Biochemistry, Biophysics, and Structural Biology Commons^™

Biochemical Investigations Of Macular Degeneration: The Significance Of Protein Oxidation Including Novel Methods For Its Study, Sarah Warburton

Theses and Dissertations

The retinal pigment epithelium (RPE) is a monolayer of cells located directly behind the photoreceptor cells in the retina. These cells are involved in a variety of functions that support the visual process in the eye, namely 1) they form a blood-retina barrier which separates the neural retina from the choroid's blood supply, 2) the apical processes of RPE cells diurnally phagocytose the outer segments of photoreceptor cells, and 3) they participate in the renewal of the photopigment 11-cis retinal. Age-related macular degneration (AMD) is the leading cause of blindness in people over the age of 50 years in North …

Fabrication Of Polymeric Microfluidic Devices For Protein Analysis, Jikun Liu

Theses and Dissertations

2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA micro capillary electrophoresis (µCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins. Both column efficiency and migration time reproducibility were one order of magnitude better with derivatized PMMA µCE devices compared to untreated versions. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA µCE chips. All analyses were completed in less than 60 seconds, and …

In Vitro Expression And Purification Of Class I Mhc Molecules, Loi Cheng

Honors Scholar Theses

The major histocompatibility complex (MHC) is a gene family responsible for many critical functions of the immune system in most vertebrates. The MHC consists of three classes differentiated by their structure and function, and MHC class I encodes antigen binding proteins as well as chaperone and accessory proteins such as tapasin. The purpose of this project is to reconstitute several human MHC class I molecules in their peptide-filled and peptide-deficient forms, and to purify these proteins for biochemical study. The expressed proteins include wild type and mutant variants of the fusion protein human leukocyte antigen HLA-B*0801-fos, and human beta-2-microglobulin (β2m). …

Membrane-Based Protein Preconcentration Microfluidic Devices, Yi Li

Theses and Dissertations

Interest in microchip capillary electrophoresis (CE) is growing due to the rapid analysis times provided and small sample input requirements. However, higher-concentration samples are typically needed because of the small (~pL) detection volumes in these devices. I have made membrane-based protein preconcentration systems in capillary and microchip designs to increase the detectability of low-concentration biological samples. A photopolymerized ion-permeable membrane interfaced with a microchannel in poly(methyl methacrylate) (PMMA) formed the preconcentrator. When a voltage was applied between the sample reservoir and the ionically conductive membrane in a capillary-based system, R-phycoerythrin was concentrated more than 1,000 fold, as determined by laser-induced …

Identification Of The Pla2 Responsible For Prostanoid Synthesis In Response To Inflammatory Cytokines, Chaminda Fernando

Theses and Dissertations

Preliminary studies from our laboratory showed that cPLA2α may be responsible for approximately 50-60% of the PGE2 production in response to inflammatory cytokines. Thus, we hypothesized that a closely-related PLA2 is responsible for 40-50% of the PGE2 produced in response to inflammatory cytokines. To this end, we utilized RNAi technology, extensively optimized, to down regulate the expression of closely-related isoforms of phospholipase A2 in A549 cells and used an enzyme linked immuno sorbent assay (ELISA) to quantitate the PGE2 produced. These studies found that cytosolic phospholipase A2α (cPLA2α) regulated 97.7% of the prostaglandin E2 (PGE2) produced in response to inflammatory …

Characterization Of 50s Ribosomal Subunit Assembly Inhibition In Erythromycin Treated Escherichia Coli Cells., Jerry Edward Usary

Electronic Theses and Dissertations

Erythromycin has long been recognized for its ability to inhibit protein synthesis by interfering with mRNA translation on the bacterial ribosome. We have recently shown that erythromycin also inhibits the assembly of the 50S ribosomal subunit in growing bacterial cells. The nature of this assembly inhibition has been investigated using ³H-uridine pulse-chase labeling of control and erythromycin treated E. coli cells.

Subunit assembly was examined by sucrose gradient centrifugation of labeled cell lysates. Normal assembly kinetics of subunit assembly were observed in control cells at 37°C. Formation of the 30S subunit was completed by 7.5 minutes and assembly of …

Dietary Protein Deficiency And Mycobacterium Bovis Bcg Affect Interleukin-2 Activity In Experimental Pulmonary Tuberculosis, David N. Mcmurray, C. L. Mintzer, R. A. Bartow, Rebecca D. Parr

Faculty Publications

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated. They were maintained for 6 weeks on defined, isocaloric diets containing either 30% (control animals) or 10% (animals receiving low protein) ovalbumin as the sole protein source. Animals were challenged by the respiratory route with a low dose of virulent M. tuberculosis H37Rv and killed 4 weeks later. Protein-malnourished animals were not protected by previous vaccination with BCG. Lymphocytes isolated from various tissues were tested in vitro for proliferative responses to mitogen (concanavalin A) and antigen (purified protein derivative [PPD]), production of interleukin-2 (IL-2), and …

Acetylation Of Synaptosomal Protein: Effect Of Na+ / Arlene Colon, Soll Berl, And Donald D. Clarke, Arlene D. Colon, Soll Berl, Donald Dudley Clarke Phd

Chemistry Faculty Publications

In a previous study it was shown that the acetyl moiety can be incorporated into the protein of purified synaptosomes (1). This process was inhibited by veratridine and the inhibitory effect was counteracted by tetrodotoxin. This suggested that the flux ofNa +may be related to the acetylation process. We now report that in a sodium free medium the amount of acetylation is increased and the inhibitory effect of veratridine (veratrine) is no longer evident. The addition ofNa +leads to a decrease in acetylation in the presence of veratrine. The presence of scorpion toxin has an effect similar to that of …

The Isolation And Characterization Of A Novel Iron Protein From Desulfovibrio Gigas, Alan Kay Smith

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. This protein was treated with acid, chelating agents, detergents and proteases in order to study their effect on the iron solubility. The results of elemental analysis of a crude protein preparation were 5.3% iron, 2.9% sulfur and 11 .9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be …