Biochemistry Commons^™

Development Of Polymer Monoliths For The Analysis Of Peptides And Proteins, Binghe Gu

Theses and Dissertations

Several novel polymer monoliths for the analysis of peptides and proteins were synthesized using polyethylene glycol diacrylate (PEGDA) as crosslinker. Photo-initiated copolymerization of polyethylene glycol methyl ether acrylate and PEGDA yielded an inert monolith that could be used for size exclusion liquid chromatography of peptides and proteins. This macroscopically uniform monolith did not shrink or swell in either water or tetrahydrofuran. More importantly, it was found to resist adsorption of both acidic and basic proteins in aqueous buffer without any organic solvent additives. A strong cation-exchange polymer monolith was synthesized by copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and PEGDA. A ternary …

Synthesis And Optical Properties Of Four Oligothiophene-Ruthenium Complexes And Synthesis Of A Bidentate Ligand For C-F Bond Activation, Joseph S. Bair

Theses and Dissertations

Photovoltaic cells and fluorescence sensing are two important areas of research in chemistry. The combination of photon-activated electron donors with electron acceptors provides a strong platform for the study of optical devices. A series of four oligothiophene-ruthenium complexes has been synthesized. Variation in oligothiophene length and bipyridine substitution allowed comparison of these variables on electronic properties. The longer oligothiophenes display lower energy absorption and emission compared to the shorter ones. Aromatic conjugation appears more complete with para-, rather than meta-, substitution. Oligothiophenes and Ru(bpy)32+ are highly fluorescent individually, but fluorescence is quenched when connected. Bonds of carbon to fluorine are …

Engineering Calpastatin To Develop A Sensor To Detect Active Calpain, Lisa M. Vanhooser

Electronic Theses and Dissertations

Calpains, Ca²⁺-activated cysteine proteases are essential for early embryonic development and function in signal transduction, cell adhesion, and apoptosis. Calpains also contribute to cataractogenesis, myocardial infarctions, and neurodegenerative diseases such as Alzheimer's. The various methods currently available to demonstrate these roles do not directly identify spatial or temporal activation of calpain in cells. Therefore, a tool to detect active calpain in situ will be useful. Calpastatin is the ubiquitous, endogenous inhibitor that specifically binds the active conformation of the conventional calpains. Calpastatin consists of four homologous domains each containing three subdomains A, B, and C. The crystal structure …

Development Of Methods For The Study Of Phosphoproteins, Zhaoyuan Chen

Theses and Dissertations

Characterization of phosphoproteins-including detection, identification of phosphoproteins and identification of phosphorylation sites-is mostly done with radiolabeling and proteomic techniques. Three main topics related to phosphoprotein characterization are included in this dissertation. First, large-scale characterization of the CHO (Chinese hamster ovary) cell phosphoproteome was done using two dimensional gel electrophoresis (2DE) separation, visualization of phosphoproteins by radiolabeling or a phosphoprotein specific dye, followed by MALDI-TOF identification. Because radiolabeling of phosphoproteins is very sensitive and straightforward to quantify, such analysis can give a clear picture of the relative phosphosphorylation of proteins present in a sample. But there are also limitations to this …

Capillary Electrophoresis Of Proteins With Selective On-Line Affinity Monoliths, Jenny Marcela Armenta Blanco

Theses and Dissertations

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, an on-line protein G monolithic preconcentrator CE system for enrichment and separation …

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 …

Characterization Of Cucurbituril Complex Ions In The Gas Phase Using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Haizhen Zhang

Theses and Dissertations

Host-guest interactions have been well studied in the new century to obtain fundamental insights into supramolecular chemistry. Most of the pioneering works have been done using techniques such as NMR, X-ray crystallography, IR spectroscopy and so on. However, none of these techniques is universal for the investigation of all types of supramolecules, and usually they have one or more limiting factors such as relatively large sample consumption, matrix effects from solvents, etc. Electrospray mass spectrometry has been widely used to investigate host-guest interactions in the gas phase. A particular advantage of gas phase host-guest research is that the experimental results …

Microfabrication And Evaluation Of Planar Thin-Film Microfluidic Devices, Bridget Ann Peeni

Theses and Dissertations

Over the past 15 years, research in the field of microfluidics has rapidly gained popularity. By seeking to miniaturize and automate separation-based analysis, microfluidic research seeks to improve current methods through decreased cost, analysis time, and sources of contamination. My work has focused on developing a novel fabrication method, based on standard microfabrication techniques, to create thin-film microfluidic devices. This microfabrication format makes it possible to generate devices that provide high efficiencies, enable mass fabrication, and provide a platform capable of integrating the microfluidic and electronic components necessary for a micro-total analysis system (μ-TAS). Device fabrication combines the processes of …

Detection Of Proteins By Two-Photon Excitation Of Native Fluorescence, Li Li

Theses and Dissertations

Proteins are of primary importance to the structure and function of all living cells. Study of proteins relies on the ability to separate a complex mixture so that individual proteins can be more easily processed by other techniques. Since protein samples often exist at low concentration in a small volume, the trend in chemical analysis is toward micro total analysis systems (µTAS) or lab-on-a-chip devices. Among µTAS separation methods, the relatively new electric field gradient focusing (EFGF) technique has shown potential. It focuses and separates analytes based on their electrophoretic migration in an opposing hydrodynamic flow. The detection principles that …

Ferritin: Mechanistic Studies And Electron Transfer Properties, Bo Zhang

Theses and Dissertations

Ferritins are ubiquitous iron storage proteins in living systems. Although much is known about the iron deposition process in ferritin and a mechanism has been developed, several important issues still remain unknown. One lingering question is the less than stoichiometric quantities of hydrogen peroxide detected in previous studies on animal ferritins. Extensive experimental data on identifying the species in competition for peroxide equivalents point to a surprising conclusion that H2O2 generated in the ferroxidase reaction is consumed by amine buffers that are commonly employed in in vitro ferritin studies, while non-nitrogen containing buffers, such as acetate, phosphate, and carbonate, do …

Semi-Global Analysis Of The Early Cold Stress Response Transcriptome Of Developing Seedlings Of Rice (Oryzasativa L.,Japonica), Chen Cheng

Electronic Theses and Dissertations

Plants are either sensitive or insensitive to low temperatures. Cool-season species acclimate to chilling and develop tolerance to freezing. Warm-season species vary in the degree of sensitivity to chilling and are not capable of acclimation. Both freezing and chilling tolerance involve the activity of an intricately coordinated network of genes. The regulatory network that defines cold acclimation and freezing tolerance is well understood in Arabidopsis. The centerpiece of this network is a group of transcriptional activators that coordinate a battery of downstream defense-related genes. In contrast, little is known about the corresponding regulon in non-acclimating species. This study utilized …

Structure-Activity Studies Of Glycosphingolipids As Antigens Of Natural Killer T Cells, Randal Donald Goff

Theses and Dissertations

Glycosphingolipids (GSLs), composed of a polar saccharide head and a lipophilic ceramide tail, are ubiquitous components of the plasma membrane of eukaryotic cells. They serve in many regulatory capacities and have antigenic properties towards natural killer T (NKT) cells of the innate immune system. Critical to the recognition of glycosylceramides by NKT cells are antigen presenting cells (APC), such as dendritic cells, which are responsible for binding, processing, and delivery of ligands to these lymphocytes. This event is mediated by CD1d, a major histocompatibility complex-like protein expressed on the surface of APCs, which binds GSL antigens by the ceramide moiety …

Dna-Templated Surface Alignment And Characterization Of Carbon Nanotubes., Huijun Xin

Theses and Dissertations

Carbon nanotubes are appealing materials for nanofabrication due to their unique properties and structures. However, for carbon nanotubes to be used in mass-fabricated devices, precise control of nanotube orientation and location on surfaces is critical. I have developed a technique to align single-walled carbon nanotubes (SWNTs) on surfaces from a droplet of nanotube suspension under gas flow. Fluid motion studies indicate that alignment is likely due to circulation of SWNTs in the droplet. My work provides a facile method for generating oriented nanotubes for nanodevice applications. I have also devised an approach for localizing SWNTs onto 1-pyrenemethylamine-decorated DNA on surfaces. …

Enhanced Electrospray Ionization For Mass Spectrometry And Ion Mobility Spectrometry, Li Zhou

Theses and Dissertations

Electrospray ionization (ESI) has become one of the most commonly used ionization techniques for mass spectrometry (MS) and ion mobility spectrometry (IMS), and efforts continue to improve its performance. ESI-MS is most recognized for its wide application to biomacromolecules where high sensitivity is of paramount importance. However, the major limitation in sensitivity with ESI-MS is due to its low ion transmission efficiency from the ESI source into the sampling orifice and through any stages utilized for transfer of ions from atmosphere to vacuum in the MS. A series of atmospheric pressure ion focusing interfaces were designed and implemented to enhance …

Electric Field Gradient Focusing-Uv Detection For Protein Analysis, Shu-Ling Lin

Theses and Dissertations

Electric field gradient focusing (EFGF) utilizes a hydrodynamic flow and an electric field gradient to focus and concentrate charged analytes and order them in a separation channel according to electrophoretic mobility. Elution can be achieved by decreasing the applied voltage or increasing the hydrodynamic flow. EFGF has the advantages of concentrating a large volume (100 micro-L) of target proteins without significant band broadening and simultaneously removing unwanted components from the sample. Two types of EFGF devices have been investigated to concentrate and separate proteins: a fiber-based EFGF device and a hydrogel-based EFGF device. Using fiber-based EFGF with UV detection, a …

Chemical Evidence For Dietary Toxin Sequestration In The Asian Snake Rhabdophis Tigrinus, Deborah A. Hutchinson

Biological Sciences Theses & Dissertations

Rhabdophis tigrinus (Colubridae: Natricinae) is an oviparous, bufophagous (toad-eating) snake from eastern Asia that possesses defensive integumentary glands on the neck known as nuchal glands. These glands are used in antipredator displays and typically contain bufadienolide toxins. Whereas toads are known to synthesize bufadienolide steroids from cholesterol precursors, we found that chemically undefended R. tigrinus must sequester bufadienolides from ingested toads in order to exhibit these compounds in their nuchal glands. Chemically defended females are capable of provisioning their embryos with these toxins so their unfed hatchlings possess defensive bufadienolides prior to consuming toads themselves. All of the hatchling R. …

Model Studies Towards The Total Synthesis Of Lyconadin A Via An Acyl Radical Cascade Reaction, Koudi Zhu

Theses and Dissertations

Lyconadin A is an alkaloid possessing a unique structure and antitumor activity. The total synthesis of Lyconadin A was proposed via an acyl radical cascade reaction. To investigate the possibility and stereoselectivity of the cascade cyclization, phenyl selenoester 16 was chosen as a model substrate to study the 7-exo-5-exo radical cyclization. A synthetic route to phenyl selenoester 16 was developed. The 7-exo-5-exo radical cyclization was found to occur with a high yield and excellent stereoselectivty. Attempts were also tried to synthesize another radical precursor 14 albeit with less success. A synthetic pathway to the synthesis of 14 as well as …

The Mechanism Of Assembly Of The G-Protein Beta Gamma Subunit Dimer By Ck2 Phosphorylated Phosducin-Like Protein And The Chaperonin Containing Tcp-1, Christine M. Baker

Theses and Dissertations

Phosducin-like protein (PhLP) binds G-protein beta gamma subunits and is thought to assist in assembly of the G-protein beta gamma dimer. Phosphorylation of PhLP at serine residues 18-20 by the casein kinase 2 (CK2) appears to play an essential role in this process. PhLP has also been shown to interact with the chaperonin containing TCP-1 (CCT) atop its apical domain, not entering the substrate folding cavity. However, the physiological role of the PhLP-CCT interaction in G-protein beta gamma dimer formation remains unclear. This study addresses the mechanism of G-protein beta gamma assembly by exploring the specific roles of CCT and …

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 …

Membrane Organization And Multimeric Interactions Of The Aer Receptor In E. Coli, Divya Dashrathkumar Amin

Loma Linda University Electronic Theses, Dissertations & Projects

Escherichia coli swims toward niches ideal for generating maximum energy by monitoring the environment with the aerotaxis sensor, Aer. This receptor has an N-terminal PAS domain, which is a sensor module present in a variety of proteins from all kingdoms of life (including humans). To date, the mechanism of signaling from a PAS sensor domain to a single signaling receiver domain has not been resolved. In Aer, the PAS sensor is separated from the signaling region by a membrane anchor. Most "transmembrane prediction" programs forecast a single membrane span for this membrane anchor, but such a topology is inconsistent our …

The Role Of Trailerhitch In Mouse Germ Cell Development, Ashley L. O'Hara

Honors Capstone Projects - All

During oogenesis in the Drosophila ovary, a 16-cell cyst develops in which one cell grows to become the oocyte. The surrounding nurse cells transport mRNAs, proteins, and organelles to the oocyte during development and must be localized properly within the oocyte. A complex of proteins is responsible for mRNA localization, including the recently discovered protein Trailerhitch. Homologs of Trailerhitch exist in other species including C. elegans, yeast, mice, and humans, but the function is unknown. We are interested in studying the function of Trailerhitch in mouse germ cell development. Mice with a nonfunctional Trailerhitch gene were synthesized using a gene …

The Response Of A Biocompatible Liquid Crystal To Ions And Chemistry On Nanostructured Surfaces, Anny Huang

Honors Capstone Projects - All

Traditional lyotropic liquid crystals are composed of amphiphilic molecules forming assemblies in water. Disodium cromoglycate (cromolyn or DSCG) – an antiasthmatic drug – is a novel and unusual lyotropic liquid crystal because it is not amphiphilic, and yet, it exhibits large birefringence (shininess) when dissolved in water forming lyotropic liquid crystal. In the work, DSCG liquid crystal is doped with different types of salt such as sodium chloride and sodium perchlorate, and the response in the changes of birefringence and liquid crystal transition temperature is studied. We find that addition of certain type of salts enhance the propensity of formation …

A Sensitive Subject: Haploinsufficiency Of The Yeast Vma13 Gene In Simple And Compound Heterozygotes, Jason Michael Rizzo

Honors Capstone Projects - All

Vacuolar-proton translocating-ATPases (V-ATPases) are the membranebound transporters responsible for controlling pH levels in intracellular compartments. The V-ATPase enzyme is a multi-subunit complex consisting of two distinct domains, a cytosolic V1 region and a membrane-bound Vo domain. While the interface between V1 and Vo is not as well understood, studies suggest the possibility of multiple peripheral stalks tethering the complex. In response to glucose deprivation, the V1 and Vo sectors of the enzyme are capable of dissociating into separate, and inactive, domains as a means of regulation. At least 4 V1 subunits (C, H, E, and G) are believed to form …

Structure-Function Analysis Of Nrage: A Protein Involved In Developmental Neural Apoptosis, Rebecca Cowling

Electronic Theses and Dissertations

The Neurotrophins and Bone Morphogenic Proteins (BMP) have both been implicated in the maintenance of cellular proliferation and apoptosis in the developing nervous system. Downstream of both signaling pathways is NRAGE, a member of the Melanoma Antigen (MAGE) gene family and, under BMP stimulation, activates p38 leading to caspase 3 cleavage. NRAGE possesses a highly conserved MAGE Homology Domain (MHD) and a second, less well conserved MHD (denoted MHD2) as well as a unique 25 tandem WQXPXX hexapeptide repeat region. Binding partners for the MHD and hexapeptide repeat regions have been identified but their cellular consequences have not been defined. …

Total Synthesis Of Pumiliotoxin 341a, Lydia Choi

Honors Capstone Projects - All

Pumiliotoxin 341 A differs from all other pumiliotoxins in that its alkylidene side chain has been cyclized to form a pyranose ring, providing another quaternary center in its indolizidine ring. Although the gross structure of this natural product has been determined, the absolute and relative stereochemistry of the tertiary alcohol on C14 is still unknown. As a result, we want to synthesize this alkaloid to prove its stereochemistry and to prepare material for further biological analysis. A rhodium-catalyzed alkyne addition reaction currently being developed in our group is employed as the key reaction in our synthesis of the indolizidine core. …

The Fate Of Iron Released From Heme By Hemeoxygenase-1, Jonathan Mark Gardner

Undergraduate Honors Capstone Projects

A strain of Escherichia coli was genetically modified to co-express human heme oxygenase-1 and ferritin. The E. coli were then grown with varying amounts of hemin to see if the iron released upon degradation of the hemin by heme oxygenase-1 is loaded into ferritin. Following incubation, the ferritin was purified and the amount of iron loaded into ferritin determined. It was found that ferritin purifed from E. coli expressing human heme oxygenase-1 contained more iron than E. coli that did not contain human heme oxygenase-1. It was concluded that some of the iron released upon degradation of hemin by heme …

Two New Resorcinarenes: A Pyridine & Acetic Acid Ligand For Metal Coordination And A Deep-Cavity Nitroquinoxaline Resorcinarene, Samantha Sizemore Vernetti

Theses and Dissertations

Functionalizing the upper rim of resorcinarene-based cavitands allows a variety of compounds to be synthesized from a single scaffold. Using the upper-rim moieties as ligands for a variety of transition metal ions further increases the versatility of this class of host compounds. A new resorcinarene-based molecule functionalized with four pyridine and acetic acid ligands has been successfully prepared to explore the properties of metal-assembled complexes. To synthesize this compound, tetra(bromomethyl)cavitand was reacted with N-(2-pyridylmethyl)-N-ethylacetate amine to give ethyl acetate pyridine resorcinarene. Hydrolysis of the ester gave acetic acid pyridine resorcinarene (APRes) in good yield. Complexes with Cu2+, Co2+, and Zn2+ …

The Study Of Nitric Oxide Synthase Expression, Function, And Regulation In The Renal Vasculature During Postnatal Renal Development, Brian Blake Ratliff

Theses and Dissertations in Biomedical Sciences

The newborn kidney is vulnerable to vasomotor acute renal failure (ARF) from adverse perinatal events or complications of prematurity. Nitric oxide (NO) vasodilation is vitally protective in this type of ARF, but its relationship with other vasoactive factors, such as angiotensin II (AII) has not been examined. In the immature kidney, nitric oxide synthase (NOS) isoforms, specifically eNOS and nNOS, are developmentally regulated, but their specific role and regulation are unknown.

The enhanced vasodilatory role of NO in the immature kidney was hypothesized to be attributed to regulatory, expressional, and functional differences in eNOS and nNOS isoforms from the adult. …

Biochemical Profiling Of Gout Patients, Tamera Fayre Schlitt

Chemistry & Biochemistry Theses & Dissertations

The purpose of the research is to investigate the biochemical cause of gout in 82 patients. The gouty patients had been diagnosed with gout according to the criterion of the American Rheumatism Association. This gout patient population consisted of 58 men and 24 women with gout whose ages ranged from 11 to 84 years of age with a mean age of 44 years. Key purine metabolism enzyme activity levels and purine metabolite concentrations in the patients' plasma and urine were compared to the mean values of a healthy control group. The control group consisted of 33 males and 25 female …

Gas Chromatography: Mass Spectrometry Of Chemical Agents And Related Interferents, Lailiang Zhai

Theses and Dissertations

One of the main problems encountered in chemical analysis operations in the field is collecting sufficient sample from the source and transferring that sample to the measurement instrument for fast separation and identification. I have been involved in developing a field-portable gas chromatography-mass spectrometry (GC-MS) system with solid phase microextraction (SPME) sampling for point detection of chemical agents. The objective is to minimize the analysis time between sampling and detection of a potential chemical threat. SPME offers a convenient means for sampling gaseous and liquid samples, concentrating the analytes, and transferring the analytes to the injection port of a GC …