Biochemistry, Biophysics, and Structural Biology Commons^™

The Role Of Bone Sialoprotein In Periodontal Tissue Development And Bone Repair, Yohannes Soenjaya

Electronic Thesis and Dissertation Repository

Bone development and repair involve complex processes that include interaction between cells and their surrounding matrix. In the body, bone sialoprotein (BSP) expression is up-regulated at the onset of mineralization. BSP is a multifunctional acidic phosphoprotein with collagen-binding, hydroxyapatite nucleating, and integrin recognition (RGD sequence, which is important for cell-attachment and signaling) regions. Mice lacking BSP expression (Bsp^-/-), exhibit a bone phenotype with reductions in bone mineral density, bone length, osteoclast activation, and impaired bone healing. This thesis examined the role of BSP in tooth development and also its potential use as a therapeutic reagent for bone …

The Role Of Thymine Dna Glycosylase (Tdg) And Dna Demethylation In Tgf Beta Signaling, Matthew E.R. Maitland

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Prompted by findings that TGFβ stimulates thymine DNA glycosylase (TDG) dependent rapid DNA demethylation and activation of the CDKN2B gene, I investigated the global role of TDG and DNA demethylation in TGFβ signaling in HaCaT cells. Using dot blot analysis, I show that TGFβ treatment increases the global levels of 5-formylcytosine, an intermediate metabolite of active DNA demethylation. Characterization of genomic regions that undergo DNA demethylation and recruitment of TDG indicate that they are both frequent events, but only overlap at 11 genomic locations. I identified 440 TGFβ upregulated genes, 40 of which were bound by TDG and 169 that …

The Rational Design And Evaluation Of Ck2alpha Mutants Bearing Inhibitor-Refractory Amino Acid Substitutions, Sam Reid Fess

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CK2 is a ubiquitously expressed and constitutively active serine/threonine protein kinase that is implicated in many cellular functions. Previous studies have indicated that the generation of mutants that are less sensitive to inhibition can be advantageous when studying protein kinases. Importantly, studies have demonstrated that mutants of CK2 rendered less sensitive to inhibition are attainable. To extend these observations, mutants of CK2α were designed and evaluated to test their effect on the inhibition of CK2 by CX-4945 using in vitro enzymatic assays followed by the development of inducible cell lines. CX-4945 is a CK2 inhibitor that has demonstrated anti-tumor activity …

Hd2d Is A Regulator Of Abscisic Acid Responses In Arabidopsis, Joshua A. Farhi

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Histone deacetylases have important roles in development and stress response in plants. To further investigate their function, the HD2D gene, of the plant specific HD2 family, was studied. An hd2d-1 mutant and two HD2D overexpression lines were used in this study. Germination was delayed in hd2d-1 and HD2D overexpression seeds only in the presence of ABA. HD2D was found to positively regulate the expression of members of the ABA-response pathway (ABI1, ABI5, and RD29A) leading to increased resistance to drought and salinity treatments. Furthermore, HD2D expression delayed flowering by positively regulating FLC expression. Using bimolecular fluorescence complementation, the HD2D protein …

Feasibility Of Using Arterial Spin Labeling For Detecting Longitudinal Changes In Cerebral Blood Flow, Tracy Ssali

Electronic Thesis and Dissertation Repository

The ability of the perfusion MRI technique, arterial spin labeling (ASL), to quantify cerebral blood flow (CBF) makes it attractive for longitudinal studies of changes in brain function, such as those related to chronic pain. However, ASL's poor spatial resolution makes image alignment between sessions difficult, leading to increased variance and greater Type-I errors. In addition, variability due to differences in basal blood flow between sessions and confounding effects such as the arterial transit time (ATT) have the potential to reduce reproducibility over time. The focus of this thesis is to investigate the ability of ASL to detect long-term changes …

Chemical Biology Approaches To Investigate The Activity And Substrate Specificity Of Protein Kinase Ck2, Michelle A. Gabriel

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Protein kinase CK2 is a constitutively active, acidophilic protein serine/threonine kinase with an extensive array of protein substrates that have been implicated in the regulation of many cellular events including those that maintain the delicate balance between proliferation and survival. Although numerous substrates of CK2 have been identified, bioinformatic analysis predicts that 20% of the phosphoproteome can be attributed to CK2, suggesting that our understanding of CK2 is greatly underappreciated. Not surprisingly, CK2 levels are elevated in multiple cancers, and therapeutics targeting CK2 are in development. In order to realize the full potential of these therapies, it is essential to …

Interdomain Interactions Of The Transactive Response Dna Binding Protein 43 Kda (Tdp-43), Karen M. Dunkerley

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Transactive response DNA binding protein, 43 kDa (TDP-43) is 416-residue RNA processing and transport protein, observed in insoluble cytoplasmic aggregates within affected neurons in neurodegenerative diseases. TDP-43 has three domains: the N-terminal (N), RNA-binding (R) and unstructured C-terminal domains (G). Unstructured domains often form intramolecular interactions regulating other domains; our goal was to determine if such an interaction occurs in TDP-43. In Far Western blots, tagged NR was observed to bind to G. A 10 residue C-terminal truncation of G virtually abolished binding and introduction of phosphomimetics at Ser409/Ser410 also reduced binding. Sedimentation velocity ultracentrifugation with tagged NR and G …

Hyperpolarized 129xe Magnetic Resonance Imaging Of Radiation-Induced Lung Injury, Ozkan Doganay

Electronic Thesis and Dissertation Repository

Lung cancer is the largest contributor to cancer-related mortality worldwide. Only 20% of stage III non-small cell lung cancer patients survive after 5-years post radiation therapy (RT). Although RT is an important treatment modality for lung cancer, it is limited by Radiation-Induced Lung Injury (RILI). RILI develops in two phases: (i) the early phase (days-weeks) referred to radiation pneumonitis (RP), and (ii) the late phase (months). There is a strong interest in early detection of RP using imaging to improve outcomes of RT for lung cancer. This thesis describes a promising approach based on ¹²⁹Xe gas as a contrast …

A Unified Framework For The Prioritization Of Variants Of Uncertain Significance In Hereditary Breast And Ovarian Cancer Patients, Natasha G. Caminsky

Electronic Thesis and Dissertation Repository

A significant proportion of hereditary breast and ovarian cancer (HBOC) patients receive uninformative genetic testing results, an issue exacerbated by the overwhelming quantity of variants of uncertain significance identified. This thesis describes a framework where, aside from protein coding changes, information theory (IT)-based sequence analysis identifies and prioritizes pathogenic variants occurring within sequence elements predicted to be recognized by proteins involved in mRNA splicing, transcription, and untranslated region binding and structure. To support the utilization of IT analysis, we established IT-based variant interpretation accuracy by performing a comprehensive review of mutations altering mRNA splicing in rare and common diseases.

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Isolation And Characterisation Of Relaxed Specificity I-Tevi Nuclease Domain Mutants, Alexander C. Roy

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Engineering nucleases is important to the advancement of genetic engineering and gene therapy approaches. Engineering requires a knowledge of which residues are contributing to each function of the nuclease. The residues which contribute to cleavage specificity of the I-TevI nuclease domain (ND) are unknown. I suspect that some of these contributions derive from the ND, thus my null hypothesis is that mutation of the ND will not alter the substrates this enzyme can cut. I have mutagenised the I-TevI nuclease domain and using directed evolution I have isolated mutations which were characterised in vivo and in vitro. These mutations permit …

Quinone Reductase 2 Is A Flavin Redox Switch, Kevin Ka Ki Leung

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Quinone reductase 2 (NQO2) is a mammalian enzyme that catalyzes the reduction of quinone using an unusual co-substrate, dihydronicotinamide riboside (NRH). In addition, NQO2 has a secondary function, which is to modulate the 20S proteasomal degradation of p53 in a redox-dependent manner. This alternate function has been characterized in its sister enzyme quinone reductase 1 (NQO1) and yeast quinone reductase Lot6p, but relatively few studies have investigated the role of NQO2 beyond quinone catalysis. From studies of Lot6p, it was proposed that quinone reductases could be categorized as flavin redox switches. In this thesis, the how NQO2 functions as a …

Characterizing The C-Terminal Region Of Human Adenovirus E1a: An Undiscovered Country, Michael J. Cohen

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Human Adenovirus (HAdV) E1A is the first protein expressed during viral infection. The primary function of E1A is to reprogram the cell for viral replication, but it is additionally capable of transforming primary rodent cells in co-operation with other oncogenes such as HAdV E1B. Despite extensive study, little is known about the function and cellular targets of the C-terminal region of E1A. Importantly, this region is required for the transforming ability of E1A with E1B, but can also suppress transformation with Ras. Previous studies showed that interaction with the C-terminal Binding Protein (CtBP) plays a role in both functions described …

Reactions Between Zinc Metallothionein And Carbonic Anhydrase, Tyler B. J. Pinter

Electronic Thesis and Dissertation Repository

More than 25% of proteins require metal ion cofactors for structure or function. The interactions between metalloproteins have largely been overlooked, though these interactions ultimately govern metal localization and control metal ion homeostasis. Mammalian metallothionein (MT) is a small, cysteine-rich metalloprotein that binds numerous metal ions per protein strand. Up to seven divalent metals, such as zinc or cadmium, are wrapped into a clustered two-domain structure. This unusually high metal content places MT as an attractive candidate for studying interactions with other metal-binding proteins. This present study investigates the metal transfer reactions between MTs and other metalloproteins, using carbonic anhydrase …

Resting And Functional Magnetic Resonance Spectroscopy Of Glutamate In Schizophrenia At 7 Tesla, Reggie E. Taylor

Electronic Thesis and Dissertation Repository

Schizophrenia is a debilitating disease that affects about 1% of the population. Current therapeutic interventions mostly target dopaminergic neurotransmission but are not effective in treating all symptoms. There is growing evidence to support involvement of glutamatergic neurotransmission, which may better account for the symptomatology of schizophrenia.

Glutamate concentrations can be measured in vivo using magnetic resonance spectroscopy (MRS). Stronger MRIs provide benefits for MRS, but they also present challenges. Simulations were designed to examine how MRI field strength influences metabolite quantification. Glutamate and its metabolic precursor, glutamine, were more reliably and independently quantified with higher MRI field strengths, showing a …

Planting The Chalcone Reductase Family Tree: Identification And Characterization Of Chalcone Reductase Genes In Soybean, Caroline Julia Sepiol

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Soybean (Glycine max [L.] Merr) is an important crop grown in Canada, generating $2.4 billion in sales. Though this number may be promising, soybean farmers lose about $50 million worth of yield annually due to root and stem rot disease caused by Phytophthora sojae. Many strategies have been developed to combat the infection; however, these methods are prohibitively expensive. A ‘cost effective’ approach to this problem is to select a trait naturally found in soybean that can increase resistance. One such trait is the increased production of root glyceollins. One of the key enzymes exclusively involved in glyceollin …

Investigating The Role Of The Extracellular Matrix Protein, Spondin 1, In Ovarian Folliculogenesis, Caitlin O'Flynn

Electronic Thesis and Dissertation Repository

Folliculogenesis is the ovarian process in which a follicle, consisting of an oocyte surrounded by granulosa cells (GCs), theca cells, a fluid-filled antrum, and a basal lamina separating GCs from TCs, grows and differentiates, culminating in development of a fertilizable oocyte. TCs under the influence of luteinizing hormone (LH) produce androgens. GCs under the influence of follicle stimulating (FSH) hormone proliferate, produce steroid hormones, and differentiate to become responsive to the surge of LH that initiates ovulation. The cellular processes of folliculogenesis require cell-extracellular matrix (ECM) interactions. Spondin 1 (SPON1) is an ECM protein primarily studied for its role in …

Analyzing A-Series Gangliosides In Neurons Following Exposure To Glutamate, Dae Hee Park

Electronic Thesis and Dissertation Repository

Neurons within different brain regions have varying levels of vulnerability to external stress and therefore respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell’s plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) technique and immunohistochemistry to evaluate the temporal changes in the expression profiles of various ganglioside species during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic …

Examining The Nucleotide Preference Of The Linker Domain In Engineered Tev-Mtalens, Brendon C. Mcdowell

Electronic Thesis and Dissertation Repository

Tev-mTALENs are genome-editing nucleases which combine the nuclease and linker domains of I-TevI with the DNA-binding domain of a TAL effector. The linker domain interacts with a portion of the Tev-mTALEN target site called the DNA Spacer, facilitating DNA cleavage. Linker-DNA Spacer interactions are poorly understood but necessary for Tev-mTALEN activity. I examined the DNA Spacer sequence requirements of the linker by assaying Tev-mTALEN activity on targets with mutated DNA Spacer sequences. I also performed activity assays using Tev-mTALENs with mutations to the I-TevI linker domain. My results indicate that the linker DNA Spacer sequence requirements are highly cryptic. No …

Characterizing The Function Of Helix Five Of The Ku70 Von Willebrand A Domain In Non-Homologous End Joining, Sarah M. Hoffer

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DNA double strand breaks (DSBs) are a toxic and dangerous form of DNA damage repaired primarily by non-homologous end joining (NHEJ) in mammals. The Ku70/80 heterodimer rapidly responds to DSBs, stimulating recruitment of downstream NHEJ factors to protect, process, and ligate broken DNA ends. Work in our lab has shown that a D192A/D195R mutation in helix five of the Ku70 von Willebrand A (vWA) domain leads to an NHEJ defect. However, little is known about the function of this region in NHEJ. We hypothesized that helix five of the Ku70 vWA domain mediates a protein-protein interaction that is crucial for …

The Role Of Bone Sialoprotein In The Tendon-Bone Insertion, Ryan M. Marinovich

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Tendons and ligaments insert into bone through a transitional tissue termed the enthesis which is susceptible to injury and difficult to repair. Entheses contain a region of calcified fibrocartilage (CFC), however mineral-associated proteins in this tissue remain poorly characterized. Bone sialoprotein (BSP) is a phosphoprotein associated with mineralizing tissues. In these studies BSP was identified in the CFC of entheses by immunohistochemistry. Analysis of the entheses of Bsp^-/- mice indicate abnormalities in the CFC. Compared to controls, the CFC of the quadriceps tendon enthesis is 28% and 41 % longer in 15 week and 14 month old Bsp^{-/- …}

Complementary Mass Spectrometry Methods For Characterizing Protein Folding, Structure, And Dynamics, Siavash Vahidi

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Proteins are involved in virtually every biochemical process. A comprehensive characterization of factors that govern protein function is essential for understanding the biomedical aspects of human health. This dissertation aims to develop complementary mass spectrometry-based methods and apply them to solve problems pertaining to the area of protein structure, folding and dynamics.

‎Chapter 1 uses fast photochemical oxidation of proteins (FPOP) to characterize partially disordered conformers populated under semi-denaturing conditions. In FPOP, ·OH generated by laser photolysis of H₂O₂ introduces oxidative modifications at solvent accessible side chains. By contrast, buried sites are protected from radical attack. Using …

Molecular Mechanisms Linking Amino Acid (Leucine) Deprivation To Igfbp-1 Hyperphosphorylation In Fetal Growth Restriction, Niyati M. Malkani

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In this study, we explore the molecular mechanisms linking amino acid (leucine) deprivation to IGFBP-1 hyperphosphorylation in vitro. During pregnancy, a maladaptive fetal response to in utero amino acid deprivation leads to Fetal Growth Restriction (FGR). FGR infants display elevated phosphorylated IGFBP-1, which is associated with decreased IGF-I bioavailability. Leucine deprivation inhibits mechanistic target of rapamycin (mTOR) signaling and stimulates the amino acid response (AAR). Using HepG2 cells, a model for fetal hepatocytes, we demonstrate that in leucine deprivation, the AAR modulates total and phosphorylated IGFBP-1 while mTOR mediates total IGFBP-1 secretion only. We also reveal that protein kinases …

The Epigenetic Regulators Atrx And Ctcf Are Required For Mouse Neuroprogenitor Cell Survival And Brain Development, Lauren Ashley Watson

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Emerging evidence implicates the regulation of higher-order chromatin structure in brain development, maturation, and function. Human mutations in two important regulators of chromatin structure, ATRX and CTCF, cause microcephaly and intellectual disability and have been identified in several cancers, suggesting an important role for these proteins in the developing brain and to suppress tumorigenesis. This thesis demonstrates that chromatin structure is critical to the differentiation and survival of neural progenitor cells, and explores the mechanisms of ATRX and CTCF function in brain development. The first chapter identifies that Atrx deficiency induces replicative DNA damage at telomeres and pericentromeric heterochromatin, and …

Novel Techniques For Investigating The Regulation Of Skeletal Muscle Hemodynamics, Baraa K. Al-Khazraji

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The effect of the sympathetic nervous system (SNS) on blood flow distribution within skeletal muscle microvasculature is conditional upon regional activation of SNS receptors. Due to a lack of appropriate experimental models and techniques, no study has systematically evaluated the effect of SNS receptor activation in continuously branching skeletal muscle arteriolar trees. In line with previous work, we hypothesize that there will be a spatially-dependent distribution of sympathetic receptor activation along the arteriolar tree. Specifically, we anticipate a progressive decrease of adrenergic activation and a progressive increase of peptidergic and purinergic activation with increasing arteriolar order. We developed a novel …

Translesion Synthesis And Mutations: On The Mutagenic Properties Of The Two Dna Lesions, 8-Oxo-G And Pt-Gg, And The Functions Of Y-Family Dna Polymerases And Rev3l On The Bypass Of Each Of The Dna Lesions In Mammalian Cells, Lizhen Guo

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I studied the capabilities of the two DNA lesions 8-oxo-guanine and cisplatin intrastrand crosslinked 1,2-d(GpG) or Pt-GG to cause mutations in mammalian cells. Using isogenic cell lines generated from mice with selective gene knockouts of distinct DNA polymerases as models, I deduced the biological functions of the translesion DNA polymerases Pol eta, Pol kappa, Pol iota, Rev1 and Rev3L on bypassing each of the lesions 8-oxo-G and Pt-GG. My study takes advantage of the Next Generation Sequencing (NGS) technology to determine mutagenic effects of the DNA lesions in vivo and effects of translesion DNA polymerases on bypassing the lesions. Through …

Towards The Development Of A Maga Reporter Gene Expression Construct For Magnetic Resonance Imaging, Karina Quiaoit

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Development of methods to monitor cellular activity with magnetic resonance imaging (MRI) provides a powerful tool for studying disease progression and monitoring therapy. This thesis investigates components necessary for development of reporter gene expression for MRI, to track Nkx2.5 transcription factor activity during tumour growth and, in the future, to calibrate MR contrast against a well-known optical reporter gene, like firefly luciferase. In human MCF-7 breast cancer cells, Nkx2.5 is induced by all-trans retinoic acid (tRA) and activates a minimal promoter of the rat sodium iodide symporter (rNISmin) gene. Here, we used firefly luciferase reporter gene expression to examine a …