Biochemistry Commons^™

Effects Of Localized Oxygen Production By Electrolysis On The First-Generation Glucose Sensor Response, Nandita Halder

Graduate Theses and Dissertations

Glucose sensors are very important for detecting blood glucose both in vitro and in vivo. First-generation glucose biosensors were based on the glucose oxidase (GOx) enzyme using molecular oxygen as the electron acceptor and therefore oxygen dependent. Unfortunately for in-vivo work, oxygen in the body is variable and limited. Alternative approaches to overcome the oxygen dependency came with their own limitations. The widely used and commercially available ex-vivo glucose test strip uses a mediator in place of oxygen to free it from oxygen dependency. The mediator-based technology, in most cases cannot be transferred to in vivo applications due to the …

Amyloidogenesis Of Β-2-Microglobulin Studied By Mass Spectrometry And Covalent Labeling, Blaise G. Arden

Doctoral Dissertations

Amyloid-forming proteins are implicated in a number of debilitating diseases. While many amyloid-forming proteins are well studied, the early stages of amyloidosis are still not well understood on a molecular level. Covalent labeling, combined with mass spectrometry (CL-MS), is uniquely well suited to provide molecular-level insight into the factors governing the early stages of amyloidosis. This dissertation leverages CL-MS techniques to examine the early stages of β-2-microglobulin (β2m) amyloidosis. β2m is the protein that forms amyloids in the condition known as dialysis-related amyloidosis. An automated CL-MS technique that uses dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide as a labeling reagent was developed and used …

Don't Sell Them Short, There's More To Bacterial Natural Products Than Antibiotics, Alison Clare Domzalski

Dissertations, Theses, and Capstone Projects

Recent genomic studies of microbiomes have revealed an overwhelming number of biosynthetic genes of unknown function. Most of these “cryptic” biosynthetic genes are not expressed in laboratory monocultures of individual microbes. Thus, there remains tremendous untapped potential for natural products discovery. Here we employ mixed microbial culture (MMC) as a simple yet powerful approach to awaken cryptic biosynthetic gene clusters. Our preliminary studies demonstrated that arrays of metabolites could be induced in MMCs upon environmental cues, such as surface adhesion. Using this system, we have screened, identified, and isolated bioactive bacterial metabolites, which were characterized structurally and biologically. Of the …

Proteomic Analysis Of Mycobacteriophage Crimd, William Moeller

Undergraduate Honors Theses

Bacteriophages represent a large portion of the biomatter on our planet, and many of them have yet to be fully characterized. Here we discuss the proteomic analysis of a particular Bacteriophage, Mycobacteriophage CrimD. This phage was discovered on the Campus of William & Mary and has had its genome characterized. We took the next logical step of proteomic analysis.

In our analyses we made use high pressure liquid chromatography paired with linear ion trap mass spectrometry to analyze the proteome of CrimD at specific time points after the infection of its host, Mycobacterium smegmatis. Additionally, we used nanospray ionization with …

Investigations Of The Structure And Protein-Protein Interactions Of Chlamydia Trachomatis Scc4, Thilini Oshadhi Senarath Ukwaththage

LSU Doctoral Dissertations

Chlamydia trachomatis (CT) is the most common, sexually transmitted bacterial disease (STD) in the world. In the developmental cycle of CT, specific chlamydia chaperone 4 (Scc4) is a unique protein with essential and multiple roles. Hence, Scc4 is significant as a virulence target for therapeutic approaches to treat chlamydial infections. A novel approach was discovered to purify tag free Scc4 by utilizing a 6X-histidine-tag on Scc1 in the co-expressed Scc4:Scc1 complex by capturing the complex on nickel-charged immobilized metal affinity chromatography resin, followed by dissociation of Scc4 with sarkosyl. Using triple resonance NMR experiments, backbone and sidechain resonances …

Using The Marcus Inverted Region And Artificial Cofactors To Create A Charge Separated State In De Novo Designed Proteins, Eskil Me Andersen

Dissertations, Theses, and Capstone Projects

To create an efficient de novo photosynthetic protein it is important to create long lived charge separated states. Achieving stable charge separation leads to an increase in the efficiency of the photosynthetic reaction which in turn leads to higher yields of end products, such as biofuels, electrical charge, or synthetic chemicals. In an attempt to create charge separated states in de novo proteins we hypothesized that we could engineer the free energy gaps in the proteins from excited primary donor (PD) to acceptor (A), and A back to ground state PD such that the forward electron transfer (ET) would be …

Mass Spectrometry-Based Strategies In Protein Higher Order Structure Analysis: Fundamentals And Applications In Protein-Ligand Interactions, Xiaoran Liu

Arts & Sciences Electronic Theses and Dissertations

Protein ligand interaction is a fundamental question in biology and biochemistry, and many approaches including X-ray crystallography, nuclear magnetic resonance, cryogenic electron microscopy, mass spectroscopy (MS), infrared spectroscopy, circular dichroism, fluorescence spectroscopy and many others have been applied to address this question. Among these techniques, mass spectroscopy has the advantage of high throughput, low sample amount requirement, and mid-to-high spatial resolution. One of the MS-based approaches is protein footprinting, which utilizes labeling reagents to map the solvent accessible surface of the protein of interest thus deliver structural information. Irreversible labeling is represented by covalent labeling and radical labeling, in which …

Flavin Modification And Redox Tuning In The Bifurcating Electron Transfer Flavoprotein From Rhodopseudomonas Palustris: Two Arginines With Different Roles, Nishya Mohamed-Raseek

Theses and Dissertations--Chemistry

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: …

Validation Of A Deployable Proteomic Assay For The Serological Screening Of Sexual Assault Samples, Catherine O'Sullivan Brown

Electronic Theses and Dissertations

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing …

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 …

Using Nmr Spectroscopy And Computational Chemistry To Confirm The Structure Of Novel Antibiotic Nocamycin O, Stephanie Lewis

CMC Senior Theses

In recent years, many medically promising antibiotics have been discovered in nature, especially in insect-microbe symbioses. One of the better-studied examples of this kind of defensive relationship is that of fungus-growing ants and the antibiotic-producing Actinobacteria. These bacteria produce several defensive chemicals with myriad uses, including one antibiotic that inhibits the growth of several bacterial strains, including other Actinobacteria. This antibiotic (known as nocamycin O) is a promising candidate for medicinal use due to its similarities to bacterial RNA polymerase inhibitors tirandamycin and streptolydigin, which inhibit several human pathogens. The determination of the structure of nocamycin O will be an …

High Throughput Analysis To Study Emerging Pollutants And Nanoparticle Fate In Biological Systems, Xiaolong He

Doctoral Dissertations

”The increasing applications of emerging and fugitive contaminants (EFCs) and engineered nanoparticles (ENPs) attract significant research interest for their potential risks to human health and the environment. In order to assess the health risks of these emerging contaminants, rapid and reliable analytical methods to measure the concentrations and fates of these contaminants are imperative. This dissertation focuses on the developments of advanced analytical methods and their applications to study those emerging contaminants in crop plant and simulated gastric fluid (SGF). Three types of mass spectrometry based methodologies have been developed, one is freeze-thaw/centrifugation extraction followed by high performance liquid chromatography …

The Quantitative Assessment Of Pond Scum: An Examination Of The Biogeochemistry Of Phosphorus Cycling In The Belgrade Lakes, Abbey M. Sykes

Honors Theses

The internal recycling phosphorus in freshwater lake bottom sediments represents a significant source of hypolimnetic phosphorus (P) release for many of Maine’s lakes. In summer months, Maine lakes often thermally stratify and the lake hypolimnion develops anoxia, leading to a reduction in redox potential at the sediment-water interface. These reducing conditions facilitate the reductive dissolution of ferric iron, and, since phosphorus is often present in freshwater lake sediments as solid FeOOH-PO₄ complexes, results in release of soluble phosphorus into the water column. Our current study presents field and laboratory data from sediment fractionation extractions designed to quantify concentrations of …

Lcms-Based Analysis Explains The Basis Of Oxidative Resistance In Selenium-Containing Thioredoxin Reductase, Daniel Haupt

Graduate College Dissertations and Theses

Selenocysteine (Sec) is referred to as the 21st proteogenic amino acid and is found in place of the redox-sensitive amino acid cysteine (Cys) in a small number of proteins. Sec and Cys carry out similar chemistry and are structural isomers save for a single atom difference; the former contains selenium (Se), while the latter contains sulfur (S) in the identical position. Sec poses a high bioenergetic cost for its synthesis and subsequent incorporation into protein not shared by Cys. Since Sec’s discovery in 1976, scientists have debated why certain proteins express Sec while others express Cys. In recent years, it …