Biochemistry, Biophysics, and Structural Biology Commons^™

Functional Consequence Of Psat1 Association On Pkm2'S Inherent Enzymatic Activity., Alexis Avidan Vega

Electronic Theses and Dissertations

Pyruvate kinase M2 (PKM2) is predominantly found in tumors, where it allows the cancer cell to adapt to metabolic conditions through allosteric regulation of its activity. We recently discovered that phosphoserine aminotransferase 1 (PSAT1) associates with and activates PKM2. Here, I sought to affirm PSAT1's ability to increase PKM2 activity through kinetic and association studies of wild-type or mutant PKM2 enzymes. I demonstrate that His-tagged WT and mutant PKM2 enzymes are active, exhibit different kinetics, yet cannot be activated by PSAT1. Comparison studies using untagged WT PKM2 suggest that inclusion of the His-tag disrupts PSAT1 association. In support, pull-down strategies …

Sip-428, A Sir2 Deacetylase Enzyme And Its Role In Biotic Stress Signaling Pathway, Bal Krishna Chand Thakuri

Electronic Theses and Dissertations

SABP2 (Salicylic Acid Binding Protein 2) plays a vital role in the salicylic acid signaling pathway of plants both regarding basal resistance and systemic acquired resistance against pathogen infection. SIP-428 (SABP2 Interacting Protein-428) is a Silent information regulator 2 (SIR2) like deacetylase enzyme that physically interacts with SABP2 in a yeast two-hybrid interaction and confirmed independently by a GST pull-down assay. We demonstrated that SIP- 428 is an NAD+ dependent SIR2 deacetylase enzyme. Transgenic tobacco plants silenced in SIP- 428 expression via RNAi showed enhanced basal resistance to microbial pathogens. Moreover, these SIP-428-silenced lines also exhibited a robust induction of …

Characterization Of Acyltransferases And Wrinkled Orthologs Involved In Tag Biosynthesis In Avocado, Md Mahbubur Rahman

Electronic Theses and Dissertations

Triacylglycerols (TAG) or storage oils in plants are utilized by humans for nutrition, production of biomaterials and fuels. Since nonseed tissues comprise the bulk biomass, it is pertinent to understand how to improve their TAG content. Typically, the final step in TAG biosynthesis is catalyzed by diacylglycerol (DAG) acyltransferases (DGAT) and/or phospholipid: diacylglycerol acyltransferases (PDAT), which also determine the content and composition of TAG. Besides enzymatic regulation of TAG synthesis, transcription factors such as WRINKLED1 (WRI1) play a critical role during fatty acid synthesis. In this study, mesocarp of Persea americana, with > 60% TAG by dry weight and oleic …

Temperature And Polyunsaturated Fatty Acid’S Effect On Daphnia Magna Reproduction, Mark Albright

Electronic Theses and Dissertations

Organisms adapt to their environments by adjusting their biochemistry and physiology; such adaptation is limited by resource availability and physiological constraints. The freshwater crustacean Daphnia magna inhabits a wide range of environments and must survive and reproduce within a range of temperatures. One limit to low-temperature adaptation is thought to be the availability of unsaturated fatty acids necessary to maintain proper fluidity of cellular membranes. D. magna maintained at 10 ºC on a diet poor in unsaturated fatty acids have been observed to produce clutches that fail to develop. However, this has not been observed on a diet rich in …

Structure And Thermodynamics Of Polyglutamine Peptides And Amyloid Fibrils Via Metadynamics And Molecular Dynamics Simulations, Riley Workman

Electronic Theses and Dissertations

Aggregation of polyglutamine (polyQ)-rich polypeptides in neurons is a marker for nine neurodegenerative diseases. The molecular process responsible for the formation of polyQ fibrils is not well understood and represents a growing area of study. To enable development of treatments that could interfere with aggregation of polyQ peptides, it is crucial to understand the molecular mechanisms by which polyQ peptides aggregate into fibrils. Many experimental techniques have been employed to probe polyQ aggregation, however, observations from these studies have not lead to a unified understanding of the properties of these systems, instead yielding competing, fragmented theories of polyQ aggregation. This …

Gq Noncanonical Roles In Translational Regulation, Brett Demarco

Electronic Theses and Dissertations

This study investigates protein nucleic acid interactions, focusing on G-quadruplex (GQ) forming DNA/RNA in human disease. GQ structures are formed in DNA/RNA, when four guanine residues form planar tetrads stabilized by Hoogsteen base pairing, that stack forming a GQ structure stabilized by potassium ions. These GQ structures are targeted by the arginine glycine-glycine (RGG) RNA-binding domain. Fragile X mental retardation protein (FMRP), a translation regulator protein implicated in the fragile X syndrome, has an RGG domain and has been previously shown to interact with neuronal GQ forming messenger RNA (mRNA). We have investigated three neuronal FMRP mRNA targets that we …

Cooperative Regulation Of Translational Repression By Fmrp And The Mirna Pathway, Navneeta Kaul

Electronic Theses and Dissertations

Fragile X syndrome (FXS) is the most common inherited monogenic cause of intellectual disability. FXS patients exhibit social and language deficits, hyperactivity, seizures, growth abnormalities, macroorchidism, anxiety, and epilepsy. FXS is caused by the transcriptional silencing of the fragile X mental retardation gene 1 (Fmr1), resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP is a selective mRNA binding protein that plays a role in translation repression. Studies suggest that FMRP utilizes the miRNA pathway to repress translation of its target mRNAs through an unknown mechanism. The aim of my thesis is to investigate …

Egfr Signaling From The Early Endosome., Julie A. Gosney

Electronic Theses and Dissertations

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR dimerizes with another ErbB family receptor, leading to kinase domain activation and transphosphorylation of C-terminus tyrosine residues. These phosphotyrosines act as crucial regulators of EGFR signaling as effector proteins dock to the receptor at these sites. The receptor undergoes clathrin-mediated endocytosis into early endosomes, where it can then be trafficked to a lysosome for degradation. However, the kinase domain of EGFR retains its activity during trafficking, suggesting that EGFR can continue …

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences …

Bayesian Analytical Approaches For Metabolomics : A Novel Method For Molecular Structure-Informed Metabolite Interaction Modeling, A Novel Diagnostic Model For Differentiating Myocardial Infarction Type, And Approaches For Compound Identification Given Mass Spectrometry Data., Patrick J. Trainor

Electronic Theses and Dissertations

Metabolomics, the study of small molecules in biological systems, has enjoyed great success in enabling researchers to examine disease-associated metabolic dysregulation and has been utilized for the discovery biomarkers of disease and phenotypic states. In spite of recent technological advances in the analytical platforms utilized in metabolomics and the proliferation of tools for the analysis of metabolomics data, significant challenges in metabolomics data analyses remain. In this dissertation, we present three of these challenges and Bayesian methodological solutions for each. In the first part we develop a new methodology to serve a basis for making higher order inferences in metabolomics, …

Deciphering The Role Of Human Arylamine N-Acetyltransferase 1 (Nat1) In Breast Cancer Cell Metabolism Using A Systems Biology Approach., Samantha Marie Carlisle

Electronic Theses and Dissertations

Background: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can additionally hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. Additionally, multiple studies have shown the knockdown of NAT1, by both small molecule inhibition and siRNA methods, in breast cancer cells leads to decreased invasive ability and proliferation and decreased anchorage-independent colony formation. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Additionally, consequences …

Canonical Nuclear Factor-Κb Signaling In Satellite Stem Cell Homeostasis And Function., Alex R. Straughn

Electronic Theses and Dissertations

Satellite cells are adult stem cells that are required for the regeneration of skeletal muscle following injury. However, the signaling mechanisms that regulate satellite stem cell homeostasis and function in adult animals remain less understood. Nuclear factor-kappa B (NF-kB) is a major nuclear transcription factor that regulates the gene expression of a plethora of molecules involved in cellular proliferation, differentiation, survival, and the inflammatory immune response. NF-kB can be activated through a canonical or non-canonical pathway. However, the role of canonical NF-kB signaling in the regulation of satellite stem cell function during skeletal muscle regeneration has not been yet investigated …

Quantitative Studies Of Amyloidogenic Protein Residue Interaction Networks And Abnormal Ammonia Metabolism In Neurotoxicity And Disease, Jeddidiah Griffin

Electronic Theses and Dissertations

Investigating similarities among neurological diseases can provide insight into disease processes. Two prominent commonalities of neurological diseases are the formation of amyloid deposits and altered ammonia and glutamate metabolism. Computational techniques were used to explore these processes in several neurological diseases. Residue interaction networks (RINs) abstract protein structure into a series of nodes (representing residues) and edges (representing connections between residues likely to interact). Analyzing the RINs of monomeric forms of amyloidogenic proteins for common network features revealed similarities not previously known. First, amyloidogenic variants of lysozyme were used to demonstrate the usefulness of RINs to the study of amyloidogenic …

Metabolic Plasticity In The Cellular Stress Response, Ying Li

Electronic Theses and Dissertations

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that ¹³C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained …

Melatonin-Micronutrients Osteopenia Treatment Study (Mots): A Translational Study Assessing The Effects Of Melatonin, Strontium Citrate, Vitamin D3 And Vitamin K2 On Bone Density, Bone Turnover Markers And Health-Related Quality Of Life In Postmenopausal Osteopenic Women Following A One-Year Double-Blind Randomized Placebo-Controlled Trial And On Osteoblast-Osteoclast Co-Cultures, Sifat Maria

Electronic Theses and Dissertations

Objective: The purpose of this study was to assess if a novel combination of melatonin and three other natural bone-aiding micronutrients: strontium citrate, vitamins D₃ and K₂ (MSDK) could improve bone health by modulating the activity of osteoblasts and osteoclasts in favor of balanced bone remodeling and by improving the overall health-related quality of life in postmenopausal osteopenic women.

Methods: The Melatonin-micronutrients Osteopenia Treatment Study (MOTS) is a translational research study that used both clinical and in vitro approaches to assess the efficacy of MSDK on bone health in women and to identify potential mechanisms for its effects. …

Biophysical Exploration Of Conformational Environments In Zymogen Prothrombin And Blood Coagulant Thrombin., Ramya Billur

Electronic Theses and Dissertations

The serine protease thrombin plays important roles in coagulation, anticoagulation, and platelet activation. Thrombin is initially expressed as the inactive zymogen prothrombin (ProT). The final cleaved and activated form of thrombin has mature anion binding exosites (ABEs) and several regulatory loops. Engagement with these exosites helps define the fate of thrombin as a procoagulant or an anticoagulant. Researchers previously reported that zymogen ProT may already bind exosite ligands. Little is known about conformational changes associated with ProT maturation, resultant ligand binding affinities, individual ligand-protein contacts, and long-range communication between thrombin exosites. Protease Activated Receptors (PARs) play critical roles in controlling …

Photonic Tools For Advanced Sensing And Imaging At The Nanoscale., Jafar Hamed Ghithan

Electronic Theses and Dissertations

This dissertation reports a novel bio-sensing strategy based on single-mode, electro-active, integrated optical waveguide (SM-EA-IOW) platforms. It also reports the development of a super-resolved far-field optical imaging tool to enable optical, electronic, and spectroelectrochemical investigations at the nanoscale. SM-EA-IOW platforms with its outstanding sensitivity for spectroelectrochemical interrogation was combined with a sandwich bioassay for the development of a novel immunosensing based strategy for label-free detection of infectious pathogens. The strategy begins with the functionalization of the electroactive waveguide surface with a capturing antibody aimed at a specific target analyte. Once the target analyte is bound to the photonic interface, it …

Functional And Structural Impact Of The Loss Of The Leucine-Rich Repeat Protein Lrit1 In The Mouse Retina., Catherine Ann Cobb

Electronic Theses and Dissertations

Mutations in genes encoding the leucine-rich repeat (LRR) proteins nyctalopin and LRIT3 lead to complete congenital stationary night blindness because they are critical to depolarizing bipolar cell function in the retina. LRIT3 has two closely related family members, LRIT1 and LRIT2. In silico analyses of publicly available RNA-Seq data showed that Lrit1 was highly expressed in the retina. Here I describe the expression pattern and impact of loss of LRIT1 on retinal function. To enable these studies, we used CRISPR/Cas9 technology to create an Lrit1^-/- mouse line. Retinal morphology and morphometry analyses showed no gross changes in retinal structure …

A Role Of Vitamin B2 In Reducing Amyloid-Beta Toxicity In A Caenorhabditis Elegans Alzheimer’S Disease Model, Muhammad Tukur Ameen

Electronic Theses and Dissertations

Alzheimer’s disease (AD) is associated with amyloid-beta peptide deposition and loss of mitochondrial function. Using a transgenic C. elegans AD worm model expressing amyloid-beta in body wall muscle, we determined that supplementation with either of the forms of vitamin B₂, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) protected against amyloid-beta mediated paralysis. FMN and FAD were then assayed to determine effects on ATP, oxygen consumption, and reactive oxygen species (ROS) with these compounds not significantly improving any of these mitochondrial bioenergetic functions. Knockdown of the daf-16/FOXO transcriptional regulator or the FAD synthase enzyme completely abrogated the …

Development Of Low Frequency Electron Paramagnetic Resonance Methods And Instrumentation For Biological Applications, Laura A. Buchanan

Electronic Theses and Dissertations

EPR is a powerful biophysical tool that can be used to measure tumor physiology. With the addition of magnetic field gradients, the spectral properties of paramagnetic species can be mapped. To facilitate EPR imaging, methods and instrumentation at frequencies between 250 MHz and 1 GHz were developed.

At low spin concentrations, the rapid scan background signal is often many times larger than the EPR signal of interest. To help remove the background contribution, a data acquisition procedure that takes advantage of a cross-loop resonator and bipolar power supplies was developed at 250 MHz. In this procedure, two scans are collected. …

Learning From Disorder And Noise In Physical Biology, Taylor Emil Firman

Electronic Theses and Dissertations

Stochasticity, disorder, and noise play crucial roles in the functioning of many biological systems over many different length scales. On the molecular scale, most proteins are envisioned as pristinely folded structures, but intrinsically disordered proteins (IDPs) have no such folded state and still serve distinct purposes within the cell. At the scale of gene regulation, realistic in vivo conditions produce stochastic fluctuations in gene expression that can lead to advantageous bet-hedging strategies, but can be difficult to characterize using a deterministic framework. Even at the organismal scale, germband extension (GBE) in Drosophila melanogaster embryos systematically elongates the epithelial tissue using …

Brønsted Acid Catalyzed Carbon-Carbon Bond Forming Reactions, Jiangyue Miao

Electronic Theses and Dissertations

The focus of this research project lies in the development of new methodology in the field of Brønsted acid catalysis enabling rapid synthesis of medicinally relevant compounds. It is foreseen that small molecule sulfonic acids evaluated in this research project will unveil new asymmetric carbon carbon bond forming reactions between substrates hitherto unexplored with Brønsted acid catalysis. It has been established that strong Brønsted acids, such as phosphoric acids, are capable of mediating highly selective transformations operating through unique mechanistic manifolds.

Specific focus for the sulfonic acid catalysts was geared towards asymmetric coupling reactions with synthetically useful precursors such as …

Tau Aggregation, Conformational Selection, And Inhibition, Michael R. Holden

Electronic Theses and Dissertations

Tau fibrils are a pathological hallmark of over 20 neurodegenerative disorders, including Alzheimer's disease. There currently is no cure for these diseases and treatments are limited. Once Tau fibrils form in the brain, they propagate down neuronal networks, and this spreading is linked to disease progression. Studying the behavior and structure of Tau monomer and Tau aggregates therefore may give insight into methods by which the spread of Tau fibrils can be inhibited. The structures of the Tau fibrils from different diseases are thought to vary, partially giving rise to the different disease phenotypes. Tau natively binds to microtubules by …

Improving The Detection Limit Of Tau Aggregates For Use With Biological Samples, Emily Rickman Hager

Electronic Theses and Dissertations

The protein Tau is found in neurofibrillary tangles in Alzheimer's disease and over 20 other neurodegenerative diseases. An assay has been developed to detect minute amounts of fibrils from human brain tissue. This assay subjects brain tissue extract and recombinant Tau to several rounds of sonication and incubation. Incubation allows recombinant Tau to add itself to the ends of the existing fibrils in brain tissue extract. Sonication breaks the existing fibrils in the brain tissue extract offering more ends for Tau to add onto. Cycles of sonication and incubation have been shown to allow for amplification of Tau fibrils from …

Relationship Between Tdp-43 Toxicity And Aggregation In Saccharomyces Cerevisiae, Martin Anthony Aguilar

Electronic Theses and Dissertations

Protein aggregation and inclusion body formation are hallmarks of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis (ALS). These neurodegenerative diseases share a common pathology in that all include accumulation of insoluble protein aggregates in the brain. TAR-DNA-binding protein (TDP-43) is the major component found in the pathological inclusions of two of these diseases, ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). This thesis focuses upon the biophysical basis for TDP-43 aggregation in S. cerevisiae. Current in vitro evidence indicates that TDP-43 is a natively dimeric protein and that binding to RNA inhibits aggregation. Corresponding …

Superresolved Three-Dimensional Analysis Of The Spatial Arrangement Of The Human Immunodeficiency Virus Type-1 (Hiv-1) Envelope Glycoprotein At Sites Of Viral Assembly, Carmen Anne Buttler

Electronic Theses and Dissertations

Human Immunodeficiency Virus type 1 (HIV-1) replicates by forcing infected host cells to produce new virus particles, which assemble form protein components on the inner leaflet of the host cell's plasma membrane. This involves incorporation of the essential viral envelope glycoprotein (Env) into a structural lattice of viral Gag proteins. The mechanism of Env recruitment and incorporation is not well understood. To better define this process, we seek to describe the timing of Env-Gag encounters during particle assembly by measuring angular positions of Env proteins about the surfaces of budding particles. Using three-dimensional superresolution microscopy, we show that Env distributions …

The Investigation Of Lactoferrin Nitration: Quantification, Function, And Inhibition, Amani Yahya Alhalwani

Electronic Theses and Dissertations

Lactoferrin (LF) is an iron-binding glycoprotein of molecular mass ca. 80 kDa that is predominantly found in mammalian body fluids. Lactoferrin is a multifunctional protein that has a wide range of properties such as antibacterial, antiviral, anti-inflammatory, and anti-allergic functions. Tyrosine residues in the protein play a part in many lactoferrin functions. Protein tyrosine nitration modification represents an oxidative and nitrosative stress process which can be caused by the exposure of proteins to oxidants from air pollution or disease. Understanding the way nitrated lactoferrin interacts with the biochemical environment of the body is thus important to the broader goal of …

Evaluating The Interaction Between The Human Melanocortin-2 Receptor And The Accessory Protein, Mrap1: Chimeric Receptor And Alanine Substitution Studies On Transmembrane Domain 4, Extracellular Loop 2, And Transmembrane Domain 5, Perry Victoria Davis

Electronic Theses and Dissertations

The melanocortin-2 receptor (MC2R) is the most complex due to its trafficking and ligand selectivity requirements for proper activation. The MC2R requires the melanocortin receptor accessory protein-1 (MRAP1) for proper trafficking and activation of the receptor by the melanocortin hormone, ACTH. MRAP1 is a single transmembrane-spanning domain protein that creates a homodimer with another MRAP1 protein. Furthermore, MRAP2 creates a heterodimer with the MC2R. Previous studies have shown that the MRAP1 protein contains an activation motif required for activation of MC2R and this activation motif located on the extracellular space side of the plasma membrane of the cell. The objective …

Engineering Of Two-Color Atp-Binding Cassette Biosensor Proteins For Discovery Of Novel Substrates And Modulators, Bremansu Osa-Andrews

Electronic Theses and Dissertations

ATP-binding cassette (ABC) transporter proteins are a vast, ubiquitous superfamily of proteins most of which unlock the energy from ATP-binding and hydrolysis to influx or efflux a wide spectrum of structurally dissimilar substrates across cell membranes. All eukaryotic ABC transporters including P-gp and MRP1 only exist as efflux pumps whose prominent expression in cells is associated with multidrug resistance of antiretrovirals, antiepileptics, antimalarials and anticancer agents. P-gp and MRP1 reduce the bioavailability and efficacy of chemotherapeutic drugs by actively pumping out these drugs, thereby, leading to poor clinical outcomes during chemotherapy. Due to their clinical importance, profiling of the interaction …

The Contributions Of Fc Gamma Receptors And Macropinocytosis To The Internalization, Sorting And Clearance Of Antibody Coated Nanovesicles In Macrophages, George Opoku-Kusi Jr.

Electronic Theses and Dissertations

Macrophages are tissue-resident phagocytes that play critical roles in immune response and tissue homeostasis. They have a tremendous capacity to internalize objects of various sizes ranging from nanoscale viral particles to micron sized bacteria and tumor cells. This phenomenon, termed phagocytosis for the uptake of large particles, or endocytosis for the uptake of small particles, is integral to the immune response in multicellular organisms. Macrophages express FcγRs, which are tyrosine kinase receptors that bind IgG on an opsonized target. Binding of IgG Fc domain to the extracellular domain of an FcγR triggers signaling cascades that coordinate internalization of the opsonized …