Biochemistry, Biophysics, and Structural Biology Commons^™

Section Abstracts: Structural Biology, Biochemistry, And Biophysics

Virginia Journal of Science

Abstracts of the Structural Biology, Biochemistry, and Biophysics Section for the 93rd Annual Meeting of the Virginia Academy of Science, May 21-23, 2015, James Madison University, Richmond, Virginia

Section Abstracts: Biology With Microbiology And Molecular Biology

Virginia Journal of Science

Abstracts of the Biology with Microbiology and Molecular Biology Section for the 93rd Annual Meeting of the Virginia Academy of Science, May 21-23, 2015, James Madison University, Richmond, Virginia

An Improved 96-Well Turbidity Assay For T4 Lysozyme Activity, Tasha B. Toro, Thao P. Nguyen, Terry J. Watt

Faculty and Staff Publications

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other …

Structural And Functional Interactions Between Bro1 Domain Of Human Alix Protein And Nucleocapsid Packaging Rna Complex From Hiv, Scott Gross

Graduate School of Biomedical Sciences Theses and Dissertations

A virus is only as powerful as its ability to spread. Enveloped retroviruses, namely HIV-1, use exocytosis pathways that normal host cells use to release particles from the plasma membrane. The main pathways of interest in this study are the Endosomal Sorting Complex Required for Transport (ESCRT) and adjacent ALIX pathways. The ESCRT pathway is especially important for degradation of receptor/cargo complexes that form Multi-Vesicular Bodies (MVBs). Currently, there is no known therapy that targets this endosomal pathway, which would prevent the spread of the virus to other cells. The virus has adapted to jump from pathway to pathway when …

Age-Dependent Breakdown Of The Blood Brain Barrier And Associated Changes In S100b Ko Mice, Eric V. Brown

Graduate School of Biomedical Sciences Theses and Dissertations

Autoantibodies play an important role in many autoimmune diseases. Recent research has shown that breakdown of the blood brain barrier (BBB) occurs concomitant to generation of brain reactive autoantibodies in many neurodegenerative diseases, which serve as biomarkers and drivers of pathology. SI00B, a calcium binding protein found most highly expressed in astrocytes which ensheathe the BBB, has many functions in neural development and signaling. Currently literature indicates that S100B KO mice develop normally, with no phenotypic abnormalities. Here, it is demonstrated that S100B KO mice seem to develop a chronic BBB breakdown similar to that seen in human neurodegenerative diseases. …

Evolution Of Sperm Activation In Nematodes, Qing Wei

Graduate School of Biomedical Sciences Theses and Dissertations

Three hermaphroditic species in the nematode genus Caenorhabditis - C. elegans, C. briggsae and C. tropicalis evolved independently from a male/female ancestor. Thus, the hermaphroditic reproductive system provides an ideal subject for studying the origin of new complex traits. Previous studies implied that this transition required two steps: a mutation that caused XY animals to make sperm, and a mutation that allowed XX spermatids to activate and fertilize oocytes. In this dissertation, I show how sperm activation is regulated in different hermaphroditic species.

First, I used TALENs to create genetic tools for C. briggsae and C. tropicalis. These tools were …

Understanding The Molecular Level Interactions Of Cancer Inhibitor Imatinib With Human Fibroblast Growth Factor-1, Tulsi Modi

Masters Theses & Specialist Projects

Fibroblast growth factors (FGFs) lack signal sequences, and are exported through endoplasmic reticulum (ER)-Golgi-independent non-classical routes. FGFs work as modulators of various cellular activities like mitosis, differentiation, survival etc. Among the FGF family, which comprises of 23 different heparin proteins, human FGF-1 (hFGF-1), a potent angiogenic factors are one of the targets in cancer inhibition, as they are involved in blood vessel formation in tissues. There has been intensive research directed at the development of drugs that could effectively inhibit angiogenesis. In this context, the purpose of this study is to fully understand the molecular principles essential to determine probability …

Biofilm-Related Materials As Total N-Nitrosamine (Tono) Precursors And Hydroxylamine-Based Interferences In Tono And N-Nitrosodimethylamine (Ndma) Measurements, David Allen Meints

Graduate Theses and Dissertations

In this study, a chemiluminescence-based total N-nitrosamine (TONO) assay was adapted to include a solid-phase extraction (SPE) step to assess the role of biologically derived materials as N-nitrosamine precursors. Methanol was determined to be a suitable solvent for the SPE-TONO assay, with N-nitrosodimethylamine (NDMA) losses estimated to be 30% by SPE alone and an additional 15% from concentration of the methanol extracts by N2-gas blowdown. Three biofilm-derived materials - poly-N-acetylglucosamine (PNAG), Pseudomonas aeruginosa, and tryptophan - were individually chloraminated and dose-response relationships were observed with the SPE-TONO assay, indicating biofilm are potential N-nitrosamine precursors. The role of hydroxylamine - a …

Scale Up Isolation Of Aaptamine For In Vivo Evaluation Indicates Its Neurobiological Activity Is Linked To The Delta Opioid Receptor, Eptisam Lambu, Nicole L. Mcintosh, Madeline Ferwerda, Allison Coker, Laura Millan-Lobo, Nicholas Lorig-Roach, Phillip Crews, Jennifer L. Whistler

Student Research Posters

Opioid receptors belong to the large superfamily of seven transmembrane-spanning (7TM) G protein-coupled receptors (GPCRs). As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones. The Mu (µ), Delta (δ) and Kappa (MOR, DOR, KOR) opioid receptors are particularly intriguing members of this receptor family as they are the targets involved in many neurobiological diseases such as addiction, pain, stress, anxiety, and depression. To date few marine natural products have been investigated for their neurobiological activities.1 One noteworthy example involves ziconotide (1) from the cone snail Conus magnus.2 Compound 1 …

Discovering The Sequence Specificity Of Human Dyrk2 And Dyrk4, Julie Klaric

University Scholar Projects

Protein phosphorylation is a post-translational modification (PTM) that is ubiquitous in regulating cellular processes. It is the most common PTM used in signal translation. Protein kinases are the class of enzymes that catalyze the transfer of a phosphate group from ATP to a specific amino acid on a substrate protein. In eukaryotes, kinases generally add a phosphate to serine, threonine, or tyrosine residues. Short linear patterns in the amino acid sequence of the substrate protein help guide the protein kinase to the correct residue to be phosphorylated. However, these patterns, or “motifs,” as well as the complete list of substrates …

Venom Peptide Induced Inhibition Of Escherichia Coli Atp Synthase, Sofiya Azim

Undergraduate Honors Theses

ATP is the main cellular energy generated by the enzyme ATP synthase in almost all organisms from bacteria to vertebrates. While malfunction of the ATP synthase complex is responsible for several disease conditions, the enzyme itself can be used as a potent molecular drug target to combat many diseases including microbial infections, cancer, tuberculosis, and obesity. Recent widespread escalation of antibiotic resistant microbes in general and E. coli in particular demands novel alternative approaches to combat microbial infections. Inhibition of ATP synthase by inhibitors such as peptides is known to deprive microbes of required energy, resulting in microbial cell death. …

Characterization Of A Novel Clade Of Transporters In Phytophthora, Stephanie Padula, Paul F. Morris Dr, Howard Casey Cromwell Dr., Menaka Ariyaratne, Andrew Wagner

Honors Projects

The oomycete Phytophthora parasitica has a worldwide distribution and is an economically important pathogen of more than 100 species⁴. RNA-seq analysis showed that one gene, PPTG_16698 has the 5^th highest level of expression of all transport proteins in the zoospore stage, and is highly conserved throughout Phytophthora species. This project attempts to characterize the important biological role that PPTG_16698 plays in P. parasitica and other oomycetes. Three strategies have been implemented to accomplish this goal: growth analysis by heterologous expression in yeast, metabolite analysis in yeast, and construction of a GFP fusion protein to enable localization of …

Design Of Antimicrobial Peptide Based On The Heparin Binding Segment Of Fgf-1, Alice Margaret Power

Chemistry & Biochemistry Undergraduate Honors Theses

Fibroblast Growth Factors (FGFs) are heparin-binding proteins known for their involvement in various biological processes such as cell differentiation and wound healing.1 The heparin binding site of FGF-1 displays a unique stretch of positive amino acid sequence and facilitates a very strong binding to negatively charged heparin due to the formation of electrostatic interaction. The aim of this study is to analyze and modify the novel antimicrobial peptide sequence (GST-HB) designed based on the heparin-binding region of FGF-1 as well as other polycationic microbial sequences. These aspects will be examined using various experimental techniques including overexpression of GST-HB, purification using …

Additional Sex Combs-Like 2 (Asxl2): A Psychological Stress-Related Gene In A Chicken Stress Model, Emily Sandra Jacobson

Chemistry & Biochemistry Undergraduate Honors Theses

Chronic stress is a problem not only in terms of its psychological effects but also due to the harmful physiological changes induced in the individual. Although chronic stress is recognized as detrimental and rampant throughout society, the stress system itself remains poorly understood in terms of comprehending which genes are involved in regulating and carrying out stress-induced signals. In a previous study, 57 candidate genes in the stress pathway had been identified based on the presence of stress-associated single nucleotide polymorphisms (SNPs) and differential expression from microarray data. The objective of this study was to investigate Additional Sex Combs-Like 2 …

Devlopment Toward Fluorescence Imaging Of Microdialysis Sampling Diffusion Profiles, Nicholas Edward Baioni

Chemistry & Biochemistry Undergraduate Honors Theses

Microdialysis is a diffusion-based method used to collect analytes from specific locations in the body. Matrix Metalloproteinases (MMPs) are important enzymes located throughout the body that play a role in the breakdown of extracellular matrix. Microdialysis sampling in conjunction with appropriate analysis methods can be used to observe the reactions of MMPs in real time. This project is aimed at determining the activity of porcine pancreatic enzyme, a known substitute for MMP-12, using nanodrop spectroscopy. This is a necessary pre-requisite toward using microdialysis in an in vivo imaging setup. These experiments were performed using Succinyl(Ala)_3-p-nitroanilide (suc(Ala)3-p-NA), 4-nitroaniline, and porcine pancreatic …

Molecular Mechanisms Of Tandem Cbd Of Clostridium Histolyticum, Dawn Weir

Chemistry & Biochemistry Undergraduate Honors Theses

In order to spread infections, bacterial collagenases methodically unravel collagen fibril in tissues. Collagen is the most abundant protein in the body, and can be found in the skin, bone and cartilage [1]. Two collagenases, ColG and ColH, synergistically dismantle collagen fibrils by seeking different weak links in the collagen structure. The collagen-binding domain (CBD) of these collagenases binds to most vulnerable regions in collagen [8]. Without CBDs, collagen fibril cannot be degraded. Cells express collagen receptors in order to anchor themselves, which is a critical step in cell proliferation. Binding sites for some collagen receptors, such as integrin and …

Efforts In Increasing Microdialysis Recovery Rates By Utilizing Bidirectional Flow Capabilites, Jacob Taylor Needham

Chemistry & Biochemistry Undergraduate Honors Theses

Microdialysis sampling involves the collection of biological fluids from tissues or fluid-filled cavities in vivo via diffusion through a semipermeable membrane probe. In order to increase the recovery of fluids for analysis from this process, a new regime was attempted that would allow perfusion fluids to make multiple passes through the probe in order to collect more analyte with each additional pass. This was dubbed the Bidirectional Flow Technique. Dextran-70 solution was used as the perfusion fluid while 100μM Methyl Orange solution was used as the analyte. The experiments were performed in vitro using a fully automated microdialysis ePump capable …

Chymotrypsin Digestion Analysis To Characterize Site-Specific Incorporation Of An Extrinsic Fluorescent Probe On A Ras-Related Protein., Akash D. Patel

Chemistry & Biochemistry Undergraduate Honors Theses

Ras Homolog Enriched in Brain (RHEB) is a member of Ras GTPase family and plays an important role in regulation of cell growth and cell cycle proliferation. RHEB is regulated by cycling between active (GTP-bound) and inactive (GDP-bound) state. Guanidine Nucleotide Exchange Factors (GEFs) and GTPase-activationg proteins (GAPs) are the key regulators of RHEB, and it is of interest to closely study their interactions with RHEB. Towards that end, this project was designed to label RHEB with an environmentally sensitive extrinsic fluorophore, succinimidyl 6-(N-(7-nitrobenz-2-oxa-1.3-diazol-4-yl)amino)hexanoate (sNBD), to be used to monitor RHEB-protein interactions. Due to its extreme sensitivity to the surrounding …

Influence Of A Potentially Destabilizing Central Tryptophan On Transmembrane Helix Domains, Vasupradha Suresh Kumar

Chemistry & Biochemistry Undergraduate Honors Theses

The vital role that membrane proteins play in cell mechanisms and the inner workings of disease in our bodies has been universally accepted. Thus, the study of protein-membrane and transmembrane interactions has become increasingly significant in understanding these membrane proteins. Synthetic model peptides, such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide)—designed at the University of Arkansas and composed of the amino acid residues of glycine (G), alanine (A), leucine (L) and tryptophan (W) —provide a favorable “host” framework for investigations of the influence of chosen “guest” amino acids. For example, it is of interest to know the consequences of having a third, centrally located, …

Percent Recovery Of Various Analytes Using A Wick Method, Patrick A. Naeger

Chemistry & Biochemistry Undergraduate Honors Theses

Finding alternate methods to measure and isolate interstitial fluid in vivo has become a consequence of the limited recovery of analytes when using a probe.1 These probes consist of an inlet constantly bringing perfusate into a semi-permeable membrane where the perfusate is replace with dialysate containing numerous analytes. The dialysate leaves the semi-permeable membrane via an outlet. A wick technique has been shown to be useful in analytical chemistry because it can be used to effectively isolate interstitial fluid and accurately measure the concentrations of various analytes. For long-term applications, wicks can be used to measure the concentrations of cytokines …

Efficient In Vitro Development Of Photoreceptors From Human Pluripotent Stem Cells, Joseph C. Reynolds

Dissertations, Masters Theses, Capstones, and Culminating Projects

Degeneration of the rod and cone photoreceptors in the human retina is among the most common causes of blindness. Replacing these damaged photoreceptors may help to restore vision. Repairing the damaged retina relies on the insertion of new, healthy cells. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are two possible sources of photoreceptors to restore vision. Previous data shows that human ES cells and iPS cells can be differentiated into photoreceptors and transplanted into the eye to restore some vision. However, this process is inefficient, and costly. Here, we show a new method for inducing photoreceptor production …

A Multilaboratory Comparison Of Calibration Accuracy And The Performance Of External References In Analytical Ultracentrifugation, Huaying Zhao, Rodolfo Ghirlando, Carlos Alfonso, Fumio Arisaka, Ilan Attali, David L. Bain, Et Al. ..., Donald F. Becker, Peter Schuck

Department of Biochemistry: Faculty Publications

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy …

Mirror Buckling Transitions In Freestanding Graphene Membranes Induced Through Scanning Tunneling Microscopy, James Kevin Schoelz

Graduate Theses and Dissertations

Graphene has the ability to provide for a technological revolution. First isolated and characterized in 2004, this material shows promise in the field of flexible electronics. The electronic properties of graphene can be tuned by controlling the shape of the membrane. Of particular interest in this endeavor are the thermal ripples in graphene membranes. Years of theoretical work by such luminaries as Lev Landau, Rudolf Peierls, David Mermin and Herbert Wagner have established that 2D crystals should not be thermodynamically stable. Experimental research on thin films has supported this finding. Yet graphene exists, and freestanding graphene films have been grown …

Exploring The Oncolytic Potentail Of A Double Knock-Out Tanapoxvirus Containing Flic Transgene, Esaw W. Kurban

Masters Theses

Since the emergence of oncolytic viruses (OVs), OVs have seen success in clinical trials and are a vital alternative treatment for cancer. Colorectal cancers are diseases which cause substantial health issues in the developed world, but current invasive treatment methods do not perform well in patients with advanced disease. We have explored the potential of tanapoxvirus (TPV) for its oncolytic virothrapy in two manners; specific gene deletion and non-targeted gene deletion. The non-targeted mutation of TPV’s genome was attempted using N-methyl-N-nitro-N-nitrosoguanidine (NTG) and N-ethyl-N-nitrosourea (ENU) as alkylating chemical. Specific gene deletion involved knocking-out a thymidine kinase gene (TPV-66R) and a …

The Use Of Shape Memory Polymers As A Tool To Study Human Fibrosarcoma And Murine Mesenchymal Stem Cell Migration, Justin N. Elkhechen

Renée Crown University Honors Thesis Projects - All

Shape memory polymers (SMPs) are a class of “smart” materials that can transform between two distinct conformations through external stimuli, such as heat or electricity. Their usage in bioengineering has led to a promising field of research that lies at the interface of cell and mechanobiology, potentially providing insight into cancer therapies and tissue development—two processes that exist in dynamic environments in vivo. The present work involves creating new, shape changing, scaffolds for studies to analyze cell migration upon changes to the environmental topography. Specifically, this Capstone has been primarily focused on the development of a “half and half” fibrous …

The Effect Of High Terperature On Mating: Developmental Buffering Of S. Cerevisiae To The Environment, Sarah Hosie

Renée Crown University Honors Thesis Projects - All

In Saccharomyces cerevisiae, cellular polarization is an essential structural and functional aspect of growth and development. It is responsible for yielding and maintaining cellular asymmetry, and allows for cells to function. Mating in S. cerevisiae is a process that incorporates cell-to-cell signaling, signal transduction, cellular polarization, plasmogamy, karyogamy, and many other cellular processes. Each of these steps is mediated by a myriad of signaling proteins that are involved in a signaling cascade that is regulated by both extracellular and intracellular signals. Much is known about the mating process and pathway in S. cerevisiae. However, this project aimed to …

Overexpression And Gene Profiling Of Asparagine Synthetase In Hybrid Poplar, Kristopher Murray

Renée Crown University Honors Thesis Projects - All

Plants with polysaccharide-rich secondary cell walls have great potential as a source of carbohydrates for bioethanol production. However, the presence of phenolic lignin inhibits the isolation of bioethanol precursors cellulose and hemicellulose from the secondary cell wall. Recent studies have linked nitrogen availability to secondary cell wall development and composition, making nitrogen metabolism genes an interesting target in the improvement of plant cell walls for biofuels production. Plants use a nitrogen assimilation pathway to convert inorganic nitrogen sources into organic sources, included amino acids, which play key roles in metabolism and cell wall development. Asparagine synthetase (AS), a key enzyme …

Characterization Of Ghrelin O-Acyltransferase Active Site, Leslie Patton

Renée Crown University Honors Thesis Projects - All

Ghrelin, first discovered in 1999, is a 28-amino acid peptide hormone involved in the regulation of appetite, insulin secretion and sensitivity, and many neurological effects such as learning, memory, and depression.^1-6 Ghrelin has been identified to have a unique posttranslational octanoylation carried out by the enzyme ghrelin O-acyltransferase (GOAT). This distinctive modification is a point of interest in studying GOAT whereby blocking the acylation of the ghrelin could potentially halt the activity of the peptide hormone and provide a means of treating obesity, diabetes, and other diseases affected by ghrelin levels. The duration of my project involved working …

Role Of Ribosomal Biogenesis In Axonal Morphogenesis., Nicholas Mcchesney Lynch

College of Arts & Sciences Senior Theses

Elongating axons require a host of macromolecules for outgrowth. The importance of lipid, protein, and general RNA synthesis for initial axonal growth has been previously investigated, but the role of rRNA synthesis and ribosome production in axonal morphogenesis remains unclear. Therefore, rat hippocampal neurons were cultured and transfected with shRNA against either TIF-IA (the activator of RNA polymerase I) or RPS6 (a small ribosomal subunit component) to determine the effects of impaired de novo ribosome synthesis on axonal development during the first three days after plating. Our data show that shRPS6 weakly inhibited total axon length, but not longest axon …

Developing Graphene-Based Supercapacitors For High Temperature Applications, Lea Rachel Rogers

Chemistry & Biochemistry Undergraduate Honors Theses

Supercapacitors are ideal tools for energy storage due to their wide range of operation temperatures, long cycle-lives, high power densities of up to 13,000 W/kg, and fast rates of charge-discharge thereby having a high specific capacitance. This research reports synthesis of grapheme-based materials using a modified Hummer’s method. Graphene oxide synthesized first and then converted to reduced graphene oxide sheets and graphene-powder, which both increase the ionic conductivity due to sp2 bonding and a high surface area. These materials were then constructed into a button-cell supercapacitor for testing. For the first supercapacitors, a porous polypropylene membrane (Celgard) that acted as …