest occurred during the research ethics review of SARS-related protocols and may be amplified during a public emergency and 3) there is a need for a multi-site review structure that could be activated on short notice to review protocols related to the emergency situation.
In vitro and In vivo High-throughput Analysis of Protein:DNA Interactions
Keywords: transcription factors
protein:DNA interactions
Yeast hybrid assays
Fluorescent proteins
Enterokinase
proteolysis specificity
DNA-binding proteins
bZIP
Affinity tag
Tag removal
Protein purification
Bioprobe

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.
Investigation of Thermal Hydraulics of a Nuclear Reactor Moderator
Keywords: Nuclear
Fluid Mechanics

A three-dimensional numerical modeling of the thermo hydraulics of Canadian Deuterium Uranium (CANDU) nuclear reactor is conducted. The moderator tank is a Pressurized heavy water reactor which uses heavy water as moderator in a cylindrical tank. The main use of the tank is to bring the fast neutrons to the thermal neutron energy levels. The moderator tank compromises of several bundled tubes containing nuclear rods immersed inside the heavy water.It is important to keep the water temperature in the moderator at sub-cooled conditions, to prevent potential failure due to overheating of the tubes. Because of difficulties in measuring flow characteristics and temperature conditions inside a real reactor moderator, tests are conducted using a scaled moderator in moderator test facility (MTF) by Chalk River Laboratories of Atomic Energy of Canada Limited (CRL, AECL). MTF tests are conducted using heating elements to heat tube surfaces. This is different than the real reactor where nuclear radiation is the source of heating which results in a volumetric heating of the heavy water. The data recorded inside the MTF tank have shown levels of fluctuations in the moderator temperatures and requires in depth investigation of causes and effects. The purpose of the current investigation is to determine the causes for, and the nature of the moderator temperature fluctuations using three-dimensional simulation of MTF with both (surface heating and volumetric heating) modes. In addition, three dimensional simulation of full scale actual moderator tank with volumetric heating is conducted to investigate the effects of scaling on the temperature distribution. The numerical simulations are performed on a 24-processor cluster using parallel version of the FLUENT 12. During the transient simulation, 55 points of interest inside the tank are monitored for their temperature and velocity fluctuations with time.
The Role of Inorganic Polyphosphate in the Formation of Bioengineered Cartilage Incorporating a Zone of Calcified Cartilage In Vitro
Keywords: Articular cartilage
Chondrocyte
Tissue engineering
Biphasic construct
Inorganic polyphosphate
Mineralization
Extracellular matrix
Fibroblast growth factor 18

The development of bioengineered cartilage for replacement of damaged articular cartilage has gained momentum in recent years. One such approach has been developed in the Kandel lab, whereby cartilage is formed by seeding primary articular chondrocytes on the top surface of a porous biodegradable calcium polyphosphate (CPP) bone substitute, permitting anchorage of the tissue within the pores of the substrate; however, the interfacial shear properties of the tissue-substrate interface of these biphasic constructs are 1 to 2 orders of magnitude lower than the native cartilage-subchondral bone interface. To overcome this limitation, a strategy was devised to generate a zone of calcified cartilage (ZCC), thereby mimicking the native architecture of the osteochondral junction; however, the ZCC was located slightly above the cartilage-CPP interface. Thus, it was hypothesized that polyphosphate released from the CPP substrate and accumulating in the tissue inhibits the formation of the ZCC at the tissue-substrate interface. Based on this information, a strategy was devised to generate biphasic constructs incorporating a properly located ZCC. This approach involved the application of a thin calcium phosphate film to the surfaces of porous CPP via a sol-gel procedure, thereby limiting the accumulation of polyphosphate in the cartilaginous tissue. This modification to the substrate surface did not negatively impact the quality of the in vitro-formed cartilage tissue or the ZCC. Interfacial shear testing of biphasic constructs demonstrated significantly improved interfacial shear properties in the presence of a properly located ZCC. These studies also led to the observation that chondrocytes produce endogenous polyphosphate and that its levels in deep zone cartilage appear inversely related to mineral deposition within the tissue. Using an in vitro model of cartilage calcification, it was demonstrated that polyphosphate levels are modulated in part by the inhibitory effects of fibroblast growth factor 18 on exopolyphosphatase activity in the tissue. Polyphosphate also appears to act in a feedback loop to control exopolyphosphatase activity. Interestingly, polyphosphate also exhibits positive effects on cartilage matrix accumulation. The potential implication of polyphosphate in the maintenance of articular cartilage homeostasis is intriguing and must be investigated further.