Biochemistry, Biophysics, and Structural Biology Commons^™

The Termite Digestome: Understanding The Digestive Physiology Involved In Lignocellulosic Biomass Degradation, Zachary John Karl

Open Access Dissertations

The purpose of this research was to advance the understanding of lower termite digestive physiology and discover potential biocatalysts that can aid in the degradation of lignocellulosic biomass. Various protein characterization and gene expression methods were used throughout this research in order to accomplish these objectives. The results of this dissertation indicate that: 1) termites and their symbionts act in a synergistic manner to degrade biomass in vitro, 2) the host fraction of the gut (i.e., foregut and midgut) is the likely site of glucose absorption, 3) the termite and its symbionts contribute specific enzymes to the digestive process, 4) …

Development Of Tyrosine Kinase Peptide Biosensors And Methods For Detection, Andrew Michael Lipchik

Open Access Dissertations

New methods to monitor tyrosine kinase activity are critical for studying kinases in cell biology, drug discovery and the clinic. Peptide-based biosensors for detection of kinase activity utilitize a kinase specific artificial peptide substrate, which can report intercellular kinase activity through the incorporation of phosphate.

An artificial Syk substrate peptide was developed and incorporated with other functional modules to produce a Syk biosensor. These modules included a biotin-tag for affinity capture, a photo-cleavable amino acid to allow release of the substrate from the delivery module and the cell penetrating peptides TAT. A live cell kinase assay utilizing this biosensor was …

Generation And Statistical Modeling Of Active Protein Chimeras: A Sequence Based Approach, Nicholas Fico

Open Access Dissertations

Generation of active protein chimeras is a valuable tool to probe the functional space of proteins. Statistical modeling is the next logical step, allowing us to build a model of gene fragment replaceability between species. In this thesis I begin to develop the statistical tools that are needed to systematically describe combinatorial protein libraries. I present three sets of diverse chimeric protein libraries developed using sequence information. The statistical model of the human N-Ras and human K-Ras-4B genes reveal a set previously unidetifed surface residues on the N-Ras G-Domain that may be involved in cellular localization. Statistical modeling of a …

Characterization Of The Specificity And Functions Of The Protein Phosphatase Cdc14, Christie Eissler

Open Access Dissertations

Protein phosphorylation is perhaps the most ubiquitous posttranslational modification in eukaryotes and recent studies suggest that upwards of 75% of human proteins are phosphorylated. Many proteins are phosphorylated at multiple sites, often controlled by multiple kinases and phosphatases. Multisite phosphorylation can differentially affect the functional and regulatory cellular outcomes. For example, dephosphorylation of a protein at a particular site may inhibit nuclear localization of a protein while dephosphorylation of a different site may be necessary for enzymatic activation of a protein. Thus, multisite protein phosphorylation can complicate our understanding of the biological significance and the functional consequences of protein phosphorylation. …

Structural And Functional Characterization Of The Endosome-Associated Deubiquitinating Enzyme Amsh, Christopher Williamson Davies

Open Access Dissertations

The endosomal sorting complexes required for transport (ESCRT) machinery is a ubiquitin-dependent molecular mechanism made of up of four individual complexes: ESCRT-0, -I, -II, III, that is necessary for regulating the degradation of cell surface receptors directed towards the lysosome. Not only are the ESCRTs implicated in endosomal sorting and trafficking of proteins, its members also have roles in other important biological processes such as: cytokinesis, HIV budding, transcriptional regulation, and autophagy. As a function of its involvement in several processes throughout the cell, the ESCRT machinery is implicated in a wide variety of diseases including cancer, neurological disease, bacterial …

Measuring And Modeling The Response Characteristics Of The Environmental Phosphate Transducer In Escherichia Coli, Chetan Sood

Open Access Dissertations

The PhoR/PhoB two-component system in Escherichia coli is a biological transducer that senses the limitation of environmental inorganic orthophosphate, the bacteria's preferred source of the essential nutrient phosphate, and transmits that information to the interior of the cell initiating a response that mitigates phosphate starvation. In the first part of this study, we present and apply a fluorescence microscopy technique to measure, in vivo, the dynamic response characteristics of the transducer with single-cell resolution. We report that the transience in the PhoR/PhoB TCS response is consistent with the transducer having a threshold sensitivity to the concentration of environmental phosphate, …

Small Molecule Antagonists Of Melanopsin-Mediated Phototransduction, Kenneth A. Jones, Megumi Hatori, Ludovic S. Mure, Jayne R. Bramley, Roman Artymyshyn, Sang-Phyo Hong, Mohammad Marzabadi, Huailing Zhong, Jeffrey Sprouse, Quansheng Zhu, Andrew T. E. Hartwick, Patricia J. Sollars, Gary E. Pickard, Satchidananda Panda

School of Veterinary and Biomedical Sciences: Faculty Publications

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several CNS responses including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. Novel sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod/cone mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses including the pupillary light reflex and …

Identification Of Cell Cycle–Regulated Genes Periodically Expressed In U2os Cells And Their Regulation By Foxm1 And E2f Transcription Factors, Gavin D. Grant, Lionel Brooks Iii, Xiaoyang Zhang, J. Matthew Mahoney, Viktor Martyanov, Tammara A. Wood, Gavin Sherlock, Chao Cheng, Michael L. Whitfield

Dartmouth Scholarship

We identify the cell cycle–regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle–regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle–regulated genes. FOXM1 is bound to cell cycle–regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly …

Size-Based Separation Of Bioparticles Using Planar Nanofluidic Devices, Jie Xuan

Theses and Dissertations

Nanofluidic devices are structures having at least one dimension in the submicron range, which is of the same order of magnitude as the sizes of biomolecules and bioparticles such as proteins and viruses. As a result, size-selective separations are important applications for nanofluidics. Well-defined micro or nano device structures fabricated via micromachining have greatly reduced sample consumption and enabled separations in a parallel fashion, promising significant speed and resolution advantages over conventional size separation techniques, such as gel electrophoresis and size exclusion chromatography. In collaboration with others, I have developed a size separation method using nanofluidic devices consisting of an …

The Tethering Complex Hops Catalyzes Assembly Of The Soluble Snare Vam7 Into Fusogenic Trans-Snare Complexes, Michael Zick, William Wickner

Dartmouth Scholarship

The fusion of yeast vacuolar membranes depends on the disassembly of cis–soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The …

A Computational Framework For High-Throughput Isotopic Natural Abundance Correction Of Omics-Level Ultra-High Resolution Ft-Ms Datasets, William J. Carreer, Robert M. Flight, Hunter N. B. Moseley

Molecular and Cellular Biochemistry Faculty Publications

New metabolomics applications of ultra-high resolution and accuracy mass spectrometry can provide thousands of detectable isotopologues, with the number of potentially detectable isotopologues increasing exponentially with the number of stable isotopes used in newer isotope tracing methods like stable isotope-resolved metabolomics (SIRM) experiments. This huge increase in usable data requires software capable of correcting the large number of isotopologue peaks resulting from SIRM experiments in a timely manner. We describe the design of a new algorithm and software system capable of handling these high volumes of data, while including quality control methods for maintaining data quality. We validate this new …

Β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic To The Plasma Membrane And Retain Functionality, Jaclyn Bubnell, Patrick Pfister, Maria L. Sapar, Matthew E. Rogers, Paul Feinstein

Publications and Research

Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane …

Differential Biosynthesis Of Antioxidant Stilbenoids In Hairy Root Cultures Of Muscadine Grape And Peanut Treated With Methyl Jasmonate And Hydrogen Peroxide, Cesar Augusto Nopo-Olazabal

Student Theses and Dissertations

Stilbenoids are polyphenolic compounds that are believed to protect plants against abiotic and biotic stresses. Importantly, an increasing number of these specialized plant metabolites have been associated with potential health benefits in humans. Therefore, understanding the molecular mechanisms involved in their biosynthesis is of importance to enhance the levels of these beneficial compounds in plants. In this research we studied the biosynthesis of stilbenoids in two economically important food crops: muscadine grape and peanut. Hairy root cultures were used as biological systems because their genetic and biochemical stability facilitates the study of specialized metabolism. Selected hairy root cultures from muscadine …

Identification Of Proteins Potentially Involved In The Formation Of Lafora Bodies, A Hallmark Of Lafora Disease, Elham Schokraie, Oliver Kötting, Matthew S. Gentry

Molecular and Cellular Biochemistry Presentations

Lafora Disease (LD) is a fatal teenage-onset progressive myoclonus epilepsy. It is characterized by the formation of Lafora bodies (LBs), deposits of abnormally branched, insoluble, hyperphosphorylated glycogen-like polymers that are generally believed to trigger the development of the clinical symptoms of LD. 58% and 35% of the LD cases are caused by mutations in EPM2A (laforin) and EPM2B (malin), respectively. However, little is known about their function in LB formation. Two different mechanisms have been proposed to explain the accumulation of insoluble LBs: first, excessive glycogen phosphorylation and, second, an imbalance between glycogen synthesizing enzymes. The present study aims at …

Computational Optogenetics: Empirically-Derived Voltage- And Light-Sensitive Channelrhodopsin-2 Model, John C. Williams, Jianjin Xu, Zhongju Lu, Aleksandra Klimas, Xuxin Chen, Christina M. Ambrosi, Ira S. Cohen, Emilia Entcheva

Department of Biomedical Engineering Faculty Publications

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silicoprediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, …

Formation, Functionalization, Characterization, And Applications Of A Mixed-Mode, Carbon/Diamond-Based, Core-Shell Phase For High Performance Liquid Chromatography, Landon A. Wiest

Theses and Dissertations

My work has focused on a variety of different types of diamond-based, core-shell particles. These particles are formed with inert cores and poly(allylamine)/nanodiamond shells. Their intended purpose is to form an LC stationary phase that is stable from pH 1 – 14 and at elevated temperatures. At the beginning of my studies, the particles that had been made in the Linford laboratory were pH stable, but irregular and had poor mechanical stability. Since that time, I have worked to improve the particles by using more spherical zirconia and carbon cores, and I have improved their mechanical stability via chemical crosslinking …

Membrane Lipid-Modulated Mechanism Of Action And Non-Cytotoxicity Of Novel Fungicide Aminoglycoside Fg08, Sanjib Shrestha, Michelle Grilley, Marina Y. Fosso, Cheng-Wei Tom Chang, Jon Y. Takemoto

Chemistry and Biochemistry Faculty Publications

A novel aminoglycoside, FG08, that differs from kanamycin B only by a C8 alkyl chain at the 4″-O position, was previously reported. Unlike kanamycin B, FG08 shows broad-spectrum fungicidal but not anti-bacterial activities. To understand its specificity for fungi, the mechanism of action of FG08 was studied using intact cells of the yeast Saccharomyces cerevisiae and small unilamellar membrane vesicles. With exposure to FG08 (30 µg mL−1), 8-fold more cells were stained with fluorescein isothiocyanate, cells had 4 to 6-fold higher K+ efflux rates, and 18-fold more cells were stained with SYTOX Green in comparison to exposure to kanamycin B …

Laboratory Characterization Of Pm Emissions From Combustion Of Wildland Biomass Fuels, S. Hosseini, S. P. Urbanski, P. Dixit, L. Qi, I. R. Burling, Robert J. Yokelson, T. J. Johnson, M. Shrivastava, H. S. Jung, D. R. Weise, J. W. Miller, D. R. Cocker Iii

Chemistry and Biochemistry Faculty Publications

[1] Particle emissions from open burning of southwestern (SW) and southeastern (SE) U.S. fuel types during 77 controlled laboratory burns are presented. The fuels include SW vegetation types: ceanothus, chamise/scrub oak, coastal sage scrub, California sagebrush, manzanita, maritime chaparral, masticated mesquite, oak savanna, and oak woodland, as well as SE vegetation types: 1 year, 2 year rough, pocosin, chipped understory, understory hardwood, and pine litter. The SW fuels burned at higher modified combustion efficiency (MCE) than the SE fuels resulting in lower particulate matter mass emission factor. Particle mass distributions for six fuels and particle number emission for all fuels …

Serdemetan Antagonizes The Mdm2-Hif1Α Axis Leading To Decreased Levels Of Glycolytic Enzymes, Jason Alexander Lehman, Paula M. Hauck, Jaimie M. Gendron, Christopher N. Batuello, Jacob A. Eitel, Allan Albig, Madhavi P. Kadakia, Lindsey D. Mayo

Biochemistry and Molecular Biology Faculty Publications

Serdemetan (JNJ-26854165), an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1), were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises …

Protein Similarity Networks Reveal Relationships Among Sequence, Structure, And Function Within The Cupin Superfamily, Richard Uberto, Ellen W. Moomaw

Faculty Articles

The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional …

Combining Orthogonal Trna/Synthatase Pair And Amber Codon Suppression To Genetically Encode Oxidative Damage In High Density Lipoproteins, Jaclyn Alatrash, Nicholas Gilliam, Alisha House, Valentin Gogonea

Undergraduate Research Posters 2013

Apolipoprotein A-I (apoA-I) is the main protein constituent of high density lipoprotein (HDL - the “good cholesterol”). Oxidatively damaged apoA-I has been isolated from circulating plasma and atherosclerosis plaque with the amino acid residue tryptophan 72 (W72) of apoA-I identified as a primary oxidation site. ApoA-I designed to include specific oxidized amino acids can be used to further investigate the role of site-specific oxidative damage in atherosclerosis. Genetic encoding of oxidized amino acids through orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs offers a reliable method for producing site-specific oxidized proteins. Our project involves the generation of Saccharomyces tryptophan-RS mutants for recognition of …

Self-Assembled Dna Origami Templates For The Fabrication Of Electronic Nanostructures, Elisabeth Pound Gates

Theses and Dissertations

An important goal of nanoscience is the self-assembly of nanoscale building blocks into complex nanostructures. DNA is an important and versatile building block for nanostructures because of its small size, predictable base pairing, and numerous sequence possibilities. I use DNA origami to design and fold DNA into predesigned shapes, to assemble thin, branched DNA nanostructures as templates for nanoscale metal features. Using a PCR-based scaffold strand generation procedure, several wire-like nanostructures with varying scaffold lengths were assembled. In addition, more complex prototype circuit element structures were designed and assembled, demonstrating the utility of this technique in creating complex templates. My …

Transformations In Matter And Energy: Student Learning And Inquiry To Inform Teaching, Jenny Dauer

Discipline-Based Education Research Group Speaker Series

Learning progressions are descriptions of increasing levels of sophistication of student reasoning about a topic based on empirical evidence. Our learning progression framework about student explanations of carbon-transforming processes (e.g. photosynthesis, cellular respiration) describes how student’s interconnected and mutually supporting ideas and practices are deeply embedded in discourse at all levels of achievement. My research is in two areas: 1) applying the learning progression framework for student explanations of carbon-transforming processes to describe the most productive pathways for student learning, 2) extending the research to student reasoning during inquiry activities about carbon-transforming processes. One finding is that students who consistently …

Demonstration Of Crispr/Cas9/Sgrna-Mediated Targeted Gene Modification In Arabidopsis, Tobacco, Sorghum And Rice, Wen Zhi Jiang, Huanbin Zhou, Honghao Bi, Michael E. Fromm, Bing Yang, Donald P. Weeks

Department of Biochemistry: Faculty Publications

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/ single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/ sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites …

Draft Genome Sequence Of A Mucoid Isolate Of Pseudomonas Aeruginosa Strain C7447m From A Patient With Cystic Fibrosis, Yeshi Yin, T. Ryan Withers, Shannon L. Johnson, Hongwei D. Yu

Biochemistry and Microbiology

Alginate overproduction by Pseudomonas aeruginosa, or mucoidy, plays an important role in the pathogenesis of chronic lung infections in cystic fibrosis (CF) patients. Here we report the draft genome sequence of a clinical isolate of mucoid P. aeruginosa strain C7447m from a CF patient with chronic lung infection.

Draft Genome Sequence Of A Stable Mucoid Strain Of Pseudomonas Aeruginosa Pao581 With A Muca25 Mutation, Yeshi Yin, T. Ryan Withers, John R. W. Govan, Shannon L. Johnson, Hongwei D. Yu

Biochemistry and Microbiology

A mutation in the mucA gene, which encodes a negative regulator of alginate production in Pseudomonas aeruginosa, is the main mechanism underlying the conversion to mucoidy in clinical isolates from patients with cystic fibrosis (CF). Here, we announce the draft genome sequence of the stable alginate-overproducing mucoid strain P. aeruginosa PAO581 with a mucA25 mutation, a derivative from the nonmucoid strains P. aeruginosa PAO381 and PAO1.

Shared Mycobacterium Avium Genotypes Observed Among Unlinked Clinical And Environmental Isolates, M. Ashworth Dirac, Kris M. Weigel, Mitchell A. Yakrus, Annie L. Becker, Hui-Ling Chen, Gina Fridley, Arthur Sikora, Cate Speake, Elizabeth D. Hilborn, Stacy Pfaller, Gerard A. Cangelosi

Chemistry and Physics Faculty Articles

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this assumption, a high-resolution PCR-based genotyping approach, large-sequence polymorphism (LSP)-mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU-VNTR), was selected and used to analyze clinical and environmental isolates of M. avium from geographically diverse sources. Among 127 clinical isolates from seven locations in North America, South America, and Europe, 42 genotypes were observed. Among 12 of these genotypes, matches were seen in …

Computational Study Of The Cyclization Of 5-Hexenyl, 3-Oxa-5-Hexenyl And 4-Oxa-5-Hexenyl Radicals, Albert Matlin, Matthew C. Leyden

Faculty & Staff Scholarship

The intramolecular cyclization of 5-hexenyl radicals continues to be an important synthetic method for the construction of five-membered rings. The synthetic utility arises from the high degree of regioselectivity to give predominantly cyclopentyl products in high yield under mild conditions. Recently we reported product cyclization studies on 4-oxa perturbed 5-hexenyl radical. In this paper we report our results from a computational study (UB3LYP and UCCSD(T)) of the cyclization of a series of 5-hexenyl and 3- and 4-oxa-5-hexenyl radicals. Three highly conserved cyclization transitions states (exo-chair, exo-boat and endo-chair) were located for 10 acyclic radicals. Activation en-ergies …

Allosteric Mechanism Of Water Channel Gating By Ca2+–Calmodulin, Steve Reichow, Daniel M. Clemens, J. Alfredo Freites, Karin L. Németh-Cahalan, Matthias Heyden, Douglas J. Tobias, James E. Hall, Tamir Gonen

Chemistry Faculty Publications and Presentations

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 …

The Creation Of Α2nc1 Transgenic Drosophila Melanogaster, Jordan Olberding

Mahurin Honors College Capstone Experience/Thesis Projects

The α2 isoform of collagen IV’s noncollagenous 1 domain (α2NC1 or canstatin) has shown promise as an anti-cancer molecule, possessing both angiostatic and pro-apoptotic properties. This work aims to further the knowledge of α2NC1 by conducting a truly in vivo study in which the Gal4-induced endogenous overexpression of α2NC1 in Drosophila melanogaster will be used to assess the effects of α2NC1 upon normal development as well as tumorigenesis. To this end, transgenic fly lines capable of overexpressing α2NC1 were created. Initial overexpression studies indicated no developmental phenotype was caused by abundance of α2NC1. However, future overexpression studies in which α2NC1 …