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Structure-Function Studies Of Self- Assembling Flagellin Proteins Fromsalmonella Typhimurium And Aquifex Pyrophilus, Venkata Raghu Ram Malapaka

Dissertations

The eubacterial flagellum is a complex structure with an elongated extracellular filament that is primarily composed of a single protein termed flagellin. The highly conserved N-and C- termini of flagellin are important in its export and assembly, whereas the middle "hypervariable" region is highly variable in size across different species and is largely dispensable. InSalmonella typhimurium phase 1 flagellin (FliC), this hypervariable region encodes two solvent-exposed D2 and D3 domains. The functional role of this knob-like structural feature in motility remains unclear. The structural and physiological role of the hypervariable region in flagellar assembly, stability and cellular motility was investigated. …

Crystal Structure Of Formate Dehydrogenase H: Catalysis Involving Mo, Molybdopterin, Selenocysteine, And An Fe4S4 Cluster, Jeffrey C. Boyington, Vadim Gladyshev, Sergei V. Khangulov, Thressa C. Stadtman, Peter D. Sun

Vadim Gladyshev Publications

Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe₄S₄ cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo( VI), Fe₄S_4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV ), Fe₄S_4(red)] form have been determined, revealing a four-domain ab structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys …

Human Frataxin: Iron And Ferrochelatase Binding Surface, Krisztina Z. Bencze, Taejin Yoon, CéSar MilláN-Pacheco, Patrick B. Bradley, Nina Pastor, J. A. Cowan, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

The coordinated iron structure and ferrochelatase binding surface of human frataxin have been characterized to provide insight into the protein’s ability to serve as the iron chaperone during heme biosynthesis.

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 …

Microwave Absorption Of Patterned Arrays Of Nanosized Magnetic Stripes With Different Aspect Ratios, Leszek M. Malkinski, Minghui Yu, Andriy Vovk, Donald J. Scherer Ii, Leonard Spinu, Weilie Zhou, Scott L. Whittenburg, Zachary Davis, Jin-Seung Jung

Chemistry and Biochemistry Faculty Publications

Arrays consisting of nanosized stripes of Permalloy with different length-to-width ratios have been fabricated using electron beam nanolithography, magnetron sputtering, and lift-off process. These stripes have a thickness of , a width of , and different lengths ranging from . The stripes are separated by a distance of . Magnetization hysteresis loops were measured using a superconducting quantum interference device susceptometer. Microwave absorption at was determined by means of ferromagnetic resonance technique. The dependence of the resonant field on the angle between the nanostructure and the in-plane dc magnetic field indicates the presence of uniaxial magnetic anisotropy associated with the …

A Highly Efficient Form Of The Selenocysteine Insertion Sequence Element In Protozoan Parasites And Its Use In Mammalian Cells, Sergey V. Novoselov, Alexey V. Lobanov, Deame Hua, Marina V. Kasaikina, Dolph L. Hatfield, Vadim Gladyshev

Vadim Gladyshev Publications

Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem–loop structure located in the 3’-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess …

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 …

Analysis Of The Tfc6 Promoter In Saccharomyces Cerevisiae: Transcriptional Regulation Through Tf Iiic Binding At Etc6, Daniel Edwards

Honors Capstones

No abstract provided.

Examination Of Microsporidia Spore Adherence And Host Cell Infection In Vitro., Timothy Robert Southern

Electronic Theses and Dissertations

Microsporidia are obligate intracellular pathogens that cause severe disease in immunocompromised humans. While albendazole is the treatment of choice, no therapy exists that effectively treats all forms or causes of human microsporidiosis. Recent studies show that the microsporidian Encephalitozoon intestinalis binds glycosaminoglycans (GAGs) associated with the host cell surface, and that the divalent cations manganese (Mn²⁺) and magnesium (Mg²⁺) augment spore adherence to host cells by activating a constituent on the spore surface. These studies also illustrate a direct relationship between spore adherence and host cell infection; inhibition of spore adherence leads to reduced host cell …

Sem Analysis Of Glue Behavior When Bonding Glass Structures With Complex Geometries, Yuri Sikorski, Robert Cunningham, Herman Orgeron, Chris Schenck, Ali Zand

Chemistry & Biochemistry Presentations And Conference Materials

Bonding of glass has been studied for many years and is a mature technology today. However, the recent advances in bio-photonics and micro-fluidics, such as lab-on-a-chip devices, accentuate a need to provide reliable adhesion and sealing of components with extremely complex surface geometries. In many cases it is necessary to prevent the adhesives from leaking into microscopic channels, capillaries and holes. We present the Scanning Electron Microscopy study of adhesion of glass samples with complex surface features. Variety of adhesion conditions and procedures were tried and studied. The results demonstrate the possibility of controlled reliable adhesion and sealing without filling/obstructing …

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.

Electronic Anisotropy Between Open Shell Atoms In First And Second Order Perturbation Theory, Gerrit C. Groenenboom, Xi Chu, R. Krems

Chemistry and Biochemistry Faculty Publications

The interaction between two atoms in states with nonzero electronic orbital angular momenta is anisotropic and can be represented by a spherical tensor expansion. The authors derive expressions for the first order (electrostatic) and second order (dispersion and induction) anisotropic interaction coefficients in terms of the multipole moments and dynamic polarizabilities of the atoms and show that a complete description of the second order interaction requires odd rank or "out-of-phase" polarizabilities. The authors relate the tensorial expansion coefficients to the adiabatic Born-Oppenheimer potentials of the molecule and show that there are linear, and in some cases nonlinear, constraints on the …

Immunoadjuvanticity Of Chlorophyll, Jocelyn Helen Parks Ramos

All-Inclusive List of Electronic Theses and Dissertations

Background: Vaccines are formulations containing antigenic determinants (from non-self organic matters) and immuno-enhancers called adjuvants. Together, they evoke protective immune responses in hosts. Adjuvants are substances that further vaccines' capability to induce a robust immune response. Common side effects with conventional adjuvants used in most vaccines are toxicity, inflammation and granulomas. In this study we investigated the pigment chlorophyll (Chl) for immunoadjuvanticity. The selection of chlorophyll is based on the fact that it is a component in foods such as spinach, and that it contains an isoprenoid structure that is a feature of many conventional adjuvants, Pristane and Squalene. Chl …

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

Renée Crown University Honors Thesis 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

Renée Crown University Honors Thesis 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 Type Ii Secretion Mutants Of Pseudomonas Aeruginosa, Sijung Suh

Renée Crown University Honors Thesis 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 …

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

Renée Crown University Honors Thesis 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 …

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 …

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 …

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 …

Stoichiometric Controls Of Mercury Dilution By Growth, Roxanne Karimi, Celia Y. Chen, Paul C. Pickhardt, Nicholas S. Fisher, Carol L. Folt

Dartmouth Scholarship

Rapid growth could significantly reduce methylmercury (MeHg) concentrations in aquatic organisms by causing a greater than proportional gain in biomass relative to MeHg (somatic growth dilution). We hypothesized that rapid growth from the consumption of high-quality algae, defined by algal nutrient stoichiometry, reduces MeHg concentrations in zooplankton, a major source of MeHg for lake fish. Using a MeHg radiotracer, we measured changes in MeHg concentrations, growth and ingestion rates in juvenile Daphnia pulex fed either high (C:P = 139) or low-quality (C:P = 1317) algae (Ankistrodesmus falcatus) for 5 d. We estimated Daphnia steady-state MeHg concentrations, using a …

Multiplex Snp Discrimination, Daniel J. Fish, M. Todd Horne, Robert P. Searles, Greg P. Brewood, Albert S. Benight

Chemistry Faculty Publications and Presentations

Multiplex hybridization reactions of perfectly matched duplexes and duplexes containing a single basepair mismatch (SNPs) were investigated on DNA microarrays. Effects of duplex length, G-C percentage, and relative position of the SNP on duplex hybridization and SNP resolution were determined. Our theoretical model of multiplex hybridization accurately predicts observed results and implicates target concentration as a critical variable in multiplex SNP detection.

Supporting Information For: A Highly Efficient Form Of The Selenocysteine Insertion Sequence Element In Protozoan Parasites And Its Use In Mammalian Cells, Sergey V. Novoselov, Alexey V. Lobanov, Deame Hua, Marina V. Kasaikina, Dolph L. Hatfield, Vadim Gladyshev

Vadim Gladyshev Publications

Selenoproteins are an elite group of proteins containing a rare amino acid, selenocysteine (Sec), encoded by the codon, UGA. In eukaryotes, incorporation of Sec requires a Sec insertion sequence (SECIS) element, a stem–loop structure located in the 3'-untranslated regions of selenoprotein mRNAs. Here we report identification of a noncanonical form of SECIS element in Toxoplasma gondii and Neospora canine, single-celled apicomplexan parasites of humans and domestic animals. This SECIS has a GGGA sequence in the SBP2-binding site in place of AUGA previously considered invariant. Using a combination of computational and molecular techniques, we show that Toxoplasma and Neospora possess …

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 …

Dna-Templated Nanomaterials, Hector Alejandro Becerril-Garcia

Theses and Dissertations

Nanomaterials display interesting physical and chemical properties depending on their shape, size and composition. Self assembly is an intriguing route to producing nanomaterials with controllable compositions and morphologies. DNA has been used to guide the self assembly of materials, resulting in: (1) metal nanowires; (2) metal or semiconductor nanorods; (3) carbon nanotubes; and (4) semiconductor, metal or biological nanoparticles. My work expands the range of DNA templated nanomaterials and develops novel ways of using DNA to pattern nanostructures on surfaces. I have performed the first synthesis of silver nanorods on single stranded DNA, an attractive material for localizing DNA coupled …

Selenoprotein H Is A Nucleolar Thioredoxin-Like Protein With A Unique Expression Pattern, Sergey V. Novoselov, Gregory V. Kryukov, Xue-Ming Xu, Bradley A. Carlson, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

The human selenoproteome consists of 25 known selenoproteins, but functions of many of these proteins are not known. Selenoprotein H (SelH) is a recently discovered 14-kDa mammalian protein with no sequence homology to functionally characterized proteins. By sensitive sequence and structure analyses, we identified SelH as a thioredoxin fold-like protein in which a conserved CXXU motif (cysteine separated by two other residues from selenocysteine) corresponds to the CXXC motif in thioredoxins. These data suggest a redox function of SelH. Indeed, a recombinant SelH shows significant glutathione peroxidase activity. In addition, SelH has a conserved RKRK motif …

Selenophosphate Synthetase 2 Is Essential For Selenoprotein Biosynthesis, Xue-Ming Xu, Bradley A. Carlson, Robert Irons, Heiko Mix, Nianxin Zhong, Vadim Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. …

Investigation Of Novel Microseparation Techniques, Yansheng Liu

Theses and Dissertations

Ultrahigh pressure liquid chromatography (UHPLC) makes it possible to use very small particles (< 2 µm) as packing materials to provide high column efficiencies. Results from a careful comparison of small porous and nonporous particles show that when the particle size is small enough (< 2 µm), both porous and nonporous particles give excellent performance, and the differences in column efficiencies between porous and nonporous particles become insignificant. Columns packed with bare diamond particles could separate small molecules, especially polar molecules, however, severe tailing occurred for less polar compounds. The polybutadiene coated diamond particles gave greater retention and better separation of small molecules compared to bare particles, although no improvement in column efficiency was observed. Changes in surface bonding of thermally hydrogenated diamond particles was achieved by chemical modification using various organic peroxides with or without reagents containing long carbon chain functional groups. It appears that the alkyl groups were attached onto the diamond surface with limited coverage. LC experiments did not demonstrate good separation; however, changes in LC behavior were observed. A repetitive solvent programming approach was successfully applied to the analysis of a continuous sample stream in microbore LC. Each analysis cycle consisted of three steps: pseudo-injection, elution and rinse. In the pseudo-injection step, elution with a non- or poor-eluting solvent produced a concentrated sample plug due to on-column focusing. Factors influencing peak symmetry, resolution and analysis cycle length were investigated. Quantitative analysis of a continuous sample stream is possible under certain operating conditions. Electric field gradient focusing (EFGF) devices with distributed resistor substrates could focus proteins in the separation channel, however, the focused bands were not stable, and the repeatability was poor due to the formation of bubbles and pH gradient in the separation channel. Both fiber-based and porous glass capillary-based planar EFGF devices with changing cross-sectional area (CCSA) channels were constructed and evaluated with the aid of a home-made scanning laser-induced fluorescence detection system. The fiber-based CCSA EFGF devices gave poorer performance compared with glass capillary based devices. Porous glass capillary-based EFGF devices could focus single proteins and separate mixtures of two to three proteins.

Caf1 Plays An Important Role In Mrna Deadenylation Separate From Its Contact To Ccr4, Takbum Ohn, Yueh-Chin Chiang, Darren J. Lee, Gang Yao, Chongxu Zhang, Clyde L. Denis

Molecular, Cellular & Biomedical Sciences

The CAF1 protein is a component of the CCR4 - NOT deadenylase complex. While yeast CAF1 displays deadenylase activity, this activity is not required for its deadenylation function in vivo, and CCR4 is the primary deadenylase in the complex. In order to identify CAF1- specific functional regions required for deadenylation in vivo, we targeted for mutagenesis six regions of CAF1 that are specifically conserved among CAF1 orthologs. Defects in residues 213 - 215, found to be a site required for binding CCR4, reduced the rate of deadenylation to a lesser extent and resulted in in vivo phenotypes that were less …