Biochemistry, Biophysics, and Structural Biology Commons^™

Applications Of Epr With An Emphasis On Tau Fibril Structure, Virginia Meyer

Electronic Theses and Dissertations

Substances containing unpaired electrons have been studied by electron paramagnetic resonance (EPR) for nearly 70 years. With continual development and enhancement of EPR techniques, questions have arisen regarding optimum method selection for a given sample based on its properties. In this work, radiation defects, natural lattice defects, solid organic radicals, radicals in solution, and spin-labeled proteins were analyzed using CW, pulse, and rapid scan EPR to compare methods. Studies of solid BDPA, E' in quartz, N_s⁰ in diamond, and a-Si:H, showed that rapid scan could overcome many obstacles presented by other techniques, cementing rapid scan as an effective …

Identification And Characterization Of Human Plasma Prekallikrein-Prolylcarboxypeptidase Interaction Sites, Jingjing Wang

Electronic Theses and Dissertations

Prolylcarboxypeptidase isoform1 (PRCP1, also known as angiotensinase C, PCP, PRCP, PrCP) is a widely distributed serine protease found throughout the human body. PRCP1 removes the C-terminal amino acid which is next to a proline residue of a peptide. Through this activity, PRCP1 is postulated to play distinct biological roles including the regulation of vascular homeostasis, the induction of inflammation and the adjustment of metabolism. Compelling evidence indicates that human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells. However, the mechanism of this activation is yet unknown. The formation of kallikrein leads to the generation of proinflammatory factors and …

Purification And Characterization Of A Novel Selenocysteine Lyase From Enterococcus Faecalis, Samantha Nelson

Electronic Theses and Dissertations

A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a …

The Anti-Toxin Properties Of Grape Seed Phenolic Compounds, Patrick Cherubin

Electronic Theses and Dissertations

Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. …

Antimicrobial Peptide Resistance And Immunomodulation By Hiv-1 Gp41, Matthew Wood

Electronic Theses and Dissertations

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide to prevent sexual transmission of HIV-1. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors …

Evaluation Of Effects Of Epigenetic Modifications, Macromolecular Crowding, Degree Of Hydration, And Transcription Factor Binding On The Thermodynamic Stability Of Dna I-Motif Structures, Yogini Bhavsar

Electronic Theses and Dissertations

No abstract provided.

Evaluation Of Wild Yam (Dioscorea Villosa) As A Demethylating Agent And Its Anti Metastatic Activity In Human Breast Cancer Cells, Pranapda Aumsuwan

Electronic Theses and Dissertations

No abstract provided.

Characterization Of Prolylcarboxypeptidase Expression Pattern In Rat Cardiomyocytes In Nutrition Overload Conditions, Tahmineh Tabrizian

Electronic Theses and Dissertations

Prolylcarboxypeptidase (PRCP) is a serine protease that cleaves the last amino acid at the carboxy-terminus of peptides with a penultimate proline such as angiotensin II (Ang II), angiotensin III (Ang III), prekallikrein (PK) and α-melanocyte-stimulating-hormone (1-13) (α-MSH1-13). By inactivating Ang II and Ang III, PRCP promotes vasodilation and insulin sensitivity. As well by activating PK, PRCP increases nitric oxide (NO) generation and vasorelaxation through bradykinin (BK) liberation from high molecular weight kininogen (HK). PK is activated to kallikrein by PRCP, implicating a role for PRCP in the coagulation pathway. The resultant kallikrein will activate FXII which in a reciprocal manner …

Role Of Cardiac Catecholamines In Embryos And Adults Under Stress, Candice Baker

Electronic Theses and Dissertations

Cardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including congenital malformations, arrhythmias, ischemic heart disease, heart failure, and sudden cardiac death. Despite intensive study, the specific roles these hormones play in the developing heart is not well-understood. Further, there is …

In Vitro Characterization Of Unmodified And Pyroglutamylated Alzheimer's Amyloid Beta Peptide, Jason Matos

Electronic Theses and Dissertations

Plaques of amyloid β peptide (Aβ) are a hallmark trait of Alzheimer’s disease (AD). However, the precise role of Aβ aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of Aβ significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated Aβ (pE-Aβ) in AD brains has been shown to reach up to 50% of total Aβ. Modified pE-Aβ co-aggregates with Aβ by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length Aβ1-42, its pyroglutamylated form AβpE3-42, their 9:1 (Aβ1-42/AβpE3-42) and 1:1 molar …

Establishment Of Methods For Isolation Of Pnmt+ Cardiac Progenitor Cells, Namita Varudkar

Electronic Theses and Dissertations

Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitor cells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful …

Genetic And Biochemical Characterization Of The Roles Of Two Putative Purine Transporters In The Infectious Cycle Of Borrelia Burgdorferi, Sunny Jain

Electronic Theses and Dissertations

Lyme disease, the most common tick borne disease in United States, is caused by the bacterial pathogen Borrelia burgdorferi. In nature, B. burgdorferi exists in an enzootic infectious cycle between an arthropod vector and mammalian hosts. Identification and characterization of the genes essential for B. burgdorferi survival throughout its infectious cycle is an important step toward understanding the molecular mechanisms involved in B. burgdorferi pathogenesis. B. burgdorferi contains a small genome, which lacks the genes encoding for the enzymes required for de novo synthesis of amino acids, fatty acids and nucleic acid precursors. Therefore, the spirochete is dependent upon the …

Validation Of A Novel Hypothesis Of Generating Foam Cells By Its Use To Study Reverse Cholesterol Transport, Bhaswati Sengupta

Electronic Theses and Dissertations

Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month storage. Macrophages incubated …

Cerium Oxide Nanoparticles: Their Phosphatase Activity And Its Control, Atul Dhall

Electronic Theses and Dissertations

Cerium oxide nanoparticles are established scavengers of reactive oxygen and nitrogen species. They have many potential biomedical applications that depend on their physicochemical properties and mode of preparation. Recent studies have found these nanoparticles possess phosphatase mimetic activity. Studying such catalytic activities will qualify their biomedical applications and render information on their bioavailability and potential toxicity. Two oxidation states of cerium exist in these nanoparticles (3+ or 4+). It is hypothesized that the oxidation state of cerium in the nanoparticles determines the amount of adsorbed water on the crystal lattices. This in turn governs their activity as phosphatases. Nanoparticles with …

Modulation Of Cholera Toxin Structure And Function By Host Proteins, Helen Burress

Electronic Theses and Dissertations

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 …

An Examination Of The Inhibitory Effects Of Antibiotic Combinations On Ribosome Biosynthesis In Staphylococcus Aureus, Justin Beach

Electronic Theses and Dissertations

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis.

To determine this we measured the effects of single and multiple antibiotics on cell growth …

New Insights Into The Roles Of Human Dna Damage Checkpoint Protein Atr In The Regulation Of Nucleotide Excision Repair And Dna Damage-Induced Cell Death, Zhengke Li

Electronic Theses and Dissertations

Integrity of the human genome is frequently threatened by endogenous and exogenous DNA damaging reagents that may lead to genome instability and cancer. Cells have evolved multiple mechanisms to repair DNA damage or to eliminate the damaged cells beyond repair and to prevent diverse diseases. Among these are ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage checkpoint and nucleotide excision repair (NER) that are the major pathways by which cells handle ultraviolet C (UV-C)- or other exogenous genotoxin-induced bulky DNA damage. However, it is unclear how these 2 pathways may be coordinated. In this study we show that ATR physically interacts …

Mechanisms Of The Anti-Pneumococcal Function Of C-Reactive Protein, Toh B. Gang

Electronic Theses and Dissertations

Human C-reactive protein (CRP) increases survival of and decreases bacteremia in mice infected with Streptococcus pneumoniae. Such protection of mice against pneumococcal infection is seen only when CRP is administered into mice 6 hours before to 2 hours after the injection of pneumococci, but not when CRP is given to mice at a later time. Our first aim was to define the mechanism of CRP-mediated initial protection of mice against infection. It was proposed that CRP binds to phosphocholine (PCh) moieties present in the cell wall and activates the complement system on the pneumococcal surface that kills the pathogen. …

Development Of A Biosensor For Investigating Membrane Curvature Sorting, Joshua C. Black

Electronic Theses and Dissertations

The physical structure of cellular membranes plays a critical role in lipid and protein sorting. A novel biosensor was developed to probe the influence of curvature on sorting. This biosensor mimics large, two-dimensional membranes in dynamic equilibrium, achieves high spatial resolution between curvature and molecules of interest, and has high sensitivity, enough for single particle detection. The biosensor consists of continuous supported lipid bilayer formed over nanoparticles (40 to 200 nm diameter) deposited on a glass substrate. The nanoparticles determine the extent of curvature. This biosensor is the first to observe large-scale 2-dimensional diffusion of biomolecules on a supported lipid …

Bioengineering The Expression Of Active Recombinant Human Cathepsin G, Enteropeptidase, Neutrophil Elastase, And C-Reactive Protein In Yeast, Eliot T. Smith

Electronic Theses and Dissertations

The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human …

Novel Roles Of Replication Protein A Phosphorylation In Cellular Response To Dna Damage, Moises A. Serrano

Electronic Theses and Dissertations

Human replication protein A (RPA) is an eukaryotic single-stranded DNA binding protein directly involved in a variety of DNA metabolic pathways including replication, recombination, DNA damage checkpoints and signaling, as well as all DNA repair pathways. This project presents 2 novel roles of RPA in the cellular response to DNA damage. The first elucidates the regulation of RPA and p53 interaction by DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) in homologous recombination (HR). HR and nonhomologous end joining (NHEJ) are 2 distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-PK, the …

Cholera Toxin Activates The Unfolded Protein Response Through An Adenylate Cyclase-Independent Mechanism, Neyda Vanbennekom

Electronic Theses and Dissertations

Cholera toxin (CT) is a bacterial protein toxin responsible for the gastrointestinal disease known as cholera. CT stimulates its own entry into intestinal cells after binding to cell surface receptors. Once internalized, CT is delivered via vesicle-mediated transport to the endoplasmic reticulum (ER), where the CTA1 subunit dissociates from the rest of the toxin and is exported (or translocated) into the cytosol. CTA1 translocates from the ER lumen into the host cytosol by exploiting a host quality control mechanism called ER-associated degradation (ERAD) that facilitates the translocation of misfolded proteins into the cytosol for degradation. Cytosolic CTA1, however, escapes this …

Bone Morphogenetic Protein-7 Attenuates Inflammation And Apoptosis And Improves Cardiac Function In Diabetes, Princess Urbina

Electronic Theses and Dissertations

Bone Morphogenetic Protein-7 (BMP-7) belongs to the transforming growth factor-β (TGFβ) family of cytokines has is known to have potent anti-inflammatory properties. It has been used in patients to treat osteoporosis clinically and has been reported to treat diabetic nephropathy in murine models. Moreover, studies show that inflammation is up-regulated in patients with pre-diabetes (PD). We, therefore, hypothesize that the administration of BMP-7 will attenuate inflammation in the heart of Streptozotocin (STZ)-induced PD mice. In this study, we divided C57Bl/6 mice into three groups: CONTROL, PD, and PD+BMP-7. CONTROL mice received intraperitoneal (i.p.) injections of Sodium Citrate Buffer while PD …

Regulation Of Actin Dynamics During Drosophila Germband Extension, Ashley M. Motlong

Electronic Theses and Dissertations

Convergent extension is a process that occurs in the development of a wide variety of organisms, including gastrulation in the Drosophila embryo to begin to lay out the adult body plan. In fly embryos, this is known as germband extension and is mainly driven by cell intercalation or neighbor exchange by planar polarized cell-cell interface contraction to shorten the tissue along the dorsal-ventral axis. In this thesis, I show that interface contraction consists of phases of fast interface shortening and intervals of stable interface size. My data also suggests that regulation of F-actin aggregates at these shrinking interfaces is important …

X-Band Rapid-Scan Epr, Deborah Gale Mitchell

Electronic Theses and Dissertations

The advantages of rapid-scan EPR relative to CW and pulse techniques for samples with long longitudinal relaxation time T₁ (N_s⁰ defects in diamond, N@C₆₀, and amorphous hydrogenated silicon), heterogeneous samples (crystalline 1:1 α,γ-bisdiphenylene-β-phenylallyl (BDPA):benzene), lossy samples (aqueous nitroxyl radicals), and transient radicals (5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)-superoxide adduct) were studied.

For samples with long relaxation times, CW (continuous wave) EPR is challenging due to power saturation and distortions from passage effects. In rapid-scan EPR, the field is swept through resonance in a time that is short relative to T₂. In rapid-scan EPR, the magnetic field is …

Biophysical Approaches To Human Genetic Disease: Adsl Deficiency As A Model, Stephen P. Ray

Electronic Theses and Dissertations

Biophysical tools are becoming more useful in approaching human genetic disease. The pathogenic mechanisms behind many disorders have been attributed to protein loss of function or gain in toxic function. For example, changes in the protein Adenylosuccinate Lyase (ADSL) lead to ADSL deficiency, a disorder that causes symptoms ranging from epilepsy to expression of autistic features. Biophysics offers different tools to study intrinsic properties of proteins. We have applied such tools to study the enzyme kinetics and protein stability of ADSL and two mutations (R426H and R303C) to formulate better hypotheses regarding the pathogenic mechanism. Enzyme kinetic findings indicate a) …

Screening Of Quantum Dots For Toxicity On The Growth And Viability Of Escherichia Coli, Jeremy Tharkur

Electronic Theses and Dissertations

Heavy metal (HM) containing quantum dots (Qdots) are increasingly used in commercial products due to their unique electronic, optoelectronic, optical and magnetic properties. Once disposed to the landfill, environmental weathering is likely to compromise HM Qdot integrity, leading to release of heavy metal ions. To minimize any negative environmental impact of HM Qdots, there is an increasing demand for developing HM free or environmentally-friendly surface modified HM Qdot alternatives. In this study, synthesis of HM free ZnS:Mn/ZnS and surface modified HM CdS:Mn/ZnS Qdots (using N-acetylcysteine, NAC, and Dihydrolipoic acid, DHLA) and their potential toxicity assessment using E. coli as a …

Molecular Considerations In The Design Of Novel Alpha/Beta Hydrolase Inhibitors, Shana Victoria Stoddard

Electronic Theses and Dissertations

Alpha/beta hydrolases (ABHs) are a superfamily of hydrolytic enzymes that process a wide variety of substrates. A subfamily of ABHs called carboxylesterases (CEs) are important enzymes that catalyze biological detoxification, hydrolysis of certain pesticides, and metabolism of many esterified drugs. The chemotherapy drug irinotecan used for treatment of colorectal cancer is metabolized to SN-38, the active drug metabolite, by two CE isozymes CES1 (localized in the liver) and CES2 (localized in the small intestines). CES2's ability to activate irinotecan at a faster rate than CES1 creates a localization of activated SN-38 in the gut epithelium, resulting in the dose limiting …

Role Of The Endocannabinoid System In Cardiac Remodeling, Monica Gole

Electronic Theses and Dissertations

The endocannabinoid system and CB1 and CB2 cannabinoid receptors have been implicated in cardiac remodeling with CB1 inhibition and CB2 activation reported to be beneficial. However, the role of these receptors in cardiac remodeling secondary to hypertension is not known. Moreover, the functions of the CB1 and CB2 cannabinoid receptors in cardiac ventricular fibroblasts and their downstream fibrotic pathways has not yet been elucidated. This study was designed to determine the temporal myocardial expression pattern of CB1 and CB2 receptors in cardiac remodeling secondary to pressure overload (PO) induced hypertension at 3, 5, 14 and 28 days. The expression of …

Target Validation For Neurofibromatosis Type 2 Therapeutics., Alejandra Guinart

Electronic Theses and Dissertations

Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets. The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. …