Physical Sciences and Mathematics Commons^™

Effects Of Allosteric Mutations On Dihydrofolate Reductase, Marian Okondo

Theses, Dissertations and Culminating Projects

Dihydrofolate reductase (DHFR) is an essential enzyme necessary in the synthesis of DNA precursors. DHFR acquires resistance to numerous known anti-folates and this has led to the continuous need to discover novel and selective DHFR inhibitors. Allosteric mutations on Bacillus stearothermophilus (Bs.) DHFR were previously found to alter inhibitor binding and specificity. In this work, we investigate how these mutations affect the conformational motions of DHFR associated with inhibitor binding. Two distal allosteric mutations, isoleucine at position 86 to alanine (I86A) and tyrosine at position 127 to alanine (Y127A) were separately introduced to C73A/S131C, a previously modified wild type DHFR …

Novel Fluorescent Sensors For Cations With Additional Pet Pathways To Suppress Signals From Protons, Lesly C. Gomez

Theses, Dissertations and Culminating Projects

A large number of fluorescent sensors for cations that use a photoinduced electron transfer (PET) process to signal cation binding have been developed over the past three decades.1 The PET process of some of these sensors depends on having a tertiary nitrogen atom as a part of the receptor for cations. While these sensors work well as cation sensors, they also generate a fluorescent signal due to protonation of these receptors. The goal of this project is to design a fluorescent sensor that uses the same cation receptors but would not generate a signal for protons. Our new sensor has …

Toward The Synthesis Of [4.2.1]Di-Azabicyclic Systems, Carlos Abel Gomez

Theses, Dissertations and Culminating Projects

Conformationally restricted bicyclic amines have been found to be very useful scaffolds in medicinal chemistry. Examples can be found in the chemical literature for the application of conformationally restricted diamines. These molecules can be used as enzyme inhibitors or GPCR ligands. Conformational restriction can improve affinity and selectivity toward receptors. The stereochemical diversity-oriented approach is a method that explores stereochemical effects in small molecule ligands for proteins. It is an effective strategy when the bioactive conformation of a ligand and the pharmacophore of the binding site are unknown. These concepts are applied in the design the target [4.2.1]di-azabicyclic compounds. The …

Testing Predicted Specificity Determinants In The Dihydrofolate Reductase Enzyme Family, Seema J. Patel

Theses, Dissertations and Culminating Projects

Dihydrofolate Reductase (DHFR) is an essential enzyme for most organisms, ranging from bacteria to humans. DHFR has essential functions in DNA biosynthesis and cell replication; as a result, cell growth can be inhibited by the inhibition of DHFR. Evaluating the use of well-known DHFR inhibitors is becoming essential in treating infections in the developing world as DHFR is a known target of antibacterial and antiparasitic drugs. Understanding determinants of DHFR inhibitor specificity in terms of amino acid sequence and structure will allow repurposing or designing of new compounds that selectively target DHFR from the pathogenic organism of interest over the …

The Role Of Stress-Activated Protein Kinases In Parasites, Tamara Kreiss

Theses, Dissertations and Culminating Projects

Lymphatic filariasis and leishmaniasis are neglected tropical diseases that are caused by nematode and protozoal parasites. These diseases cause disfiguration, leaving their host socially marked, and in some cases cause more severe disease that can lead to death. These infections, which tend to persist for long periods of time, also lead to bacterial and fungal co-infections, which further exacerbate the disease. Currently there are insufficient treatment options. Current therapies are often too expensive, have toxicity associated with them and are subjected to growing resistance amongst parasite populations. In this thesis I investigated two potential drug targets. The first, is the …

Elucidation Of The Substrate Recognition Mechanism Of Vccry1, Rohe Ahmad

Theses, Dissertations and Culminating Projects

Vibrio cholerae cryptochrome 1 (VcCryl), a DNA repair enzyme, uses a lightdriven electron transfer to repair UV damaged single strand DNA but not double strand DNA. VcCryl has an active site FAD which is required for substrate binding and repair. Size exclusion chromatography (SEC) was used to observe changes in oligomer structure when changes in the oxidation state of the FAD cofactor were made. SEC studies found the oxidized state exists as a dimer with an observed mass of 163 kDa in solution, in dynamic equilibrium with a 67 kDa fragment. The reduced state exists as a monomer in …

Graphene-Like 2-D Nanomaterials And Their Properties, Diana Daikie Aggudey

Theses, Dissertations and Culminating Projects

Graphene, unlike other carbon allotropes has recently been the subject of interest in the field of nano material research. Graphene is being utilized as the building block for graphitic materials such as the 0-D fullerenes, 1-D nanotubes and 3-D graphites. Graphene is a single layer material, made up of interconnected hexagons of sp bonded carbon atoms arranged in a honeycomb crystal lattice. Graphene and its derivatives have been shown in previous studies/research to exhibit exceptional high electronic and thermal conductivity, optical transparency and high specific surface area additional to outstanding mechanical flexibility and environmental stability. Graphene is amongst the most …

High-Level Expression Of The Brugia Malayi Protein Kinase, Bm-Mpk 1, In Insect Sf9 Cells, Arti Rana

Theses, Dissertations and Culminating Projects

Lymphatic Filariasis (elephantiasis) is a neglected tropical disease caused by the filarial nematodes Brugia malayi (B. malayi), Brugia timori, and Wuchererici Bancrofti (1,8). These parasites are present in over 83 counties in the tropics and sub-tropics where more than 1.4 billion people are at risk of infection and 130 million people are presently infected (1,7). Previous work from our lab has led to the identification and expression of a B. malayi stress-activated protein kinase, Bm-MPKl (a human p38/C. elegans PMK-1 ortholog). Bm-MPKl plays an important role in the parasites’ protection against oxidative stress (1), and as such is a potential …

Spectroscopic Investigation Of The Interaction Of Fadh In Dna Photolyase With Uv-Damaged Dna, Kyle Luke Williams

Theses, Dissertations and Culminating Projects

Skin cancer is the most prevalent malignancy found in humans, with the diagnosis rate continuing to steadily increase. The primary cause of this disease is overexposure to harmful ultraviolet (UV) radiation from sunlight, which can induce damage to the nitrogenous bases in DNA via dimerization. The most prevalent UV-induced photoproducts in DNA are cyclobutane pyrimidine dimers (CPDs), most commonly between adjacent thymidines.

Organisms have implemented methods by which to repair these CPDs, the primary of which are nucleotide excision repair (NER) and photoreactivation by photolyases. Photolyases are blue-light activated flavoproteins that are more efficient at recognizing and repairing CPDs than …

Modeling Amyloid-Ss Self-Assembly : Stability Of On-Pathway Aggregate Formation, Andrew Kevin Mauro

Theses, Dissertations and Culminating Projects

Protein misfolding and concomitant aggregation towards amyloid formation is the underlying biochemical commonality among a wide range of human pathologies. Amyloid formation involves the conversion of proteins from their native monomeric states (intrinsically disordered or globular) to well-organized, fibrillar aggregates in a nucleation-dependent manner. Understanding the mechanism of aggregation is important not only to gain better insight into amyloid pathology but also to simulate and predict molecular pathways. One of the main impediments in doing so is the highly stochastic nature of interactions that complicates the development of meaningful insights. In this study, we have utilized a well-characterized intermediate along …

Decoration Of Graphene With Metal Nanoparticles And Its Application For Bioanalysis, Keun Soo Kwon

Theses, Dissertations and Culminating Projects

Graphene is a two-dimensional monolayer of tightly packed sp² hybridized Carbon atoms. Formed from graphite, the theoretical surface area of the monolayer is about 2630 m²/g, and the initial formation of graphene oxide from graphite oxide introduces a variety o f functional groups, such as -COOH, -C=0, and -OH, on its surface. The high surface area and the rich presence of functional groups, along with the potential formation o f sp² bonding networks within graphene oxide, all serve as critical factors allowing for additional chemical modifications. There are extensive research attempts being performed to explore and …

A Leishmania Mitogen Activated Protein Kinase As A Potential Anti-Parasitic Drug Target : Purification, Characterization And Inhibitor Interactions, Aysenur Sayakci

Theses, Dissertations and Culminating Projects

The disease, Leishmaniasis, is caused by the protozoal parasite Leishmania, which is transmitted by the bite of an infected Phlebotomine female sandfly. It is a significant health problem in tropical regions of the world and new therapeutic approaches for treating this disease are urgently needed.

Protozoal MAPKs (Mitogen Activated Protein Kinases) play important roles in parasite viability and infectivity and as such, represent viable drug targets. It has been demonstrated that LmxMPKl is an essential MAPK required for the parasite to establish infection and for proliferation of the amastigote stage (the mammalian stage of the parasite) of the parasite. In …

Investigation Of Autoregulation Of Icer And Electrochemical Properties Of Tryptophan, Mark Demilio

Theses, Dissertations and Culminating Projects

A relatively new development in the field of research-based chemistry is to study biomolecules, their interactions, and a biochemical mechanisms by examination of their physical properties and application of laboratory techniques rooted in concepts of physical chemistry. Both projects that are encompassed within this master's thesis indeed fall under the umbrella of biophysical chemistry, as they apply physical chemical techniques study particular biomolecular interactions.

The first of these two projects is the study of a leucine zipper protein, Inducible cAMP Early Repressor [ICER], which is a product of the cAMP Responsive Element Modulator [CREM] gene. ICER functions as a transcriptional …

Conformational Motions Associated With Ligand Binding In Dihydrofolate Reductase From Bacillus Stearothermophilus, Mayam Tobilola Alapa

Theses, Dissertations and Culminating Projects

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently attached fluorophore, N-[2-(lmaleimidyl) ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR …

Characterization Of A C-Jun N-Terminal Kinase Pathway In The Parasitic Nematode, Brugia Malayi, Agnieszka N. Chojnowski

Theses, Dissertations and Culminating Projects

Lymphatic filariasis (elephantiasis) is a major neglected disease caused by filarial nematodes including; Wuchereria bancrofti, Brugia malayi {B. malayi) and Brugia timori. Over 120 million individuals are infected and more than 1.3 billion people are at risk of infection in 81 endemic countries. Current treatments are limited, toxic and fail to kill adult parasites. There is a need for new target and treatments. One potential novel drug target for this disease is stress-activated protein kinases. It has been previously demonstrated in our laboratory that a B. malayi stress-activated protein kinase, Bm- MPK1, an ortholog of the human p38/Caenorhabditis elegans (C. …

Combining Bioinformatics And Chemical Biological Approaches To The Study Of Signaling Pathways In Parasitic Nematodes, William R. De Martini

Theses, Dissertations and Culminating Projects

Lymphatic filariasis (elephantitis) is a disfiguring disease caused by thread-like nematodes. This disease affects the lives of over 120 million people and over one billion people are at risk for infection in endemic regions. Drugs used to treat this disease suffer from toxicity and emerging resistance and new therapies need to be identified. Our laboratory has been studying the filarial parasite, Brugia malayi (B. malayi), one of the causative agents of this disease. The laboratory is focused on the study of critical protein kinase signaling pathways, necessary for parasite protective anti-oxidative responses, as potential therapeutic targets. We have previously determined, …

The Applications Of Graphene And Its Derivatives For Heavy Metal Removal, Clemonne John Madarang

Theses, Dissertations and Culminating Projects

Heavy metal pollution is very prevalent in our environment. The entire removal of heavy metal and organic contaminants in natural water resources will not only affect the environment itself, but also can block the toxic contaminant transfers in food chains. Currently, there are many studies being done to remove these heavy metal ions from our water, such as reduction reactions, chemical precipitation, reverse osmosis and electrolytic recovery. One of the promising techniques is adsorption. The focus of this research is using graphene and its derivatives, such as silylated graphene oxide (GO), to adsorb heavy metals, specifically, lead. This research describes …

Investigation Of Charge Recombination In Escherichia Coli Photolyase And Vibrio Cholera Cryptochrome Dash, Agnieszka Zieba

Theses, Dissertations and Culminating Projects

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp‘). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH') results in the formation of FADH and Trp . Charge recombination between these two intermediates requires O A/T # the uptake of a proton by ~ Trp . The …

Preparation And Crystal Structures Of Chiral And Non-Chiral Mixed Ligand Copper Complexes Containing N-Methyl Imidazole And Various N-Phthaloylalanines, Hiral Patel

Theses, Dissertations and Culminating Projects

A quasiracemate is the cocrystallization of two similarly shaped but chemically distinct molecules of opposite handedness. There has been extensive work done in the field of organic quasiracemates by Dr. Kraig A. Wheeler. Limited work has been done in inorganic quasiracemates. The goal of this study was to prepare various metal based enantiomers, racemates, and quasiracemates using A-phthaloylalanines, and TV-methylimidazole as ligands bound to copper arranged in a centrosymmetric pattern. In this study, we developed a method to prepare A-phthaloylalanines and its copper complexes. Three complexes have yielded crystal structures: a racemate, bis(7V-methylimidazole) bis(D,L-3-chloro-7V-phthaloylalanato)copper(II); a quasiracemate, bis(A-methyhmidazole)(L-3-chloro-iV-phthaloylalanato)(D-3-methyl-7V-phthaloylalanato) copper(II); and an …

Conformational Motion Associated With Catalysis In Indole-3-Glycerol Phosphate Synthase From S. Solfataricus, Tomasz Kurcon

Theses, Dissertations and Culminating Projects

Indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (SsIGPS) belongs to a broad family of (βα)₈-barrel enzymes. It catalyzes the fifth step in tryptophan biosynthesis, converting l-(o-carboxylphenylamino)-l-deoxyribulose-5-phosphate (CdRP) to indole-3-glycerol phosphate (IGP). Site selective mutagenesis was used to introduce a single cysteine in two loops near the active site, generating two recombinant proteins, each containing a single cysteine handle. The two construct were labeled, each with two different thiol-reactive probes generating four labeled constructs that were used for this study. Steady-state kinetic parameters of the labeled mutants and the wild type SsIGPS were well characterized using fluorescence spectroscopy. Subsequent experiments …

Vibrational Spectroscopy Of Isotopically Labeled Indoles And Electron Transfer Involving Indole (Tryptophan) Radicals, Senghane Dominique Dieng

Theses, Dissertations and Culminating Projects

Vibrational spectroscopy of tryptophan can be used as a probe for local protein conformation and to study protein electron transfer. For the interpretation of the vibrational spectra, the proper assignment of the vibrational bands to normal modes is essential. We report the experimentally measured infrared and Raman spectra of tryptophan, indole and 3-methylindole as well as of several of their isotope substituted forms; tryptophan indole-d₅ and -¹⁵N₂, and indole 2-¹³C. The effect of the exchange of the indole NH to ND on the vibrational spectra of these molecules was also determined. The measurements …

A Fluorescent Photoinduced Electron Transfer (Pet) Sensor For Cations With A Separate Pet Channel To Suppress Proton Signals, Supun Pathirana

Theses, Dissertations and Culminating Projects

This thesis presents A-((10-((2-methoxy-4-methylphenoxy)methyl)anthracen-9- yl)methyl)(pyridin-2-yl)-A-((pyridin-2-yl)methyl)methanamine, 1, a photoinduced electron transfer (PET) sensor that acts as a fluorescent ‘Off-On’ switch in the presence of Zn ions and not sensitive to protons.

Optimization Of Expression And Purification Of Acetoacetyl-Coa Thiolase From Sunflower, Anthony Muriithi Maina

Theses, Dissertations and Culminating Projects

The [beta]-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the existence of two different thiolase isoforms, Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9 AACT) and Thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16 OACT). Glyoxysomal AACT is the last enzyme in the p-oxidation of fatty acids in plant glyoxysomes. Glyoxysomal AACT has been successfully cloned, expressed and purified from sunflower cotyledons (Dyer et al., 2006). In this paper we investigate the optimal conditions for both expression and purification. These include the choice of vector, the growth conditions and the purification parameters for the maximum possible yield of AACT protein.

Identification Of Metallo-Ss-Lactamase Inhibitors Via Enzyme Assay And Hplc-Ms Monitoring, Peggy Ann Sanchez

Theses, Dissertations and Culminating Projects

The vast overuse of antibiotics along with extrachromosomal gene insertion between different bacterial species has led to the Darwinian consequence of antibiotic resistance. Amongst the contributing factors to the rise of bacterial resistances are the production of ß-lactamases, which pose a major impediment to treatment with ß-lactam drugs such as penicillins and carbapenems. SPM-1 is a metallo-ß-lactamase (MBL) that confers antibiotic resistance to bacteria through zinc-mediated hydrolysis of ß-lactams. Bacteria that express the SPM-1 metalloenzyme show reduced sensitivity to ß-lactam drugs. One method to defeat this form of resistance is to administer a ß-lactam antibiotic with a potent ß-lactamase inhibitor …

Fluorescent Photoinduced Electron Transfer (Pet) Proton Sensors, Risper Nyabeta

Theses, Dissertations and Culminating Projects

Two new fluorescent photoinduced electron transfer (PET) proton sensors based on anthracene and pyrazoline chromophores have been prepared. The anthracene sensor was prepared through alkylation of 2-pyridylcarbinol with 9-chloromethylanthracene and the pyrazoline sensor was prepared through an aldol condensation of 4- carboxybenzaldehyde and acetylpyridine followed by reaction with phenylhydrazine. The fluorescence of these sensors is modulated by the initiation or quenching of a PET process between the chromophore and a receptor due to protonation. The synthesis, characterization and proton binding studies of the new sensors are reported in this thesis.

Fluorescent Photoinduced Electron Transfer (Pet) Cation Sensors Derived From Natural Products, Kenny Loo

Theses, Dissertations and Culminating Projects

Two new second generation fluorescent switches that are based on the chromophore-spacer-receptor architecture are reported in this thesis. The fluorescence modulation of these switches is a result of quenching or initiating photoinduced electron transfer (PET) between the chromophore and the receptors due to cation binding. These two molecules utilize an alkaloid, anabasine, as the proton receptor, and function as fluorescent off-on-off switches with decreasing pH. One of these molecules also utilizes a coumarin as the chromophore, making it the first rationally designed fluorescent PET system based on natural products. Further studies of two well-known first generation fluorescent switches to achieve …