Molecular Biology Commons^™

Studies On Inositol Polyphosphates And Their Role In Programmed Cell Death, Rakhee Agarwal

Theses and Dissertations

The agents that induce apoptosis in cancerous cells are considered as potential candidates for cancer therapy. Inositol polyphosphates, the naturally occurring compounds, regulate diverse cellular processes including induction of apoptosis in certain cancerous cells and hence can be considered as potential anticancer agents. The aim of this research dissertation was to establish firmly the role of inositol polyphosphates in induction of apoptosis by examining their mechanism of action. The overall hypothesis was that alterations in intracellular levels of inositol polyphosphates would bring about apoptotic changes. The hypothesis was tested by changing endogenous inositol polyphosphates by way of chemical treatments known …

Computational Prediction Of The Agregated Structure Of Denatured Lysozyme, Pongsathorn Chotikasemsri

Masters Theses & Specialist Projects

Mis-folded proteins and their associated aggregates are a contributing factor in some human diseases. In this study we used the protein lysozyme as a model to define aggregation structures under denaturing conditions. Sasahara et al. (2007), Frare et al. (2009, 2006), and Rubin et al. (2008) observed conditions where heat denatured lysozyme formed fibril structures that were observed to be 8-17 nanometers in diameter under the electron microscope. Even though the crystal structure of lysozyme is known, the denatured form of this protein is still unknown. Therefore, we used Rosetta++ protein folding and blind docking software to create in silico …

Low-Dose Of Sodium Arsenite Causes Delayed Differentiation In C2c12 Mouse Myoblast Cells Through The Repression Of The Transcription Factor Myogenin, Amanda Steffens

All Theses

A number of epidemiological studies have correlated arsenic exposurwith cancer, skin diseases, cardiovascular diseases, and adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, which would indicate that arsenic has the ability to act as a developmental toxicant. Myoblast cells were exposed to 20nM sodium arsenite and allowed to differentiate into myotubes and expression of the muscle-specific transcription factor myogenin, along with the expression of myosin light chain …

Applications Of Variable Number Tandem Repeat Genotyping In The Validation Of An Animal Medical Model And Gene Flow Studies In Threatened Populations Of Reptiles, Candace D. Smith

Graduate Theses and Dissertations

We used variable number tandem repeats (VNTR) to validate the chicken as a human medical model for Pulmonary Arterial Hypertension. We identified seven regions on four chromosomes and interrogated for VNTR markers that significantly associate with Pulmonary Hypertension Syndrome/ascites. In those regions, we identified 7 candidate genes; AGTR1, ACE, p38MAPK, SST, 5HT2B, NET1, and CALM3 for further analysis as significantly contributing QTL for ascites/PHS. We also used variable number tandem repeats to measure gene flow and gather evidence for multiple paternity in a population of Timber rattlesnakes, Crotalus horridus. We were able to verify 1 VNTR that can be used …

Cloning And Expression Of Porcine Dicer And Argonaute-2, Heather Stowe

All Theses

In vitro-produced embryos exhibit aberrations in development, but the reasons for these developmental problems are unknown. Recently, a class of small non-coding RNA called microRNA (miRNA) has been described and reported to have roles in normal mammalian embryonic development. These miRNAs are encoded in the genome, transcribed by RNA pol II and processed into fragments approximately 22 nt in length by ribonuclease enzymes, the final one being a protein called Dicer. miRNA work through the RNA-induced silencing complex (RISC), of which the argonaute gene family are key proteins. Argonaute-2 (Ago2) has been identified as the only member possessing endonuclease activity, …

The Role Of Multiple Ccaat-Binding Factors In Candida Albicans Gene Expression, Lashall Lynn Bates

Graduate Theses and Dissertations

The CCAAT-binding factor is a heterooligomeric transcription factor that is evolutionarily conserved in eukaryotes. In yeast, the DNA-binding component that interacts with the CCAAT consensus sequence in promoters consists of the subunits termed Hap2p, Hap3p and Hap5p. In yeast and fungi, a fourth subunit, Hap4p, is required for regulating gene expression. The goal of this research is to understand the function of the Candida albicans CCAAT-binding factor and how it relates to virulence and pathogenicity. C. albicans is a human opportunistic pathogen responsible for a variety of mucosal and systemic infections that result in significant morbidity and mortality, particularly in …

Characterization And Functional Regulation Of Bioactive Peptides In Avian Macrophages And Heterophils, Lakshmi Kannan

Graduate Theses and Dissertations

Oligopeptides and low molecular weight polypeptides play central roles as effectors and signal transducers acting as hormones, neurotransmitters, growth factors, toxins, and antimicrobial factors that are important for the survival of the organism. Owing to the ubiquitous involvement of peptides in many key regulatory processes, we have been interested to identify native peptides in different cells and tissues and understand their functions. To conduct our studies, we used avian macrophages and heterophils as models of specialized cells which constitute central components of innate immunity. These studies involved (a) qualitative identification and characterization of the peptides associated with high intensity mass …

Quantifying And Resolving Multiple Vector Transformants In S. Cerevisiae Plasmid Libraries, Thomas C. Scanlon, Elizabeth C. Gray, Karl E. Griswold

Dartmouth Scholarship

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the …

Trnas: Cellular Barcodes For Amino Acids, Ranat Banerjee, Shawn Chen, Kiley Dare, Marla Gilreath, Mette Praetorius-Ibba, Medha Raina, Noah M. Reynolds, Theresa E. Rogers, Hervé Roy, Srujana S. Yadavalli, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl‐tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of …

Venn, A Tool For Titrating Sequence Conservation Onto Protein Structures, Jay Vyas, Michael R. Gryk, Martin R. Schiller

Life Sciences Faculty Research

Residue conservation is an important, established method for inferring protein function, modularity and specificity. It is important to recognize that it is the 3D spatial orientation of residues that drives sequence conservation. Considering this, we have built a new computational tool, VENN that allows researchers to interactively and graphically titrate sequence homology onto surface representations of protein structures. Our proposed titration strategies reveal critical details that are not readily identified using other existing tools. Analyses of a bZIP transcription factor and receptor recognition of Fibroblast Growth Factor using VENN revealed key specificity determinants. Weblink: http://sbtools.uchc.edu/venn/.

Broad Range Amino Acid Specificity Of Rna-Dependent Lipid Remodelling By Multiple Peptide Resistance Factors, Hervé Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacylphosphatidylglycerol synthases (aaPGSs) are multiple peptide resistance factors that transfer amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is used by bacteria to decrease the net negative charge of the cell envelope, diminishing affinity for charged molecules and allowing for adaptation to environmental changes. Lys-PGS, which transfers lysine to PG, is essential for the virulence of certain pathogens, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. Ala-PGS was also recently described, but little is known about the possible activities of other members of the highly diverse aaPGS family of proteins. Systematic …

Self-Assembly And Disassembly Of The Snare Complex: Examined Using Circular Dichroism And Atomic Force Microscopy, Jeremy D. Cook, Won Jin Cho, Timothy L. Stemmler, Bhanu P. Jena

Biochemistry and Molecular Biology Faculty Publications

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF-ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure.

The Cca Anticodon Specifies Separate Functions Inside And Outside Translation In Bacillus Cereus, Sandro F. Ataide, Theresa E. Rogers, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Bacillus cereus 14579 encodes two tRNAs with the CCA anticodon, tRNA^Trp and tRNA^Other. tRNA^Trp was separately aminoacylated by two enzymes, TrpRS1 and TrpRS2, which share only 34% similarity and display different catalytic capacities and specificities. TrpRS1 was 18-fold more proficient at aminoacylating tRNA^Trp with Trp, while TrpRS2 more efficiently utilizes the Trp analog 5-hydroxy Trp. tRNA^Other was not aminoacylated by either TrpRS but instead by the combined activity of LysRS1 and LysRS2, which recognized sequence elements absent from tRNA^Trp. Polysomes were found to contain tRNA^Trp, consistent with its role in …

A Proposed Syntax For Minimotif Semantics, Version 1., Jay Vyas, Ronald J. Nowling, Mark W. Maciejewski, Sanguthevar Rajasekaran, Michael R. Gryk, Martin R. Schiller

Life Sciences Faculty Research

BACKGROUND:

One of the most important developments in bioinformatics over the past few decades has been the observation that short linear peptide sequences (minimotifs) mediate many classes of cellular functions such as protein-protein interactions, molecular trafficking and post-translational modifications. As both the creators and curators of a database which catalogues minimotifs, Minimotif Miner, the authors have a unique perspective on the commonalities of the many functional roles of minimotifs. There is an obvious usefulness in standardizing functional annotations both in allowing for the facile exchange of data between various bioinformatics resources, as well as the internal clustering of sets of …

Differential Expression Of Collagen Type V And Xi Α-1 In Human Ascending Thoracic Aortic Aneurysms, Julia Thom Oxford

Biomedical Research Institute Publications and Presentations

Background—The molecular mechanisms leading to ascending thoracic aortic aneurysms (ATAAs) remain unknown. We hypothesized that alterations in expression levels of specific fibrillar collagens occur during the aneurysmal process.

Methods—Surgical samples from ascending aortas from patients with degenerative ATAAs were subdivided by aneurysm diameter: small, 5 to 6 cm; medium, 6 to 7 cm; and large, greater than 7 cm; and compared with nonaneurysmal aortas (mean diameter, 2.3 cm).

Results—Histology, immunofluorescence, and electron microscopy demonstrated greater disorganization of extracellular matrix constituents in ATAAs as compared with control with an increase in collagen α1(XI) within regions of cystic medial …

Geometric Build-Up Solutions For Protein Determination Via Distance Geometry, Robert Tucker Davis

Masters Theses & Specialist Projects

Proteins carry out an almost innumerable amount of biological processes that are absolutely necessary to life and as a result proteins and their structures are very often the objects of study in research. As such, this thesis will begin with a description of protein function and structure, followed by brief discussions of the two major experimental structure determination methods. Another problem that often arises in molecular modeling is referred to as the Molecular Distance Geometry Problem (MDGP). This problem seeks to find coordinates for the atoms of a protein or molecule when given only a set of pair-wise distances between …

Cloning And Expression Of A Putative Exported Protein Coding Gene Sas1089 From An Invasive Community Associated Staphylococcus Aureus Strain Mssa476, Nailah Alzaki

All-Inclusive List of Electronic Theses and Dissertations

Originally, methicillin resistant Staphylococcus aureus (MRSA) occurred primarily in the hospital setting and was designated hospital- associated MRSA (HA-MRSA). In recent years, isolates of Staphylococcus aureus from the community (CA-MRSA), found to be methicillin resistant, are not as resistant tooth~ antibiotics as HA-MRSA. However, community strains are noted for their invasiveness causing soft tissue infections with propensity for invasion to deeper sites.

Nudibranchs Of The Ross Sea, Antarctica: Phylogeny, Diversity, And Divergence, Christopher Shields

All Theses

The Southern Ocean (SO) surrounding Antarctica is extremely cold and geographically isolated. The phylogenetic affinities of only a few SO taxa have been examined in detail; in these, a high degree of endemism and radiation within the SO has been established using molecular phylogenetic methods. In order to address these Antarctic paradigms, we used Bayesian inference to construct phylogenetic trees of nudibranch molluscs based on mitochondrial cytochrome-c oxidase I (COI) and 18S ribosomal DNA. We gathered sequences from temperate (COI n=37; 18S n=31) and polar (COI n=21; 18S n=22) species and then combined them with sequences retrieved from GenBank (COI …

Genomic, Proteomic And Metabolomic Approaches To Study Drought Responses In Aquilegia, David Henry

All Dissertations

Global population is expected to increase 30% by 2040, which will result in an increased need for crop production to feed the growing population. Combined with projected increased drought conditions worldwide, plant genetic research is necessary to gain a deeper knowledge of the molecular factors involved in plant drought response in order to engineer crop species with improved drought tolerance. Aquilegia has been recently developed as a model species for gene exploration based on its ability to thrive in a wide variety of environments including arid locations. An attractive asset of Aquilegia is its evolutionary position, equidistant between rice and …

Effect Of Chronic Treatment With A Cyclooxygenase Inhibitor On Reproductive Parameters In Male Rat, Sheikh A Saeed, Naheed Anwar, Khalid M Khan, Noorjehan Sarfraz

Department of Biological & Biomedical Sciences

BACKGROUND: Indomethacin is a member of non-steroidal anti-inflammatory drugs (NSAIDs) commonly used for treatment of gout, arthritis, and other inflammatory conditions. It has been shown to inhibit ovarian prostaglandins synthesis in mammals, birds, fish and reptiles. However, the effects of its chronic administration on male reproductive functions remain largely unknown. Using rat as a model, we studied the effect of chronic treatment with indomethacin on the male reproductive system.

METHODS: Testosterone was measured in the serum, testicular tissue, and testicular interstitial fluid by radioimmunoassay. Moreover, we also studied the direct effect of indomethacin in vitro on luteinizing hormone stimulated testosterone …

Prostate Cancer Biomarker Identification: A Comparative Study, Wenjuan Jiang

Computational Modeling & Simulation Engineering Theses & Dissertations

With the current development of proteomics techniques, the discovery of potential molecular biomarkers for early detection of prostate cancer has been greatly improved. In this thesis, we implemented five classifiers including the support vector machine (SVM), Bayesian Classifier, Decision Tree, Random Forest and the Multilayer perceptron (MLP) to test their effectiveness in prostate cancer biomarker identification using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) tissue imaging data. The classifiers were utilized to discriminate cancer mass spectra from normal ones for the data collected at the Eastern Virginia Medical School (EVMS). There are 94 7 spectra from normal tissues and 27 from …

Inhibition Of Yeast Hexokinase By The Antimalarial Drug Artemisinin: Probing Mechanism Of Action With A Model Enzyme, Jennifer S. Spence

Biological Sciences Theses & Dissertations

A leading infectious cause of death, malaria threatens approximately half of the world's population, and drug-resistant strains of Plasmodium falciparum have created immense difficulty in chemotherapy of the disease. The artemisinin (ART) class of antimalarials may represent a powerful solution. In addition to their safety, effectiveness, and moderate cost, they are the only drugs in use for which there has been no widespread evidence of clinical resistance. The exact parasiticidal mechanism of ART is highly contested, but evidence suggests that protein alkylation may play a role in cytotoxicity. in vitro essays were performed using yeast hexokinase (HK) to demonstrate a …

Section Abstracts: Biology And Microbiology & Molecular Biology

Virginia Journal of Science

Abstracts of the Biology Section and Microbiology & Molecular Biology for the 87th Annual Meeting of the Virginia Academy of Science, May 27th-29th, 2009, Virginia Commonwealth University, Richmond, VA.

Lipid Targets Of The Antimalarial Trioxanes In Plasmodium Falciparum, Carmony Leah Hartwig

Biological Sciences Theses & Dissertations

Malaria is among the most debilitating diseases of man. The protozoan parasite, Plasmodium falciparum, causes over a million annual fatalities. The antimalarial trioxanes, exemplified by artemisinin, are among the few pharmaceuticals for which clinical resistance has not become widespread. Artemisinin is a naturally occurring sesquiterpene lactone, containing a unique endoperoxide pharmacophore. Despite extensive study, the precise antimalarial mechanism of action of trioxanes remains elusive. Heme iron-mediated cleavage of the endoperoxide within the parasite digestive vacuole is hypothesized to generate cytotoxic metabolites capable of alkylating heme and damaging cellular macromolecules. The hypothesis of this research is that the endoperoxide pharmacophore …

Effects Of Estrogen On Muscle Damage In Response To An Acute Resistance Exercise Protocol, Megan R. Wolf

Honors Scholar Theses

Creatine Kinase (CK) is used as a measure of exercise-induced muscle membrane damage. During acute eccentric (muscle lengthening) exercise, muscle sarcolemma, sarcoplasmic reticulum, and Z-lines are damaged, thus causing muscle proteins and enzymes to leak into the interstitial fluid.

Strenuous eccentric exercise produces an elevation of oxygen free radicals, which further increases muscle damage. Muscle soreness and fatigue can be attributed to this membrane damage. Estradiol, however, may preserve membrane stability post-exercise (Brancaccio, Maffulli, & Limongelli, 2007; Carter, Dobridge, & Hackney, 2001; Tiidus, 2001). Because estradiol has a similar structure to Vitamin E, which is known to have antioxidant properties, …

Analysis Of The Phosphorylated Forms Of Protein Kinase R, Christine Quartararo

Honors Scholar Theses

Protein Kinase R (PKR) is induced by interferon and activated by dsRNA. Subsequent autophosphorylation and phosphorylation of eIF2alpha inhibits viral replication. In the latent state PKR exists as an unphosphorylated monomer. Work in the Cole laboratory has shown two additional states, a phosphorylated monomeric state (pPKRm) and a phosphorylated dimeric state (pPKRd). RNA serves as a scaffold bringing two PKRs together allowing dimerization and autophosphorylation to occur. The contribution of each state to the function of PKR remains unclear. Western blots were performed to examine the phosphorylation states of the essential residues,

T446 and T451. Activity assays have shown activation …

The Role Of Raf Kinase Inhibitor Protein In Cell Motility, Kathryn Grive

Honors Scholar Theses

Raf Kinase Inhibitor Protein (RKIP) has been identified as a phosphatidylethanolamine-binding protein capable of inhibiting Raf-1 kinase, an enzyme significant in cell proliferation and cancer development. When properly functioning, RKIP can mediate the expression of Raf-1 kinase and help prevent uncontrolled cell division. RKIP also has suggested, but unclear, roles in spindle fiber formation during mitosis, regulation of apoptosis, and cell motility. The Fenteany laboratory in the Chemistry Department identified a new small molecule, named Locostatin, as a cell migration inhibitor in mammalian cells, with RKIP as its primary molecular target.

Dictyostelium discoideum possess two RKIP proteins, RKIP-A and RKIP-B. …

Characterizing The Role Of Phaeobacter In The Mortality Of The Squid, Euprymna Scolopes, Brian Shawn Wong Won

Honors Scholar Theses

The subject of our study is the Hawaiian bobtail squid, Euprymna scolopes, which is known for its model symbiotic relationship with the bioluminescent bacterium, Vibrio fischeri. The interactions between E. scolopes and V. fischeri provide an exemplary model of the biochemical and molecular dynamics of symbiosis since both members can be cultivated separately and V. fischeri can be genetically modified 1. However, in a laboratory setting, the mortality of embryonic E. scolopes can be a recurrent problem. In many of these fatalities, the egg cases display a pink-hued biofilm, and rosy pigmentation has also been noted in the deaths of …

Characterization Of A Hypothetical Protein Critical For The Symbiotic Interaction Of Aeromonas Veronii And Hirudo Verbana, Kaitlin Vaughan

Honors Scholar Theses

The digestive tract symbiosis of the medicinal leech, Hirudo verbana, is a model system for studying the genes required for microbial colonization of digestive tracts, as H. verbana has only two species of bacteria that dominate the crop microbiota, Aeromonas veronii and a Rikenella-like bacterium. Signature-tagged mutagenesis (STM) of the A.

veronii strain, HM21R, revealed genes required for the colonization of the digestive tract. One of these mutants, JG573, has an interrupted gene that is predicted to encode a hypothetical protein. The region flanking the transposon insertion of this mutant was sequenced by primer walking.

Comparison of the flanking DNA …

Contribution Of The Novel C-Terminal Domain To The Ribosome Binding Activities Of Virulence Regulator Bipa, Heeren Makanji

Honors Scholar Theses

Bacterial GTPases regulate many cell functions, including the stress response, signal recognition, protein synthesis, and cell differentiation, through a molecular switch that is activated and deactivated depending on their nucleotide bound state (1). A member of the translational family of bacterial GTPases along with LepA and EF-G, BipA is a 67 kD protein that is essential for virulence and the stress response. Crystal structures from the Robinson lab have shown a unique C-terminal domain on BipA that has been implicated in ribosome binding. Using N-terminal deletion constructs, we have shown that the C-terminal domain is necessary, but not sufficient, to …