Biochemistry Commons^™

Preparation Of Divalent And Trivalent Antigens For Immunogical Studies On Degranulation Of Mast Cells And Preparation Of Ceragenins For Antiviral Studies Against Vaccinia Virus., Dianliang Geng

Theses and Dissertations

CHAPTER 1

Aggregation of receptors for IgE (Fc RI) causes mast cells and basophils to release preformed contents of granules, including histamine and a variety of enzymes. This process, called degranulation plays a central role in allergic reactions. Methods to study this process are to create multivalent ligands which can interact with the receptors and, in turn, lead to aggregation of the receptors. We prepared a series of fluorophore-labeled divalent and trivalent antigens to study the degranulation of mast cells. Trivalent antigens proved to be much better stimulators for degranulation of mast cells than divalent antigens. These results indicate that …

Pyridinium Bisretinoids: Synthesis And Photoactivated Cytotoxicity, Junping Gao

Theses and Dissertations

This thesis discusses pyridinium bisretinoid compounds (PBRs), which were prepared for two purposes: 1) to use them as standards for detection of novel fluorophores in human RPE cells, which may be involved in age-related macular degeneration (AMD), and 2) to use them in the development of a targeted and triggered drug delivery system for cancer therapy. We prepared a selection of PBRs using a one-pot biomimetic method; synthesis, mechanisms for formation, and characterization of these compounds is described. We also explored the photoreactivity of three novel PBR compounds and found that these PBRs form oxidation products under blue-light irradiation. The …

Study Of The Reproducibility Of Proteomics Methods And Variability Of Fruit Fly Proteomes., Thomas Franklin Culwell

Theses and Dissertations

The reliability of biomarker discovery by means of proteomics has been called into question. It was speculated that "background noise" variation resulting from differences in preparation and handling of samples and proteome dynamics may mask subtle, yet important, differences due to the biological condition. Little is understood about complex proteomes and their variability. A critical aspect of proteomic biomarker research that is largely unexplored is the comparative reproducibility of certain methods such as two-dimensional gel electrophoresis and liquid chromatography/mass spectrometry. In particular, with liquid chromatography/mass spectrometry, it is not known whether variability in peptide quantitation is dependent on any of …

Pyridinium Bis-Retinoids A2-Dopamine And A2-Cadaverine: Implications In Age-Related Macular Degeneration And Cancer, Mckenzie Ruth Pew

Theses and Dissertations

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States of America. The pyridinium bis-retinoid A2-ethanolamine (A2E) has been implicated to play a role in AMD. We have observed novel pyridinium bis-retinoids through melanolipofuscin and human RPE extractions that may also play a role in the pathology of AMD. We have begun the construction of an amino-retinoid library in order to identify these ocular compounds. The compounds from the amino-retinoid library are also used in a targeted and triggered drug delivery system for treating cancer. Folic acid is coupled with the amino-retinoids to specifically target cancer …

Studies In The Biochemistry And Cell Biology Of Trypanosoma Brucei Hexokinases, Jeremy Chambers

All Dissertations

Trypanosoma brucei expresses two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 did not, a finding attributed to differences in the C-termini of the proteins. We found that the C-terminal tails of TbHK contained a novel motif specific for eukaryotic hexokinases found within the final helix of an ATP-binding domain. By altering the residues in the C-terminal tail of TbHK1 to resemble the residues found in TbHK2, we found that D454, F462, M466, and N469 were essential to TbHK1 catalysis. Additionally, we found …

Effects Of Water Vapor On The Kinetics Of The Methylperoxy Radical Self-Reaction And Reaction With Hydroperoxy, Alecia Mower

Theses and Dissertations

The gas phase reactions of CH3O2 + CH3O2, HO2 + HO2, and CH3O2 + HO2 in the presence of water vapor have been studied at temperatures between 263 and 303 K using laser flash photolysis coupled with UV time-resolved absorption detection at 220 nm and 260 nm. Water vapor concentration was quantified using tunable diode laser spectroscopy operating in the mid-IR. The HO2 self-reaction rate constant is significantly enhanced by water vapor, consistent with what others have reported, whereas CH3O2 self-reaction and the cross-reaction (CH3O2 + HO2) rate constants are nearly unaffected. The enhancement in the HO2 self-reaction rate coefficient …

Microchip Liquid Chromatography And Capillary Electrophoresis Separations In Multilayer Microdevices, Hernan Vicente Fuentes

Theses and Dissertations

In this dissertation, several microfabricated devices are introduced to develop new applications in the area of chemical analysis. Electrochemical micropumps, chip-based liquid chromatography systems and multilayer capillary electrophoresis microdevices with crossover channels were fabricated using various substrates such as poly(dimethylsiloxane) (PDMS), glass, and poly(methyl methacrylate) (PMMA). I have demonstrated pressure-driven pumping of liquids in microfabricated channels using electrochemical actuation. PDMS-based micropumps were integrated easily with channel-containing PMMA substrates. Flow rates on the order of ~10 µL/min were achieved using low voltages (10 V). The potential of electrolysis-based pumping in microchannels was further evaluated for pressure driven microchip liquid chromatography (LC). …

Microfluidic Electro-Osmotic Flow Pumps, John Mason Edwards

Theses and Dissertations

The need for miniaturized, portable devices to separate and detect unknown compounds has greatly multiplied, leading to an increased interest in microfluidics. Total integration of the detector and pump are necessary to decrease the overall size of the microfluidic device. Using previously developed thin film technologies, an electroosmotic flow (EOF) pump was incorporated in a microfluidic liquid chromatography device. An EOF pump was chosen because of its simple design and small size. EOF pumps fabricated on silicon and glass substrates were evaluated. The experimental flow rates were 0.19-2.30 microliters/minute for 40-400 V. The theoretical pump efficiency was calculated along with …

Counter-Flow Ion Mobility Analysis: Design, Instrumentation, And Characterization, Nosa Agbonkonkon

Theses and Dissertations

The quest to achieve high resolution in ion mobility spectrometry (IMS) has continued to challenge scientist and engineers in the field of separation science. The low resolution presently attainable in IMS has continued to negatively impact its utility and acceptance. Until now, efforts to improve the resolution have mainly focused on better instrumentation and detection methods. However, since the resolution of IMS is diffusion limited, it makes sense to address this limitation in order to attain high resolution. This dissertation presents a new IMS technique, which utilizes a high electric field and opposing high gas flow velocity with the aim …

Gene Synthesis, Cloning, Expression, Purification And Biophysical Characterization Of The C2 Domain Of Human Tensin, Kiran Sukumar Gajula

Doctoral Dissertations

Tensin is a large "docking" protein found in the adhesive junctions of animal cells and recruited early in the development of cell-substrate contacts. There it binds to the cytoplasmic domain of integrin β1 and caps the barbed ends of filamentous actin. This forms a rational basis for its implication in a direct role in the mechanics of membrane-cytoskeleton interactions. Tensin provides a physical link between the actin cytoskeleton, integrins, and other proteins at the cell-substrate contacts. Its overall biochemical properties are a function of its domain composition and architecture, i.e., the domains that are present and their relative positions in …

Compartmentalization Of Hiv-1 In The Secondary Lymphoid Tissues, James Peter Gregson

Theses and Dissertations

Follicular dendritic cells (FDCs) reside in the lymphoid follicles of the secondary lymphoid tissues (sLTs). Following the infection of an individual with human immunodeficiency virus type 1 (HIV-1), viral particles are trapped in massive quantities on the surfaces of FDCs. HIV-1 viral compartments are cell types or tissues between which there is a restriction of virus flow. Compartmentalization of HIV-1 creates numerous sites within the body in which the virus can undergo independent evolution, giving rise to a more diverse total viral population. Given the sessile nature of the FDC, I hypothesized that contrary to common assumptions, FDC-trapped HIV-1 is …

Fluorescent Photoinduced Electron Transfer (Pet) Proton Sensors, Risper Nyabeta

Theses, Dissertations and Culminating Projects

Two new fluorescent photoinduced electron transfer (PET) proton sensors based on anthracene and pyrazoline chromophores have been prepared. The anthracene sensor was prepared through alkylation of 2-pyridylcarbinol with 9-chloromethylanthracene and the pyrazoline sensor was prepared through an aldol condensation of 4- carboxybenzaldehyde and acetylpyridine followed by reaction with phenylhydrazine. The fluorescence of these sensors is modulated by the initiation or quenching of a PET process between the chromophore and a receptor due to protonation. The synthesis, characterization and proton binding studies of the new sensors are reported in this thesis.

Studies On The Mechanisms Of Homolog Pairing And Sister Chromatid Cohesion During Drosophila Male Meiosis, Jian Ma

Masters Theses

Meiosis is a complex process involving one round of DNA replication followed by two rounds of cell divisions. The proper segregation of homologs at meiosis I and sister chromatids during meiosis II is essential for the survival of the offspring. Aberrant chromosome segregation at any stage of meiosis can lead to aneuploidy. Meiotic chromosome segregation without crossing over or chiasmata is a widespread but poorly understand chromosome segregation pathway. In male Drosophila meiosis the absence of recombination in chromosomes makes it easier to identify mutations which influence homologous chromosome pairing and segregation.

Modifier of Mdg4 in Meiosis (MNM), a protein …

Development Of Chemomechanical Functionalization And Nanografting On Silicon Surfaces, Michael Vernon Lee

Theses and Dissertations

Progress in chemomechanical functionalization was made by investigating the binding of molecules and surface coverage on the silicon surface, demonstrating functionalization of silicon with gases by chemomechanical means, analyzing atomic force microscopy probe tip wear in atomic force microscopy (AFM) chemomechanical nanografting, combining chemomechanical functionalization and nanografting to pattern silicon with an atomic force microscope, and extending chemomechanical nanografting to silicon dioxide. Molecular mechanics of alkenes and alkynes bound to Si(001)-2x1 as a model of chemomechanically functionalized surfaces indicated that complete coverage is energetically favorable and becomes more favorable for longer chain species. Scribing a silicon surface in the presence …

Synthesis Of Resveratrol And Its Analogs, Phase-Transfer Catalyzed Asymmetric Glycolate Aldol Reaction, And Total Synthesis Of 8,9-Methylamido-Geldanamycin, Jing Liu

Theses and Dissertations

The phytoalexin resveratrol and its acetyl analogs have been made using a decarbonylative Heck reaction. The acid chloride derived from 3,5-dihydroxybenzoic acid was coupled with suitable protected 4-hydroxystyrene in the presence of palladium acetate and N,N-bis-(2,6-diisopropylphenyl)-4,5-dihydro imidazolium chloride to give the substituted stilbene in good yield as the key step. Human HL-60 cell assays showed the 4'-acetyl resveratrol variant improved activity (ED50 17 μM) relative to resveratrol (24 μM). Cinchona phase-transfer catalysts (PTC) were developed for glycolate aldol reactions to give differentially protected 1,2-diol products. Silyl enol ether of diphenylmethoxy-2,5-dimethoxyacetophenone reacted to generate benzhydryl-protected products. O-Allyl trifluorobenzyl cinchonium hydrodifluoride (20 …

Synthesis And Antiviral Evaluation Of Some 3'-Carboxymethyl-3'-Deoxyadenosine Derivatives, Houguang Shi

Theses and Dissertations

3'-Carboxymethyl-3'-deoxyadenosine derivatives were prepared from 2'-O-TBDMS-3'-deoxy-3'-[(ethoxycarbonyl)methyl]adenosine (1) via simple and efficient procedures. Conversion of 1 to 5'-azido-2'-O-TBDMS-3', 5'-dideoxy -3'-[(ethoxycarbonyl) methyl] adenosine (4) was accomplished via a novel one-pot method employing 5'-activation (TosCl) followed by efficient nucleophilic displacement with tetramethylguanidinium azide. Compound 4 was converted to a 5'-[(N-methylcarbamoyl)amino] derivative (5) via one-pot reduction/acylation employing H2/Pd-C followed by treatment with p-nitrophenyl N-methylcarbamate. The latter step of this two-step process required an efficient source of p-nitrophenyl N-methylcarbamate, thus a highly efficient new method for preparing p-nitrophenyl N-alkylcarbamate was developed. N6-phenylcarbamoyl groups were introduced by treatment with phenylisocyanate, and an efficient new method for …

Asymmetric Dihydroxylation And Aziridination Of Allenes And Related Chemistry, Renmao Liu

Theses and Dissertations

A novel method for asymmetric synthesis of α-hydroxy ketone with excellent regio- and stereoselectivity has been established by the systematic investigation of asymmetric dihydroxylation of allenes. The efficiency of kinetic resolution of racemic allenes was also investigated by using the AD reaction on both 1,3-disubstituted and trisubstituted allenes. Steric effects, electronic effects and allene substitution are also discussed. Aziridines were formed by copper-catalyzed intramolecular nitrene addition to alkenes. The carbamate group was used as the tether between the alkene and the nitrene. Subsequent nucleophilic attack of the aziridine was accomplished using RSH, R2NH, N3-,or ROH as the nucleophile. This addition …

Towards Early State Disease Detection In Microdevices: Fabrication And Testing Of Micro Total Analysis Systems For Bioanalytical Applications, Tao Pan

Theses and Dissertations

The past few years have seen a rapid expansion in interest in the characterization of the entire complement of proteins, or proteome. Micro total analysis systems (μTAS) are an emerging promising method, offering rapid, sensitive and low sample consumption separations. I have demonstrated microchip capillary electrophoresis (CE) devices made of CaF2. New methods have been developed for micromachining enclosed capillaries in CaF2. CE analysis of fluorescently labeled amino acids was used to illustrate bioanalytical applications of these microdevices. Initial on-chip infrared spectroscopy results for qualitative analyte identification were achieved in microfluidic CaF2 channels. I have also shown the evaluation of …

Early Stage Detection Of Marine Oil Oxidation, Loren Lapointe

Honors Theses

A method involving overnight stir bar sorptive extraction (SBSE) headspace gas chromatography/mass spectrometry (GC/MS) in negative ion chemical ionization (NICI) mode was developed to determine the presence of aldehydes (cis-4-heptenal, trans,trans-2,4-heptadienal, trans-2-octenal, cis-6-nonenal, trans-2-cis-6-nonadienal, trans-2-nonenal, and trans,trans-2,4-decadienal) in fresh and aged commercial fish oil (untreated and treated with antioxidant blends). Aldehyde standards were quantifiable to >100ppb levels using this method. Only trans,trans-2,4-heptadienal was shown to be present in the commercial fish oil and its presence was used to compare the performance of the two different antioxidant treatments in the fish oil. Includes Powerpoint.

Biochemical Characteristics Of Inconsistent Free T₄ Assays, Kristofer S. Fritz

Loma Linda University Electronic Theses, Dissertations & Projects

Serum levels of free thyroxine (T₄) are helpful in the clinical evaluation of patients who are suspected of having thyroid disease, since free T₄ (unbound) is known to closely relate to hyper, eu and hypothyroidism. There are documented inconsistencies among commercially available direct free thyroxine immunoassays. The biochemical basis for these inconsistencies is not understood and has not been characterized. Direct free T₄ measurements have been linked to both T₄-binding serum protein concentrations and protein-bound T₄ concentrations. A free T₄ reference method using direct equilibrium dialysis radioimmunoassay has been well characterized. New …

Notch Regulation Of Human Breat Cancer Progression: Contrasting Roles For Notch Signaling, Christine F. O'Neill

Electronic Theses and Dissertations

Notch signaling is associated with activation of either oncogenic or tumor suppressor activities. The human mammary adenocarcinoma cell line, MDA-MB-231, was characterized in vitro and in xenografts in vivo to test the hypothesis that activation of Notch signaling regulates mammary tumor phenotype. Notch 1, Notch2, and Notch4 signaling was compared by stable expression of their constitutively active intracellular domains (ICD). Notch4 activation led to enhanced tumorigenicity, in addition to increased cell proliferation and survival in vitro, whereas the activation of Notch 1 or Notch2 decreased cell proliferation and survival, in which Notch2 increased apoptosis. Stably transfected cell lines were …

Iron Dysregulation And Inflammation In Alzheimer’S Disease, Shino D. Magaki

Loma Linda University Electronic Theses, Dissertations & Projects

Alzheimer’s disease (AD) is the most common form of senile dementia in the US and worldwide but the causes of its pathogenesis are currently unknown. In this study, we examined two processes that have been implicated in the early stages of AD and other forms of neurodegeneration, iron dysregulation and inflammation, both of which can promote the increased production of amyloid precursor protein (APP). We have measured different pools of brain iron in transgenic iron regulatory protein 2 knockout (IRP2-/-) mice in the early stages of neurodegeneration and in affected brain regions from AD patients at different stages of the …

Radiation-Induced Increases In Pkc Modulate Integrin Expression And Contribute To Fibrotic Changes, Pinal Rushikesh Pandya

Loma Linda University Electronic Theses, Dissertations & Projects

The sequences of events in fibrosis are similar to those in wound healing; however, the normal termination and resolution stages do not take place. The initial cellular response following ionizing radiation involves accumulation of the ECM (extracellular matrix) including collagen, fibronectin and the interaction of many growth factors (cytokines) with their receptors. There are a number of unanswered questions regarding many aspects of radiation-induced fibrosis, including the initial triggers and physical changes that initiate the process. We have confirmed that elevated pKC and other cellular changes following radiation are similar to those found at sites of inflammation. This information leads …

Characterizing Ntrc-Like Activators Affecting Myxococcus Xanthus Development, Jessica Eisenstatt

Honors Capstone Projects - All

Myxococcus xanthus is a soil bacterium that is a member of a group of organisms known as the myxobacteria. M. xanthus cells live in biofilms and feed on other bacteria to obtain nutrients. During times of inadequate nourishment, M. xanthus cells aggregate, build fruiting bodies, and fruiting body cells differentiate into spores that are highly resistant. In order to form the fruiting bodies, M. xanthus cells must be able to move across solid surfaces. There are two motility systems, the adventurous system (A-motility) and the social system (S-motility), used by M. xanthus cells to navigate across surfaces. The adventurous system …

Compensation Mechanisms For Altered Membrane Sterol Compositions In The Yeast: Saccharomyces Cerevisiae, David M. Dewolfe

Honors Capstone Projects - All

Cell Membranes are composed of several different lipid and sterol products. Among these are, chiefly, phospholipids, glycolipids, sphingolipids, various proteins and sterols. The sterol that is prevalent in fungi, including yeast, is ergosterol. It plays the same physiological role as cholesterol in mammalian cells. That is, mainly, to control membrane fluidity. Membranes in general are extremely important to the normal functioning of any cell and its sub-cellular compartments. The primary factor in the normal functioning of a membrane is the relative composition of the previously mentioned components. Even though there is a high amount of traffic between different membranes within …

Characterization Of Hypersensitive Response Related Genes Of Arabidopsis Thaliana, Keluo Yao

Honors Capstone Projects - All

Like most complex living organisms, plants have many mechanisms to prevent disease by microbial pathogens. One of the most important and well developed defense systems that involve recognition, identification and systematic response is the hypersensitive response.

The hypersensitive response is a complex, early defense response against pathogens that causes necrosis and cell death at the site of infection to restrict the spread of pathogen. Hypersensitive response is a type of programmed cell death, and its activation usually happens when the plant recognizes a pathogen through an elicitor. This recognition triggers a series of signal transductions events which end in the …

Characterization Of Type Ii Secretion Mutants Of Pseudomonas Aeruginosa, Sijung Suh

Honors Capstone Projects - All

Pseudomonas aeruginosa mutants PA0686, PA3102, PA3103, and PA5210 carry transposon insertions in uncharacterized genes in the PA01 background. The biofilms formed by these mutants show different phenotypes than PA01, their wild-type counterpart. BLAST searches and phylogenomic mapping tools indicate that protein products of the mutated genes are involved in P. aeruginosa type-II secretion, which is linked to twitching motility. In this study, 96-well microtitre plate biofilm assays, UV/visible spectrophotometer analysis, and motility assays were used to demonstrate a link between type-II secretion, twitching motility and biofilm formation. The results show that the PA5210 mutant forms a biofilm that is less …

The Effects Of Acute Ethanol Treatment On The Suprachiasmatic Nucleus In Adult Male Mice, Charles Andrew Mangrum

Masters Theses

Light is the primary entraining signal for the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Light entering the eye leads to release of glutamate directly onto SCN neurons where it binds to N-methyl D-aspartate (NMDA) receptors initiating a cascade of cellular processes that ultimately modulates clock phase. SCN neurons show a 24-hour rhythm in neuronal activity that peaks in the middle of the day when isolated in a brain slice preparation. Treatments that phase-shift the SCN clock in vivo have been shown similarly to shift this rhythm of neuronal activity in vitro. Here, I have investigated …

The Development Of A Gc/Ms Protocol For The Analysis Of Polysaccharides In Echinacea Purpurea, Michaela Knapp

Undergraduate Theses and Capstone Projects

Echinacea purpurea is an herbal supplement used to reduce symptoms of common colds and flu-like illnesses. As of right now, it is not well understood what chemical components in the plant are immunologically active. The four main potentially immunologically active chemical components in E. purpurea are caffeic acid derivatives, alkamides, polyacetylenes, and polysaccharides. In this investigation, a protocol was developed in an attempt to identify polysaccharides present in Puritan’s Pride E. purpurea (aerial) “non-irradiated” herbal supplement First, an estimate of the crude total polysaccharide content in Puritan’s Pride and in a ChromaDex certified botanical standard (E. purpurea -powdered root) was …

Kinetic Studies Of 6-Halopurine Nucleoside In Snar Reactions; 6-(Azolyl, Alkylthio And Fluoro)-Purine Nucleosides As Substrates For Suzuki Reactions, Jiangqiong Liu

Theses and Dissertations

In chapter 1, we describe development of a mild and efficient method for SNAr iodination of 6-chloropurine 2'-deoxynucleosides and nucleosides. Our studies demonstrate that 6-iodopurine nucleosides are excellent substrates for certain transition metal-catalyzed cross-coupling reactions. In chapter 2, we describe synthesis of protected 6-fluoro, 6-chloro, 6-bromo and 6-sulfonylpurine nucleosides. Comparisons among 6-fluoro-, 6-chloro-, 6-bromo, 6-iodo and 6-sulfonylpurine nucleosides for SNAr reactions with various N, O and S nucleophiles were investigated. Our results demonstrate that the 6-fluoropurine nucleoside is the best substrate for SNAr reactions among the four 6-halopurine nucleosides with oxygen, sulfur and aliphatic amine nucleophiles, and also with an …