Physical Sciences and Mathematics Commons^™

Artemisinin And Its Derivatives Reactions: Characterization Of The Reaction Products Using Lc/Tof Ms, Kogila Vijayan

Theses and Dissertations

Artemisinin (ART) is a sesquiterpene lactone and a popular malaria drug with potential anticancer properties. In this work, LC/TOF MS was used to investigate the reaction of ART with DNA bases and estradiol. ART-deoxyadenosine and ART-deoxycytidine interactions were studied in the presence of Fe (II) ions. ART-deoxyadenosine and ART-deoxycytidine reaction mixtures gave chromatographic signatures that remained unchanged at room temperature but grew after incubation at 37°C. The change in temperature from room temperature to 37°C was the main driver of adduct formation in these reactions. ART was found to react with Fe (II) ions as observed from several new chromatographic …

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 …

Dynamics Of Redox-Driven Molecular Processes In Local And Systemic Plant Immunity, Philip Berg

Theses and Dissertations

The work here presents two main parts. In the first part, chapters 1 – 3 focus on dynamical systems modeling in plant immunity, whereas chapters 4 – 6 describe contributions to computational modeling and analysis of proteomics and genomics data. Chapter 1 investigates dynamical and biochemical patterns of reversibly oxidized cysteines (RevOxCys) during effector-triggered immunity (ETI) in Arabidopsis, examines the regulatory patterns associated with Arabidopsis thimet oligopeptidase 1 and 2’s (TOP1 and TOP2), roles in the RevOxCys events during ETI, and analyzes the redox phenotype of the top1top2 mutant. The second chapter investigates the peptidome dynamics during ETI …

Classification Models For 2,4-D Formulations In Damaged Enlist Crops Through The Application Of Ftir Spectroscopy And Machine Learning Algorithms, Benjamin Blackburn

Theses and Dissertations

With new 2,4-Dichlorophenoxyacetic acid (2,4-D) tolerant crops, increases in off-target movement events are expected. New formulations may mitigate these events, but standard lab techniques are ineffective in identifying these 2,4-D formulations. Using Fourier-transform infrared spectroscopy and machine learning algorithms, research was conducted to classify 2,4-D formulations in treated herbicide-tolerant soybeans and cotton and observe the influence of leaf treatment status and collection timing on classification accuracy. Pooled Classification models using k-nearest neighbor classified 2,4-D formulations with over 65% accuracy in cotton and soybean. Tissue collected 14 DAT and 21 DAT for cotton and soybean respectively produced higher accuracies than the …

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 …

Characterization Of Ph – Responsive Nanocage Based On The Ferritin Iron Storage Protein, Satyam Singh

Theses and Dissertations

The iron-storage protein ferritin (Ftn) assembles into a protein cage structure with 24 subunits and octahedral (4-fold, 3-fold, 2-fold) symmetry. Each monomeric subunit contains a robust four-helix bundle fold. The fully assembled Ftn structure has a high degree of thermal stability (up to 100°C), a mono dispersed size (12 nm in diameter), and a large central cavity (7-8 nm in diameter). The central cavity stores ferric iron in phylogenetically diverse group of organisms, including humans. The central cavity has been used for encapsulation of cargoes such as other metals, contrast agents for imaging, small molecule drugs for therapy, …

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 …

Single Molecule Investigations Of Holliday Junction Binding Protein Ruva, Dalton Reed Gibbs

Theses and Dissertations

DNA breaks are inevitable as they mainly occur due to cells’ own reactive oxygen species (ROS). While DNA breaks can be single-stranded or double-stranded, the double-stranded DNA (dsDNA) breaks are more dangerous. If such damage is not repaired, it can lead to genetic instability and serious health issues including cancers. One way dsDNA breaks can be repaired is via a process called homologous recombination (HR), which involves several DNA-binding proteins. Therefore, to have a better insight into the repair mechanism and origin of repair defects, we need a better understanding of how these proteins interact with DNA itself and DNA …

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 …

Adsorption Behavior Of Chemically / Charged Modified Antibodies On Gold Nanoparticles, Samuel Okyem

Theses and Dissertations

Gold nanoparticles (AuNPs) have been exploited in the various domains of science such as drug delivery, bio-sensing, immunoassays and environmental sensors, due to their optical properties and intriguing surface chemistry. Different scientific procedures have been used to effectively immobilize antibodies onto AuNPs. Although acceptable outcomes have been achieved in the immobilization of antibodies onto AuNPs, the sensitivity of these immobilized antibodies to target antigen or binding sites is limited due to improper orientation of the antibodies. Also, the possibility of nanoparticle aggregation when exposed to proteins limits its biomedical applicability.

There is some evidence that the surface charge of antibodies …

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 …

3d Printed Microfluidic Devices For Bioanalysis, Michael J. Beauchamp

Theses and Dissertations

This work presents the development of 3D printed microfluidic devices and their application to microchip analysis. Initial work was focused on the development of the printer resin as well as the development of the general rules for resolution that can be achieved with stereolithographic 3D printing. The next stage of this work involved the characterization of the printer with a variety of interior and exterior resolution features. I found that the minimum positive and negative feature sizes were about 20 μm in either case. Additionally, micropillar arrays were printed with pillar diameters as small as 16 μm. To demonstrate one …

Dna Capture Via Magnetic Beads In A Microfluidic Platformfor Rapid Detection Of Antibiotic Resistance Genes, David Hyrum Harris

Theses and Dissertations

Antibiotic resistant infections are a growing health care concern, with many cases reported annually. Infections can cause irreversible bodily damage or death if they are not diagnosed in a timely matter. To rapidly diagnose antibiotic resistance in infections, it is important to be able to capture and isolate the DNA coding for the resistance genes. This is challenging because bacteria are present in blood in minute concentrations. To enrich the DNA to detectable levels, I modified magnetic microbeads with ssDNA sequences complementary to the target DNA to capture the DNA via hybridization. I compared DNA capture efficiency in three different …

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 …

Effects Of Electrical Stimulation On Glioma Cells In Vitro With Implications For Treating Chronic Pain: Development Of A Model System, David C. Platt

Theses and Dissertations

Glial cells comprise over 70% of the central nervous system cells and exhibit diverse functions including regulation of synaptic transmission, neuron protection/repair, maintenance of neuronal metabolism, and are implicated in the development of persistent neuropathic pain. In addition, a perturbation in the concentration of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) has likewise been associated with the development of a chronic pain state. This perturbation in ROS/RNS creates an environment of oxidative stress. However, the mechanism by which the pain signal transmission is modulated, and the roles ROS play in the perpetuation of the pain state are …

Design And Synthesis Of Ceragenins–Cationic Steroid Antimicrobial Compounds, Structural Improvement And Synthesis Of Cyclopentenone Prostaglandins And Modification And Synthesis Of Derivatives Of Ribityllumazines: Potential Antigens For Activation Of Mait Cells, Yubo Li

Theses and Dissertations

Antimicrobial peptides (AMPs) are ubiquitous and display broad-spectrum antimicrobial activity that can control bacterial colonization of surfaces. Ceragenins are small-molecule mimics of AMPs and have several advantages over AMPs, including cost of manufacture and stability. A ceragenin, CSA-120, modified with an acrylamide group was directly incorporated into fluoropolymer coatings as a means of inhibiting bacterial biofilm formation. The ceragenin-containing coatings displayed improved performance. By conjugating a copper chelating group to the ceragenin, chelation of 64Cu by the conjugate was effective and provided a stable complex that allowed in vivo imaging. This conjugate may provide a means of identifying infection sites …

Total Synthesis Of Anticancer Agent Deoxypodophyllotoxin And Antiviral F4-4 Demonstrating The Utility Of The Intramolecular Styryl Diels-Alder (Isda) Reaction, Diana Isabel Saavedra Nova

Theses and Dissertations

The intramolecular styryl Diels – Alder (ISDA) reaction is a rare and unique [4+2] cycloaddition with potential in the syntheses of polycycles. Its utility is based on the formation of two rings and one stereocenter in a single step, making it an efficient method for the construction of lignan-type natural product targets. Detailed mechanistic studies with complex esters and the application to natural product synthesis has been limited due to drawbacks including the loss of aromaticity, producing slow reactivity, a potentially problematic thermal [1,3]-hydrogen shift, and electronic mismatch related to the substituents on the aryl functional groups. In this research, …

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 …

Kinetic Characterization Of Listeria Monocytogenes 2-C- Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase ( Cms ) Enzymes Using High Performance Liquid Chromatography ( Hplc ), Mark Oblazny

Theses and Dissertations

Infectious diseases, with increasing prevalence of antibiotic resistant bacteria, coupled with the declining rate in discovery of antimicrobial agents, impose one of the most significant threats to human health. Here we identify 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS) as a valid target for antibiotic development which is an enzyme in pathogenic organisms that leads to the biosynthesis of isoprene precursor molecules. Isoprene molecules are one of nature’s most common building blocks that are vital to many biological metabolic processes and are synthesized via the mevalonic acid dependent (MVA), or methylerythritol phosphate (MEP) pathway. Vertebrates utilize the MVA pathway, while many pathogenic bacteria …

The Biochemical Assessment Of Two Secreted Acid Phosphatases From Leishmania Tarentolae, Their Response To Electric Fields, Glycosidase Incubation, And / Or Vanadium, Benjamin M. Dorsey

Theses and Dissertations

Leishmaniasis, as defined by the Center for Disease Control and Prevention, is a neglected tropical disease with 1.6 million new cases reported each year. However, there is yet to be safe, effective, and affordable treatments provided to those affected by this disease1. Still underappreciated as a potential pharmaceutical targets, especially for cutaneous leishmaniasis infections, are the two isozymes of secreted acid phosphatase (SAP); secreted acid phosphatase 1 (SAP1) and secreted acid phosphatase 2 (SAP2). These enzymes are involved in the survival of the parasite in the sand fly vector, and the prevention of host macrophages from forming parasitophorous vacuole and …

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 …

Cytotoxic And Antimicrobial Effects Of Silver-Containing Surfaces, Sarah Goderecci

Theses and Dissertations

This study examines applications of sputtered silver coatings as alternatives to traditional antibiotic treatments. Given the increase in reports of antibiotic-resistant bacteria, new treatments and coatings for in-dwelling medical devices such as catheters and orthopedic implants are necessary. Silver oxide films were deposited onto Ti surfaces to examine the efficacy of such coatings against a variety of bacterial species both in vitro and in vivo. Bacterial growth studies showed that coatings exhibited antimicrobial activity against a range of bacterial species acting either in a bacteriostatic or bactericidal mechanism, depending on the target. Limited toxicity to in vitro mammalian cells was …

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 …

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 …