Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

Direct Comparison Of Feature Tracking And Autocorrelation For Velocity Estimation, Gregory R. Bashford, Derek J. Robinson

Biomedical Imaging and Biosignal Analysis Laboratory

Feature tracking is an algorithm for estimating tissue motion and blood flow using pulse-echo ultrasound. It was proposed as a computationally simpler alternative to other techniques such as autocorrelation and time-domain cross correlation. The advantage of feature tracking is that it selectively extracts easily identifiable parts of the speckle signal (e.g., the local maxima), reducing the amount of information being processed. Studies on feature tracking to date have used stationary, specklegenerating targets to simulate blood flow. Also, feature tracking has not been compared with accepted commercial methods. This study directly compares feature tracking performance with the complex autocorrelation method, which …

Novel Metabolism In Chlamydomonas Through The Lens Of Genomics, Arthur R. Grossman, Martin Croft, Vadim N. Gladyshev, Sebeeha S. Merchant, Matthew C. Posewitz, Simon Prochnik, Martin H. Spalding

Vadim Gladyshev Publications

Chlamydomonas has traditionally been exploited as an organism that is associated with sophisticated physiological, genetic and molecular analyses, all of which have been used to elucidate several biological processes, especially photosynthesis and flagella function and assembly. Recently, the genomics of Chlamydomonas has been combined with other technologies to unveil new aspects of metabolism, including inorganic carbon utilization, anaerobic fermentation, the suite and functions of selenoproteins, and the regulation of vitamin biosynthesis. These initial fi ndings represent the first glimpse through a genomic window onto the highly complex metabolisms that characterize a unicellular, photosynthetic eukaryote that has maintained both plant-like and …

The Purification Of Helicobacter Pylori Urease Β Subunit Protein And Dna For Vaccine Production, Alyson Moll

Honors Capstones

No abstract provided.

Molecular Phylogenetics Of Perciform Fishes Using The Nuclear Recombination Activating Gene 1, Andrew R. Mahon

Biological Sciences Theses & Dissertations

The order Perciformes contains one-third of all extant fishes in twenty different suborders and over 10,000 species. Few systematic investigations have been performed on this large group of fishes at the suborder level and their evolutionary history is widely recognized as problematic. This dissertation presents three studies: a molecular phylogenetic analysis of the putative suborders of the order Perciformes, an analysis of interrelationships of the families of the perciform suborder Percoidei, and a multi-gene investigation of the percoid superfamily Sparoidea.

The taxa sampled in this dissertation represent one of the most inclusive molecular datasets, to date, testing the monophyly of …

The Role Of Phosducin-Like Protein As A Co-Chaperone With The Cytosolic Chaperonin Complex In Assembly Of The G Protein Βγ Subunit Dimer, Paul Jayson Ludtke

Theses and Dissertations

Phosducin-like protein (PhLP) has been shown to interact with the cytosolic chaperonin containing TCP-1 (CCT), and the βγ subunit dimer of heterotrimeric G proteins (Gβγ). Here we provide details obtained from cryo-electron microscopic and biochemical studies on the structure of the complex between the cytosolic chaperonin CCT and PhLP. Binding of PhLP to CCT occurs through only one of the two chaperonin rings, making multiple contacts with CCT through both its N- and C-terminal domains. In addition, we show that PhLP acts as a co-chaperonin along with CCT in mediating the assembly of the G protein βγ subunit and that …

Size Dependence Of Static And Dynamic Magnetic Properties In Nanoscale Square Permalloy Antidot Arrays, Minghui Yu, Leszek M. Malkinski, Leonard Spinu, Weilie Zhou, Scott L. Whittenburg

Chemistry and Biochemistry Faculty Publications

Permalloy antidot arrays with different square hole sizes ( , , and ) have been fabricated by means of electron-beam lithography and lift-off techniques. The smaller square hole size results in enhanced remanence and reduced coercivity in the antidot array. Multiple resonance modes were clearly observed for the magnetic field applied normal to the array plane, and double uniform resonance modes occurred when the field deviated more than 30° from the normal to the plane. Two distinct dipolar field patterns with different orientations and magnitudes split the uniform resonance into double resonance modes. The double resonance modes show uniaxial in-plane …

The Niemann-Pick Type C2 Protein Loads Isoglobotrihexosylceramide Onto Cd1d Molecules And Contributes To The Thymic Selection Of Nkt Cells, Paul B. Savage, Nicolas Schrantz, Yuval Sagiv, Yang Liu, Albert Bendelac, Luc Teyton

Faculty Publications

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Valpha 14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-gamma in vivo and in vitro after activation with alpha-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Valpha 14 hybridoma cells. Importantly, we determined that similar …

Hiv/Aids In Pakistan: The Battle Begins, Mohammad A. Rai, Haider J. Warraich, Syed H. Ali, Vivek R. Nerurkar

Department of Biological & Biomedical Sciences

Pakistan, the second most populous Muslim nation in the world, has started to finally experience and confront the HIV/AIDS epidemic. The country had been relatively safe from any indigenous HIV cases for around two decades, with most of the infections being attributable to deported HIV positive migrants from the Gulf States. However, the virus finally seems to have found a home-base, as evidenced by the recent HIV outbreaks among the injection drug user community. Extremely high-risk behavior has also been documented among Hijras (sex workers) and long-distance truck drivers. The weak government response coupled with the extremely distressing social demographics …

Pressure Dissociation Of Integration Host Factor-Dna Complexes Reveals Flexibility-Dependent Structural Variation At The Protein-Dna Interface, Donald F. Senear, Vira Tretyachenko-Ladokhina, Michael L. Opel, Kimberly A. Aeling, G. Wesley Hatfield, Laurie M. Franklin, Reuben C. Darlington, J. B. A. Ross

Chemistry and Biochemistry Faculty Publications

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236-39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences …

Dynamic Polarizabilities Of Rare-Earth-Metal Atoms And Dispersion Coefficients For Their Interaction With Helium Atoms, Xi Chu, Alexander Dalgarno, Gerrit C. Groenenboom

Chemistry and Biochemistry Faculty Publications

The dynamic scalar and tensor polarizabilities of the rare-earth-metal atoms are calculated with time-dependent density functional theory. The frequency-dependent polarizabilities at imaginary frequencies are used to determine the isotropic and orientation-dependent van der Waals coefficients for the interactions of the rare-earth-metal atoms with helium atoms. The static polarizabilities are compared with other theoretical values and with experimental results. The agreement is satisfactory in most cases but there are some exceptions where the discrepancy between theory and experiment is significant. The derived isotropic van der Waals coefficients range between 37 and 50 E(H)a(0)(6) and the orientation-dependent coefficients between 2 and -1 …

Atp Activates A Reactive Oxygen Species-Dependent Oxidative Stress Response And Secretion Of Pro-Inflammatory Cytokines In Macrophages, Christiane M. Cruz, Alessandra Rinna, Henry Jay Forman, Ana L. M. Ventura, Pedro M. Persechini, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN …

Cost-Effective Engineering Of A Small-Scale Bioreactor, John Tansey, Sadie R. Bartholomew

Chemistry Faculty Scholarship

Several methods exist for increasing the scale of cell culture in the laboratory. While these methods provide significant increases in biomass, they are often prohibitively expensive for many laboratories. We have engineered a small-scale bioreactor with a novel means of introducing oxygen through the catalytic breakdown of hydrogen peroxide using a manganese oxide catalyst. We have also adapted and modified an existing assay for dissolved oxygen to be compatible with culture conditions. In this system we have been able to culture CHO cells at densities of up to 10⁷ cells/mL without the use of automated feedback systems.

Total Parotidectomy Under Local Anesthesia: A Novel Technique, Kamran Shahid, Bilal Karim Siddiqui, Muhammad Hammad Tahir, Salman Bin Ayub, Ghulam Murtaza Memon, Adnan Yousuf, Arif Hussain, Osmani, Abdul Karim Siddiqui

Department of Biological & Biomedical Sciences

Parotidectomy is a common procedure usually done for a parotid mass necessitating a histological diagnosis. Operation is normally performed under General anesthesia with a nerve stimulator to facilitate facial nerve stimulation. We describe a new technique with reports of three cases, making total parotidectomy under local anesthesia possible. The ascending cervical branch of cervical plexus and the auriculotemporal nerve were anesthetized by bupivacaine 0.25% (2mg/kg) and lignocaine with adrenaline 7 mg/kg. Effective onset of anesthesia was within 15-25 minutes and the operations lasted between 2-3 hours without any complications. This offers advantage in high-risk patients where general anesthesia is contraindicated. …

Optimization Of Cutaneous Electrically Mediated Plasmid Dna Delivery Using Novel Electrode, L. C. Heller, M. J. Jaroszeski, D. Coppola, A. N. Mccray, J. Hickey, R. Heller

Bioelectrics Publications

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo. A critical component of this technique is the electrode configuration. Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode. The highest transgene expression and efficiency of individual cell transformation with minimal damage was produced with eight 150 ms pulses at field strength of …

Purification Method Directly Influences Effectiveness Of An Epidermal Growth Factor-Coupled Targeting Agent For Noninvasive Tumor Detection In Mice, Joy L. Kovar, William M. Volcheck, Jiyan Chen, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled …

The Structure And Function Of Small Nucleolar Ribonucleoproteins, Steve Reichow, Tomoko Hamma, Adrian R. Ferré-D’Amaré, Gabriele Varani

Chemistry Faculty Publications and Presentations

Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequencespecific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein–RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose- 20-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the …

The Yeast Orthologue Of Grasp65 Forms A Complex With A Coiled-Coil Protein That Contributes To Er To Golgi Traffic, Rudy Behnia, Francis A. Barr, John J. Flanagan, Charles Barlowe, Sean Munro

Dartmouth Scholarship

The mammalian Golgi protein GRASP65 is required in assays that reconstitute cisternal stacking and vesicle tethering. Attached to membranes by an N-terminal myristoyl group, it recruits the coiled-coil protein GM130. The relevance of this system to budding yeasts has been unclear, as they lack an obvious orthologue of GM130, and their only GRASP65 relative (Grh1) lacks a myristoylation site and has even been suggested to act in a mitotic checkpoint. In this study, we show that Grh1 has an N-terminal amphipathic helix that is N-terminally acetylated and mediates association with the cis-Golgi. We find that Grh1 forms a complex with …

A Proposal For Robust Temperature Compensation Of Circadian Rhythms, Christian I. Hong, Emery D. Conrad, John J. Tyson

Dartmouth Scholarship

The internal circadian rhythms of cells and organisms coordinate their physiological properties to the prevailing 24-h cycle of light and dark on earth. The mechanisms generating circadian rhythms have four defining characteristics: they oscillate endogenously with period close to 24 h, entrain to external signals, suffer phase shifts by aberrant pulses of light or temperature, and compensate for changes in temperature over a range of 10°C or more. Most theoretical descriptions of circadian rhythms propose that the underlying mechanism generates a stable limit cycle oscillation (in constant darkness or dim light), because limit cycles quite naturally possess the first three …

High-Throughput Identification Of Catalytic Redox-Active Cysteine Residues, Dmitri E. Fomenko, Weibing Xing, Blakely M. Adair, David J. Thomas, Vadim Gladyshev

Vadim Gladyshev Publications

Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redoxactive Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification …

Supporting Online Material For “High-Throughput Identification Of Catalytic Redox-Active Cysteine Residues”, Dmitri E. Fomenko, Weibing Xing, Blakely M. Adair, David J. Thomas, Vadim Gladyshev

Vadim Gladyshev Publications

Identification of redox-active Cys by homology to selenoproteins
Identification of Cys/Sec pairs
Statistical analysis of redox-active Cys neighborhoods
AdoMet-dependent methyltransferase
Activity of AS3MT and its Cys-to-Ser mutants

A Cadmium-Transporting PIb-Type Atpase In Yeast Saccharomyces Cerevisiae, David J. Adle, Devis Sinani, Heejeong Kim, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P_IB-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense …

Identification Of Genes Encoding Acyl-Coa Reductases And Aldehyde Reductases In Mycobacterial Genome By Characterization Of The Reductases Expressed In E. Coli, Harminder Singh

Electronic Theses and Dissertations

Mycobacterium tuberculosis has been long known to produce wax esters. However, the enzymes involved in their biosynthesis have not been identified. Here we report the identification of Rv3391 and Rv1543 as genes that encode fatty acyl-CoA reductases and Rv1544 as one that encodes an aldehyde reductase. When expressed in E.coli, the products of Rv3391 and Rv1543 catalyzed reduction of fatty acyl-CoA to fatty alcohol with the corresponding aldehyde as an intermediate with an optimal pH of 7.0. Both enzymes showed a strong preference for NADPH over NADH as a reductant. Apparent Km for NADPH was 38 [micro]M for Rv3391 product …

Correlation Of Rpob Gene Mutation With Clinical Rifabutin And Rifampicin Resistance For Treatment Of Crohn's Disease, Daniel Beckler

Electronic Theses and Dissertations

Emerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 10 MAP isolates in attempt to seek correlation with rpoB sequences. We determined that MAP strain …

Mechanism Of Action And Regulation Of Membrane Serine Protease Prostasin In The Prostate And Prostate Cancer, Mengqian Chen

Electronic Theses and Dissertations

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation …

A Member Of The Novel Fikk Family Of Plasmodium Falciparum Putative Protein Kinases Exhibits Diacylglycerol Kinase Activity And Is Exported To The Host Erythrocyte, David Floyd Curtis

Electronic Theses and Dissertations

Plasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for malaria is extremely urgent. The recent identification of a group of novel protein kinases within the Plasmodium falciparum genome has provided …