Biochemistry Commons^™

Spectroscopic Analysis Of Polymerization And Exonuclease Proofreading By A High-Fidelity Dna Polymerase During Translesion Dna Synthesis, Babho Devadoss, Irene Lee, Anthony J. Berdis

Chemistry Faculty Publications

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient …

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading …

Periplasmic Binding Proteins In Thermophiles: Characterization And Potential Application Of An Arginine-Binding Protein From Thermotoga Maritima: A Brief Thermo-Story, Alessio Ausili, Maria Staiano, Jonathan D. Dattelbaum, Antonio Varriale, Alessandro Capo, Sabato D'Auria

Chemistry Faculty Publications

Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima …

Structurally Diverse Hamigerans From The New Zealand Marine Sponge Hamigera Tarangaensis: Nmr-Directed Isolation, Structure Elucidation And Antifungal Activity, A. Jonathan Singh, Jonathan D. Dattelbaum, Jessica J. Field, Zlatka Smart, Ethan F. Woolly, Jacqueline M. Barber, Rosemary Heathcott, John H. Miller, Peter T. Northcote

Chemistry Faculty Publications

The NMR-directed investigation of the New Zealand marine sponge Hamigera tarangaensis has afforded ten new compounds of the hamigeran family, and a new 13-epi-verrucosane congener. Notably, hamigeran F (6) possesses an unusual carbon–carbon bond between C-12 and C-13, creating an unprecedented skeleton within this class. In particular, the structural features of 6, hamigeran H (10) and hamigeran J (12) imply a diterpenoid origin, which has allowed the putative biogenesis of three hamigeran carbon skeletons to be proposed based on geranyl geranyl pyrophosphate. All new hamigerans exhibited micromolar activity towards the HL-60 …

Oligodeoxynucleotide Synthesis Using Protecting Groups And A Linker Cleavable Under Non-Nucleophilic Conditions, Xi Lin

Dissertations, Master's Theses and Master's Reports - Open

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process.

To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. …

An Acid Catalyzed Reversible Ring-Opening/Ring-Closure Reaction Involving A Cyano-Rhodamine Spirolactam, H. Li, H. Guan, X. Duan, J. Hu, Guiren Wang, Qian Wang

Faculty Publications

Cyanamide was introduced into the rhodamine spirolactam framework to produce a colorless and non-fluorescent compound RBCN. It shows a reversible ring-opening/ring-closure process in response to the solution pH, which exhibits an “ON/OFF” switching in its fluorescence. Different from other rhodamine-type dyes, the ring-open form of RBCN is stable in protic solvents under neutral, near neutral and basic conditions, showing a pink color and very strong fluorescence. We also demonstrated the potential of RBCN in live cell imaging.

Hiv-1 Frameshift Efficiency Is Primarily Determined By The Stability Of Base Pairs Positioned At The Mrna Entrance Channel Of The Ribosome, Kathryn Mouzakis

Chemistry and Biochemistry Faculty Works

The human immunodeficiency virus (HIV) requires a programmed -1 ribosomal frameshift for Pol gene expression. The HIV frameshift site consists of a heptanucleotide slippery sequence (UUUUUUA) followed by a spacer region and a downstream RNA stem-loop structure. Here we investigate the role of the RNA structure in promoting the -1 frameshift. The stem-loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. No correlation between overall stability and frameshift efficiency is observed. In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3-4 bp in …

Optimizing Electrode Design For Microbial Fuel Cells Used For Wastewater Treatment, Lindsay Nichols, John A. Hogan

STAR Program Research Presentations

Microbial fuel cells (MFC) utilize bacteria to generate an electrical current that can be used in the decomposition of sludge and human urine. In a MFC there is an anode (for oxidation of organic compounds), cathode (reduction of oxygen or carbon dioxide), and a proton exchange membrane (PEM, allows protons to migrate); reduction-oxidation reactions between the anode and cathode produce a measurable current. Bacteria that are found in sludge can be used to produce electrons in a voltaic cell, but optimizing conditions for harnessing the energy is crucial to making a MFC efficient. Research has shown that the ratios of …

Determination Of Structural Stabilities Of Metal-Free Porphyrin Cytochrome C, Zinc Cytochrome C, And Cobalt Cytochrome C, Usha K. Tottempudi

Masters Theses

No abstract provided.

A Comparison Of Boltzmann And Gibbs Definitions Of Microcanonical Entropy For Small Systems, Randall B. Shirts

Faculty Publications

Two different definitions of entropy, S= klnW, in the microcanonical ensemble have been competing for over 100 years. The Boltzmann/Planck definition is that W is the number of states accessible to the system at its energy E (also called the surface entropy). The Gibbs/Hertz definition is that W is the number of states of the system up to the energy E (also called the volume entropy). These two definitions agree for large systems but differ by terms of order N^-1 for small systems, where N is the number of particles in the system. For three analytical …

Microfluidic Devices With Integrated Sample Preparation For Improved Analysis Of Protein Biomarkers, Pamela Nsang Nge

Theses and Dissertations

Biomarkers present a non-invasive means of detecting cancer because they can be obtained from body fluids. They can also be used for prognosis and assessing response to treatment. To limit interferences it is essential to pretreat biological samples before analysis. Sample preparation methods include extraction of analyte from an unsuitable matrix, purification, concentration or dilution and labeling. The many advantages offered by microfluidics include portability, speed, automation and integration. Because of the difficulties encountered in integrating this step in microfluidic devices most sample preparation methods are often carried out off-chip. In the fabrication of micro-total analysis systems it is important …

Anti-Germinants As A New Strategy To Prevent Clostridium Difficile Infections, Amber Janece Howerton

UNLV Theses, Dissertations, Professional Papers, and Capstones

Clostridium difficileinfections (CDI) have emerged as a leading cause of hospital-associated complications. CDI is the major cause of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. The infective form of C. difficileis the spore, a dormant and hardy structure that forms under stress. Germination of C. difficile spores into toxin producing bacteria in the GI tract of susceptible patients is the first step in CDI establishment. Patient susceptibility occurs with a disruption of the natural gut microbiota by broad-spectrum antibiotics. Antibiotic treatments usually resolve CDI but refractory cases are on the rise. Of great concern is the …

Microfabrication, Characterization, And Application Of Carbon Nanotube Templated Thin Layer Chromatography Plates, And Functionalization Of Porous Graphitic Carbon, David S. Jensen

Theses and Dissertations

This dissertation contains the following sections. Chapter 1 contains a detailed description of the theory of thin layer chromatography (TLC). Chapter 2 describes the benefits and practical considerations of elevated temperatures in liquid chromatography (LC). The porous graphitic carbon (PGC) I modified as part of my work is often used in elevated temperature LC. Chapter 3 shows a thermodynamic analysis of chromatographic retention at elevated temperature, and Chapter 4 contains a closer look at the van 't Hoff equation in LC and how it can be used in retention modeling. In Chapter 5, I describe a new procedure for microfabricating …

Deposition And Characterization Of Hydrophobic Coatings, Vipul Gupta

Theses and Dissertations

Hydrophobic coatings find application in various sectors of the economy including to electronics, textiles, optical devices, and in scientific and commercial equipment. These different applications demand that different hydrophobic coatings posses a range of properties that may include smoothness or roughness, thicknesses on the order of a monolayer or a micron, robustness or the ability to dissolve quickly, transparency or opacity, water resistance or water permeability, electrical conductivity, oleophobicity, etc. However, whatever the final/desired properties, deposition via dry-deposition processes offers significant advantages, including greater reproducibility, increased environmental friendliness, and cost effectiveness on an industrial scale. Herein I explore the chemical …

Proposed Coherent Trapping Of A Population Of Electrons In A C60 Molecule Induced By Laser Excitation, Thomas George, G.P. Zhang

Chemistry & Biochemistry Faculty Works

This Letter demonstrates the possibility of generating coherent population trapping in C60. Similar to a three-level Λ system, C60 has a forbidden transition between the highest occupied molecular orbital (HOMO) (|a⟩) and the lowest unoccupied molecular orbital (LUMO) (|c⟩), but a dipole-allowed transition between HOMO and LUMO+1 (|b⟩) and between |b⟩ and |c⟩. We employ two cw laser fields, one coupling and one probe. The strong coupling field is switched on first to resonantly excite the transition between |b⟩ and |c⟩. After a delay, the probe is switched on; the coherent interaction between the coupling and probe fields traps the …

Mechanism Of Action Studies On A New Class Of Anticancer Nucleosides, Megan E. Browning

Theses and Dissertations

We have completed mechanism of action studies on a new class of anticancer nucleosides typified by a novel nucleoside discovered in our lab, MAP-870. In order to study the mechanism of MAP-870, several experiments were completed on a colorectal adenocarcinoma cell line, HT-29, including trypan blue cell count, sulforhodamine B assays, flow cytometry of cell cycle, propidium iodide incorporation, and phosphatidylserine externalization, Caspase-Glo3/7 assays, DNA fragmentation gel, cyclophilin A release gel, PAMPA, and confocal imaging. Sulforhodamine B assays show that MAP-870 does indeed cause growth inhibition and cell death in the model tested. PAMPA assays show that MAP-870 does not …

Differential Effects Of The Hydrophobic Surfactant Proteins On The Formation Of Inverse Bicontinuous Cubic Phases, Mariya Chavarha, Ryan W. Loney, Kamlesh Kumar, Shankar B. Rananavare, Stephen B. Hall

Chemistry Faculty Publications and Presentations

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce formation of the bicontinuous structures and faster adsorption by similar mechanisms, then they should also have differential ability to form the cubic phases. To test this hypothesis, we measured small angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the doserelated …

Exploring The Role Of A Conserved Class A Residue In The Ω-Loop Of Kpc-2 Β-Lactamase: A Mechanism For Ceftazidime Hydrolysis

Chemistry Faculty Publications

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we …

Breast Tumour Initiating Cell Fate Is Regulated By Microenvironmental Cues From An Extracellular Matrix, Sharmistha Saha, Pang-Kuo Lo, Xinrui Duan, Hexin Chen, Qian Wang

Faculty Publications

Cancer stem cells, also known as tumour-initiating cells (TICs), are identified as highly tumorigenic population within tumours and hypothesized to be main regulators in tumour growth, metastasis and relapse. Evidence also suggests that a tumour microenvironment plays a critical role in the development and progression of cancer, by constantly modulating cell–matrix interactions. Scientists have tried to characterize and identify the TIC population but the actual combination of extracellular components in deciphering the fate of TICs has not been explored. The basic unanswered question is the phenotypic stability of this TIC population in a tissue extracellular matrix setting. The in vivo …

Gas Phase Characterization Of Supramolecules Using Cross-Sectional Areas By Fticr And Sustained Off-Resonance Irradiation Collision Induced Dissociation Techniques In A Fourier Transform Ion Cyclotron Resonance Mass Spectrometer, Fan Yang

Theses and Dissertations

In my dissertation, I use a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR-MS) to investigate supramolecules. Cross-sectional areas by Fourier transform ICR (CRAFTI), a novel technique for measurements of collision cross sections by FTICR, is demonstrated for the first time. The CRAFTI method measures the total "dephasing cross section" for removal of the ions from the coherent packet in the FTICR cell, including contributions not only from momentum transfer but also from reactive collisions including those leading to collisional dissociation. Experimental CRAFTI collision cross sections correlate linearly with theoretically computed results and with results obtained using ion mobility measurements. …

Flow Valve Diagnostics For Label-Free, Quantitative Biomarker Detection: Device Fabrication, Surface Modification, And Testing, Danielle Scarlet Mansfield

Theses and Dissertations

Diseases are often diagnosed by detection of disease-specific biomarkers in fluid samples. However, many state-of-the-art detection methods require a lab with complex machinery, trained operators, and/or lengthy analysis time. In contrast, point-of-care (POC) devices are brought to the patient's location, they are easy to use, and results are obtained almost immediately. Many current POC devices are too difficult to be used without a skilled assistant, and although many are able to detect analytes above a threshold value, they give little or no quantitative information. This work presents the development of polymer-based microfluidic devices capable of sensing and quantifying biomarkers in …

Synthesis And Biological Evaluation Of Various Derivatives Of A Broad-Spectrum Anticancer Nucleoside, Jadd R. Shelton

Theses and Dissertations

Recently the Peterson lab discovered a promising anticancer adenosine derivative-- 2´,3´-bis-O-tert-butyldimethylsilyl-5´-deoxy-5´-[N-(methylcarbamoyl)amino]-N6-(N-phenylcarbamoyl)adenosine. This compound showed selective toxicity against human colon cancer cells in vitro with LC50's = 6--10 µM. It was hypothesized that the lead compound exerted its cytotoxic effects by interacting with a protein kinase. A systematic Structure Activity Relationship (SAR) was undertaken in an attempt to increase the kinase-binding affinity of the lead compound. Many regions of the lead compound were examined: the N6-phenyl urea moiety, the 5´-N-methyl urea group, the 2´,3´-bis-O-TBS groups, the nucleobase, and the ribose sugar. Results of these studies produced some promising new derivatives. In …

Understanding The Molecular Determinants Of Substrate And Inhibitor Specificities In The Carbapenemase Kpc-2: Exploring The Roles Of Arg220 And Glu276

Chemistry Faculty Publications

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions …

Part I - A Study Of The Formation Of Carbenes By Elimination Of Α-Bromosilanes And Application Toward The Synthesis Of Transition Metal Complexed Quinone Methide Analogs. Part Ii - Development Of Novel 7-Membered Ring Carbene Ligands For Palladium Catalyzed Cross Coupling Reactions., Christian Michael Loeschel

Graduate Theses and Dissertations

In part I, we wish to report our approaches toward transition metal complexed ortho-quinone methide analogs. ortho-Quinone Methides are a class of highly reactive compounds with a wide range of chemical and biological applications. Previously, a stable iron complexed benzannulated 5-membered ring quinone methide analog was reported by Allison and Neal27. Herein, we report our approaches to improve the reactivity of that system by removing benzannulation as well as changing the metal from iron to manganese and rhenium.

Furthermore, a methodological study on generating carbenes under mild conditions by elimination of α-halosilanes and its application towards metal complexed quinone methide …

Discrimination Of Colon Cancer Stem Cells Using Noncanonical Amino Acid, Xinrui Duan, Honglin Li, Hexin Chen, Qian Wang

Faculty Publications

Cancer stem cells (CSCs) may be responsible for tumor recurrence. Metabolic labelling of newly synthesized proteins with non-canonical amino acids allows us to discriminate CSCs in mixed populations due to the quiescent nature of these cells.

The Synthesis And Structural Characterization Of Metal Oxide Nanoparticles Having Catalytic Applications, Stacey Janel Smith

Theses and Dissertations

Nanotechnology is blossoming into one of the premiere technologies of this century, but the key to its progress lies in developing more efficient nanosynthesis methods. Variations in synthetic technique, however, can cause variations in size, structure, and surface characteristics, thereby altering the physical properties and functionality of the particles. Careful structural characterizations are thus essential for understanding the properties and appropriate applications for particles produced by new synthetic techniques.In this work, a new ‘solvent-deficient’ method is presented for the synthesis of an unprecedentedly wide range of metal oxide nanomaterials including at least one metal oxide from each group in Groups …

Identification Of Persistent Long Range Interactions In GA95 And GB95 Through Thermal Unfolding Simulations, Milen Redai Tesfamariam

Chemistry & Biochemistry Theses & Dissertations

For over five decades, different experiments have been performed to research how proteins attain their native three dimensional structures. However, the folding problem continues to be a puzzle in modern science. The design of two proteins that have maximal sequence identity but different folds and functions is one method that is being used to study the relationship between protein structure and amino acid sequence. In particular, mutant proteins of Streptococcus protein G, G_A and G_B, have 95% sequence identity and a 3a helix fold and β4/a fold, respectively. Molecular dynamics simulations of G_A95 …

Synthesis Of S-Ribosyl-L-Homocysteine And Analogs Modified At The Homocysteine-C3 Position, Ruoyi Liu

Master's Theses

Quorum sensing (QS) is a process of bacterial cell-to-cell communication that conveys population density information in order to coordinate gene expression to produce synchronized behaviors. QS regulates the expression of virulence genes in many species of bacteria; hence, the manipulation of QS pathways may lead to treatment options against many bacterial diseases. The LuxS enzyme converts S-ribosyl-L-homocysteine (SRH) into homocysteine (HCys) and 4(S),5-dihydroxypentane-2,3-dione (DPD), which is the precursor of autoinducer-2 (AI-2). Thus, inhibitors of LuxS could prevent QS by halting the conversion of SRH to AI-2 rendering the cell “uncommunicative”. This work shows the successful chemical synthesis …

Synthesis Of Fluorogenic Substrates For The Enzymatic Characterization Of Rv0045c From M. Tuberculosis, Kelly Jo Mckenna

Undergraduate Honors Thesis Collection

Mycobacterium tuberculosis is the pathogenic bacterial agent commonly responsible for tuberculosis, or TB. Although treatment exists for the active form of tuberculosis, no method has been developed for eliminating M tuberculosis in its dormant state. One hypothesized method for the elimination of dormant TB is to develop an inhibitor specific for M tuberculosis esterases and lipases, as these esterases and lipases are essential to the survival of dormant TB infection. In this research, the substrate specificity of the Rv0045c esterase from M tuberculosis was studied due to the essential role of Rv0045c in TB metabolism and its dissimilarity to other …

Refining Vibrationally-Resonant Sum Frequency Generation Spectroscopy For Studies Of Interfacial Interactions, Alexander D. Curtis

Theses and Dissertations

Many phenomena of interest to science and engineering occur at interfaces; however, access to, or discrimination of, interfacial interactions has been challenging, especially at buried interfaces. Vibrationally resonant sum-frequency generation (VR-SFG) spectroscopy is a powerful tool for investigating the molecular structure of free or buried interfaces, but spectral analysis has relied on many assumptions. To claim accurate new insights, practitioners must be able to make unique determinations from the data without experimental artifacts affecting the final results. For example, two independent and overlapping studies for the polystyrene/air interface were carried out, but reported different surface structures. Initially, this difference was …