Physical Sciences and Mathematics Commons^™

Impact Of The Pre-A Motif On Truncated Hemoglobin N Activity, Alexander Shayne Drena

Theses and Dissertations

Tuberculosis (TB) remains the leading cause of death by an infectious agent and therefore a global health crisis, according to the most recent report by the World Health Organization. This is due, in part, to Mycobacterium tuberculosis’ impressive defensive mechanisms against immune response, as well as the rise of Multi-Drug Resistant strains that have recently developed. Towards the turn of the century, a small heme protein called truncated hemoglobin N (trHbN) was discovered to protect the bacteria against reactive nitrogen species by converting nitric oxide (NO) to nitrate at rates far exceeding those of myoglobin and closer to those of …

The Effects Of An R103q Mutation On The Chemical And Physical Properties Of The Enzyme Cytochrome C Nitrite Reductase (Ccnir), Shahama Alam

Theses and Dissertations

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. The project described herein explored the properties of the Shewanella oneidensis R103Q ccNiR variant and compared them to the properties of the wild type enzyme. Under standard assay conditions, which use the strong reducing agent methyl viologen monocation radical (MVred) as an electron source, the R103Q variant still catalyzes reduction of nitrite to ammonium, albeit with an MVred turnover rate that is 20% of the wild type’s (780 ± 50 s-1 …

Structure & Function Of Enzymes In Two Uncharacterized Gene Clusters From Pseudomonas Brassicacearum & Streptomyces Griseofuscus, Lamia Tabassum Badhon

Theses and Dissertations

Pyridoxal 5’-phosphate (PLP)-dependent enzymes harness this versatile cofactor to catalyze a variety of reactions including transamination, decarboxylation, racemization and various elemination/subsitution reactions. Several years ago, a new class of PLP-dependent enzymes was discovered that uses PLP and molecular oxygen to catalyze the 4-electron oxidation of L-arginine to 4-hydroxy-2-ketoarginine. Work with the prototypical enzyme of this class, MppP from Streptomyces wadayamensis (SwMppP), showed that the dioxygen consumed during the reaction is reduced to hydrogen peroxide, and that the hydroxyl group installed in the product derives from water. Thus, SwMppP is an L-arginine oxidase, and not an oxygenase. This was surprising given …

Part I: Development Of Small-Molecule-Based Probes For The Vitamin D Receptor; Part Ii: Development Of A Scalable Manufacturing Process For Orcein Dye, Tania Roseann Mutchie

Theses and Dissertations

PART I:The vitamin D receptor (VDR) is a ligand-dependent transcription factor and member of the nuclear hormone receptor superfamily. VDR is expressed in the epithelia of endocrine organs, digestive system, bronchi, kidneys, and thymus, as well as being present in leukocytes and bone cells. Cell proliferation, cell differentiation, and immunomodulation, along with calcium and phosphate homeostasis, are all processes regulated by the receptor. Within the cell, VDR can be membrane-bound or located in the nucleus. Nuclear localization of VDR transpires following the binding of vitamin D metabolites, the most active of which is 1α,25-dihydroxyvitamin D3 (calcitriol). Within the nucleus, interactions …

Investigation Of G Protein-Coupled Receptor Quaternary Structure Through Fluorescence Micro-Spectroscopy And Theoretical Modeling: Interdependence Between Receptor-Receptor And Receptor-Ligand Interactions, Joel David Paprocki

Theses and Dissertations

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal …

A Mechanistic Investigation Of Cytochrome C Nitrite Reductase Catalyzed Reduction Of Nitrite To Ammonia: The Search For Catalytic Intermediates, Shahid Shahid

Theses and Dissertations

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were …

Zinc Proteomics: Interactions Of Zn2+, Cd2+, And Metal-Binding Ligands With Zn-Binding Sites In The Proteome, Eric Daniel Lund

Theses and Dissertations

Cadmium ion causes toxicity in humans, most prominently in the kidney. This thesis focuses on mechanisms by which Cd2+ harms kidney proximal tubule cells. Previous experiments have shown the time-dependent distribution of Cd2+ and Zn2+ within supernatant of pig kidney, LLC-PK1 cells after exposure to Cd2+ plus the ionophore pyrithione1. The first part of this research describes the trafficking of cadmium within LLC-PK1 proximal tubule cells, including the time dependent distribution, speciation, and quantification of Cd2+ and Zn2+ in cells exposed to Cd2+ and Cd2+ plus pyrithione. The latter treatment introduces Cd2+ into cells at time zero and permits the …

The Curious Case Of Nad-Cleaving Nudix Hydrolases, Atreyei Ray

Theses and Dissertations

Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds (x). These enzymes have catalytic activity on a wide range of substrates such as dNTPs (both canonical and their oxidized forms), nucleotide sugars, alcohols, dinucleoside polyphosphates, dinucleotide cofactors, and nucleoside diphosphates linked to RNA. The bacterial genome encodes around 13, while the human genome encodes for 22 such nudix proteins.

The E. coli genome encodes for a mutT mutator gene, the progenitor of the family expressing the MutT pyrophosphohydrolase (NudA) protein (Treffers et al., 1954; Bhatnagar et al., 1988). The enzyme …

Design And Synthesis Of Achiral And Chiral Imidazodiazepine (Imdz) Gaba(A)R Subtype Selective Ligands For The Treatment Of Cns Disorders, As Well As Asthma, Guanguan Li

Theses and Dissertations

Gamma-aminobutyric acid type A receptors (GABAAR) are transmembrane pentameric ligand-gated chloride ion channels that respond to GABA, the major inhibitory neurotransmitter in the central nervous systems (CNS). The benzodiazepines (BZDs) bind at the extracellular interface of the α+γ2-subunits of GABAAR. The binding of ligands at different subunits of GABAA receptors specifically at α1-6β2/3 γ2 ion channels, can affect a wide variety of brain functions. The α1-subtype selective ion channels of GABAARs are involved in the sedative, ataxic, amnesic, anticonvulsant and addictive effects, which should be avoided, with the exception of the anticonvulsant effects, when designing ligands for this BZ allosteric …

Part I: An Investigation Of Calcitroic Acid And Its Phase Ii Conjugates. Part Ii: Toward The Development Of A Novel Orally-Available Asthma Treatment Targeting Gabaa Receptors In The Lungs, Olivia B. Yu

Theses and Dissertations

PART I

Calcitroic acid (CTA) was isolated and characterized more than four decades ago.5 Radiolabeled calcitriol (1,25-dihydroxyvitamin D3) was used at that time to enable the identification of radioactive CTA formed in vivo, which was subsequently extracted and characterized by derivatization.7 CTA was found to be predominantly formed in the liver and secreted into the gut through the bile duct via enterohepatic circulation, leading to fairly high concentrations of this metabolite of vitamin D in the intestine.8 However, assuming it was only a catabolic product of calcitriol, it was ignored thereafter.

Recently, novel experiments showed that CTA can bind the …

Part I: An Investigation Of Calcitroic Acid And Its Phase Ii Conjugates. Part Ii: Toward The Development Of A Novel Orally-Available Asthma Treatment Targeting Gabaa Receptors In The Lungs, Olivia B. Yu

Theses and Dissertations

PART I

Calcitroic acid (CTA) was isolated and characterized more than four decades ago.5 Radiolabeled calcitriol (1,25-dihydroxyvitamin D3) was used at that time to enable the identification of radioactive CTA formed in vivo, which was subsequently extracted and characterized by derivatization.7 CTA was found to be predominantly formed in the liver and secreted into the gut through the bile duct via enterohepatic circulation, leading to fairly high concentrations of this metabolite of vitamin D in the intestine.8 However, assuming it was only a catabolic product of calcitriol, it was ignored thereafter.

Recently, novel experiments showed that CTA can bind the …

Studies In Molecular Recognition: Non-Proteogenic Amino Acids For Antibiotic Studies And Chemosensors For Recognition And Reporting Of Metal-Ions, Sarah Anne Oehm

Theses and Dissertations

The field of molecular recognition focuses on the selective and reversible binding of small guest molecules to larger host molecules. This dissertation describes synthesis of small molecules as binding guests for enzymatic substrates as well as molecules as host chemosensors to detect and identify metal ions. Two approaches to new antibiotic drugs have been explored, and an array of sensors for the quantitation of aqueous metals is being commercialized.

As strains of deadly bacteria emerge with evolved resistance to known antibiotics, new drugs are needed with novel mechanisms of action. Natural product antibiotics containing enduracididine, a non-canonical amino acid derived …

Interaction With Nitric Oxide Of The Nitrosomonas Europaea Tetraheme Protein Cytochrome C554, And Two Of Its Variants, In Increasingly Reducing Environments, Jennifer M. Mcgarry

Theses and Dissertations

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the 2-electron or 4-electron reduced protein will nitrosylate reversibly. The nitrosylation process was found to be first order in C554, first-order in NO, and second-order overall. The rate constant for NO binding to the heme was determined to be 3000 ± 140 M-1s-1, while the rate constant for dissociation was 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report, …

Part – I: Development Of A Two-Step Regiospecific Synthetic Route For Multigram Scale Synthesis Of Β-Carboline Analogs For Studies In Primates As Anti-Alcohol Agents,Part – Ii: Design And Synthesis Of Novel Antimicrobials For The Treatment Of Drug Resistant Bacterial Infections Part – Iii: A Novel Synthetic Method For The Synthesis Of The Key Quinine Metabolite (3s)-3-Hydroxyquinine, Veera Venkata Naga Phani Babu Tiruveedhula

Theses and Dissertations

PART – I

Development of a Two-Step Regiospecifc Synthetic Route for Multigram-Scale Synthesis of β-Carboline Analogs for Studies in Primates as Anti-Alcohol Agents

β-Carboline and their derivatives are important structural motifs in synthetic organic and medicinal chemistry because of their novel biological activity, especially in regard to the reduction of alcohol self-administration [binge drinking (BD)], a major problem increasing day by day in modern society. This anti-alcohol effect is proposed to be due to the activity of ligands at the benzodiazepine site of the GABAA receptor in the central nervous system acting as antagonists at the α1 subunit. The past …

Structural And Functional Characterization Of Acetoacetate Decarboxylase-Like Enzymes, Lisa Mueller

Theses and Dissertations

The acetatoacetate decarboxylase-like superfamily (ADCSF) is a largely unexplored group of enzymes that may be a potential source of new biocatalysts. Bioinformatic analysis has grouped these approximately 2000 enzymes into seven different families based on comparison of predicted active site residues. To date, only the prototypical ADCs (Family I) that catalyze the decarboxylation of acetoacetate have been studied. Analysis of gene context suggests that Family V contains predominantly enzymes predicted to be involved in secondary metabolism. On average, these share about 20% sequence identity to the true ADCs. To learn more about the diversity of chemistries performed by members of …

The Function Of Renalase, Brett Allen Beaupre

Theses and Dissertations

Renalase was originally reported to be an enzyme secreted into the blood by the kidney to lower blood pressure and slow heart rate. Despite multiple reports claiming to confirm this activity in vivo there has been considerable discord in regards to the reaction catalyzed by renalase. The structural topology of renalase resembles that of known flavoprotein oxidases, monooxygenases and demethylases, but the conserved active site residues are unique to renalase. It has been reported that the catalytic function of renalase is to oxidize circulating catecholamines, however in vitro studies have failed to demonstrate a catalytic activity in the presence of …

The Role Of Proteome In Cellular Zn2+ Trafficking And In The Ability Of The Fluorescent Zinc Sensors To Image Intracellular Zn2+, Mohammad Rezaul Karim

Theses and Dissertations

Zinc is an essential biological trace metal used in as many as 3000 Zn-proteins, about 10% of the eukaryotic proteome, as either a structural constituent or a catalytic cofactor. These proteins include the zinc fingers, the most prevalent transcription factors that bind a wide range of gene promoters and thus regulate gene expression. A eukaryotic cell contains several hundred micromolar of Zn2+- almost all of it is bound to specific Zn-proteins. Recently, Zn2+ has been reported to serve as a regulatory signal and a neurotransmitter, suggesting that there also exists a dynamic Zn2+ pool in cells. These findings led to …

Arylboronates As H2o2 Or Photo-Inducible Dna Cross-Linking Agents: Design, Synthesis, Mechanism, And Anticancer Activity, Yibin Wang

Theses and Dissertations

Interest in the development of cancer therapies with improved selectivity and reduced host toxicity has been growing. In this thesis, we designed and synthesized a series of novel non-toxic arylboronic ester and biarylboronic ester derivatives that can be activated by hydrogen peroxide (H2O2) to induce DNA interstrand cross-link formation. The mechanism of DNA cross-linking induced by these arylboronates involves generation of phenol intermediates 1 followed by departure of leaving group (L) leading to quinone methides (QMs) 2, which directly cross-link DNA via alkylation. The QM formation is the rate-determining step for DNA cross-linking. The activity and selectivity of these compounds …

Mechanistic Study Of Heme Protein-Mediated Nitric Oxide Dioxygenation Using Photolytically Produced Nitric Oxide, Karl Joseph Koebke

Theses and Dissertations

The previously reported NO precursor [Mn(PaPy2Q)(NO)]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was synthesized and proven capable of producing as much as 180 µM NO when irradiated by a single 3 mJ 500nm laser pulse, in a 0.15 cm path cell, without the need for additional sacrificial reductants or oxidants. Species 1 was first used to study the reaction of nitric oxide with oxy-myoglobin (oxyMb) to form ferric myoglobin (metMb) and nitrate. This reaction had long been assumed to proceed via the same iron-bound peroxynitrite intermediate (metMb(OONO)) as the metMb-catalyzed isomerization of peroxynitrite to nitrate. Recent research showed that the metMb-catalyzed …

Application Of Coumarin Derivatives In Dna-Associated Study: Mutation Detection, Site-Specific Labeling, Photoinduced Interstrand Cross-Links And Ligation Reactions, Huabing Sun

Theses and Dissertations

Coumarin derivatives have been widely utilized as cross-linking agents in polymer science, being fluoroprobes in biochemistry and as medicines in pharmacy. But the coumarin's fluorogenic properties and reactivities in DNA were rarely reported and unclear, which limits its bioapplications due to possible side reactions towards biomolecules. In this thesis, we investigated the activity of coumarin moiety towards natural DNA and expanded its application in DNA-associated study. We have found that coumarin derivatives can serve as perfect DNA cross-linking agents, as alkylation agents for site-specific labeling, and fluoroprobes for single nucleotide polymorphism (SNP) analysis, which provided a novel insight of biotoxicity …

Cellular Zinc Trafficking: The Zinc Proteome And Its Reactions With Cadmium, Mohammad Ali Namdarghanbari

Theses and Dissertations

Metals play a crucial role in living systems. Iron, zinc, copper, molybdenum, and manganese are involved in many essential biological activities. Among transition metals, zinc after iron is the most abundant transition metal in the human body and the most abundant in the brain. It exists in more than 3000 proteins, which comprise about 10% of the human proteome. Zn2+ dyshomeostasis is associated with chronic diseases such as metabolic syndrome, diabetes and related complications, bone loss, growth retardation in young children, and neurological and behavioral problems. Despite a good knowledge obtained for metabolism of some metal ions such as copper, …

Evaluation Of Vdr-Coactivator Inhibitors Using Biochemical And Cell-Based Assays, Athena Marie Baranowski

Theses and Dissertations

ABSTRACT

EVALUATION OF VDR–COACTIVATOR INHIBITORS USING BIOCHEMICAL AND CELL–BASED ASSAYS

by

Athena Baranowski

The University of Wisconsin–Milwaukee, 2013

Under the Supervision of Dr. Alexander Arnold

The vitamin D receptor (VDR) is a ligand–dependent transcription factor, which belongs to the nuclear receptor superfamily. VDR–mediated gene regulation is governed by coregulators (coactivators and corepressors). VDR coregulator binding inhibitors (CBIs), which were discovered using high throughput screening (HTS), were evaluated using cell–based assays and biochemical assays to determine their ability to inhibit the interaction between VDR and steroid receptor coactivator–2 (SRC–2). Determining their ability to inhibit the VDR–SRC–2 interaction can lead to the …

Zinc Chemical Biology: The Pursuit Of The Intracellular Targets Of Zinquin, Andrew Nowakowski

Theses and Dissertations

Fluorescent sensors have been a main microscopic tools used to understand Zn2+ physiology on a cellular level. The use of the fluorescent Zn2+ sensor Zinquin (ZQ) and its analogues have revealed that transient Zn2+ is a chief component in a variety of biochemical pathways. Yet, little work has been performed to validate the exact targets of Zinquin in a cellular environment. The goals of this investigation are to determine the types of Zinquin reactions that take place in the cell as well as the identities of its cellular targets.

It has been hypothesized that Zinquin reacts with free Zn2+ within …

Towards The Use Of Time-Resolved X-Ray Crystallography In Mechanistic Studies Of Cytochrome C Nitrite Reductase From Shewanella Oneidensis, Matthew David Youngblut

Theses and Dissertations

A high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "Small Tetra-heme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-C strain, generated approximately 20 mg crude ccNiR/L culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR exhibited nitrite and hydroxylamine reductase activities comparable to those of E. …

High-Throughput Approaches For The Assessment Of Factors Influencing Bioavailability Of Small Molecules In Pre-Clinical Drug Development, Megan Marie Mccallum

Theses and Dissertations

A bioactive molecule must pass many hurdles to be designated as a "good" pharmaceutical lead or hit compound. It should have a significant activity, selectivity, bioavailability, and metabolic half-life. Many factors have been identified that influence the free drug concentration or bioavailability of orally administered drugs in the earliest development stages. In vitro pre-clinical assays have been developed to measure these parameters. The small molecule properties that are investigated here include aqueous solubility, permeability, reactivity (electrophilicity), small molecule-protein binding, and displacement of protein-bound molecules (drug-drug interactions). The development of rapid and miniaturized assays to quantify these factors is presented herein. …

Methods In Metallomics, Proteomics, And Toxicology: Development And Applications Of Laser Ablation Inductively Coupled Plasma Mass Spectrometry And Native Sds-Page, William John Wobig

Theses and Dissertations

Polyacrylamide gel electrophoresis (PAGE) is a bio-analytical method used to separate proteins in solution into an array of individual bands of proteins in a gel matrix. Current PAGE methods, however, have severe limitations in simultaneously maintaining a protein's native structure and association with transition metals while providing adequate resolution. Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) provides a means to perform trace to ultra-trace level inorganic analysis of solid samples such as dried PAGE gels containing metallo-protein arrays. Current LA-ICP-MS methods involving the analysis of PAGE gels, however, have been limited in their effective use by inadequate limits of …

Part 1. Design And Synthesis Of Cysteine/Cystine Prodrugs And Bioisosteres Including Symmetrical And Unsymmetrical Disulfides Designed To Increase Cystine Levels In The Cns In Order To Drive The Cystine/Glutamate Antiporter: A Novel Treatment For Schizophrenia And Drug Addiction. Part 2. Design And Synthesis Of Subtype Selective Ester Bioisosteres Of Bzr Ligands For Gabaa/Benzodiazepine Receptors To Enhance Metabolic Stability, Edward Merle Johnson Ii

Theses and Dissertations

Part 1. Schizophrenia is a debilitating disorder that affects almost 1% of the world's population; pharmacotherapy expenditures for this disorder exceed $10 billion dollars even though existing medications exhibit a poor safety/efficacy profile. It is estimated that 75% of patients discontinue drug treatment, in part due to poor safety/efficacy. The current data set demonstrates that cysteine prodrug NAC reverse the behavioral and neurochemical effects of PCP used to model schizophrenia.

As a result cysteine prodrugs represent a highly novel approach to treating schizophrenia; indeed, these compounds may ultimately be more effective than existing medications because these drugs target the pathology …