Molecular Biology Commons^™

Binding Of Maize Necrotic Streak Virus (Mnesv) 3’ I-Shaped Structure (3’ Iss) To Eukaryotic Translation Factors (Eifs) And Implication In Eif4f Mediated Translation Initiation, Qiao Liu

Dissertations, Theses, and Capstone Projects

5' m⁷GpppN cap and the 3' poly adenosine (A) tail of eukaryotic mRNAs are key elements for recruiting translation initiation machinery in canonical translation initiation. Unlike host mRNAs, many viruses lack these elements and yet they are translated efficiently. Plant viruses, in particular, have complex structures within their untranslated regions (UTR) that allow them to bypass some cellular translation control steps. In Maize necrotic streak virus (MNeSV) 3' UTR, an I-Shaped RNA Structure (ISS) has been reported to mediate the virus translation initiation progress. 3’ ISS binding with eIF4F has been shown to facilitate translation. 5’ -3’ kissing …

Regulation Of The Tubulin Homolog Ftsz In Escherichia Coli, Monika S. Buczek

Dissertations, Theses, and Capstone Projects

Escherichia coli is a well-known pathogen, and importantly, a widely used model organism in all fields of biological sciences for cloning, protein purification, and as a model for Gram-negative bacterial species. And yet, researchers do not fully understand how this bacterium replicates and divides. Every year additional division proteins are discovered, which adds complexity to how we understand E. coli undergoes cell division. Due to their specific roles in cytokinesis, some of these proteins may be potential targets for development of antibacterials or bacteriostatics, which are much needed for fighting the current global antibacterial deficit. My thesis work focuses on …

Phase Diagram Of Water Confined By Graphene, Zhenghan Gao, Nicolas Giovambattista, Ozgur Sahin

Publications and Research

The behavior of water confined at the nanoscale plays a fundamental role in biological processes and technological applications, including protein folding, translocation of water across membranes, and filtration and desalination. Remarkably, nanoscale confinement drastically alters the properties of water. Using molecular dynamics simulations, we determine the phase diagram of water confined by graphene sheets in slab geometry, at T = 300 K and for a wide range of pressures. We find that, depending on the confining dimension D and density σ, water can exist in liquid and vapor phases, or crystallize into monolayer and bilayer square ices, as observed in …

Mechanisms For Survival And Drug Resistance In Cancer Cells, Matthew B. Utter

Dissertations, Theses, and Capstone Projects

PART I

Prostate cells are hormonally driven to grow and divide. Typical treatments for prostate cancer involve blocking the hormone androgen from activating the androgen receptor (AR) and thus inhibit growth and proliferation of the cancer. Androgen deprivation therapy (ADT) can lead to the selection of cancer cells that grow and divide independently of androgen receptor activation. Prostate cancer cells that are insensitive to androgens commonly display metastatic phenotypes and reduced long-term survival of patients. In this study, we provide evidence that androgen-insensitive prostate cancer cells have elevated phospholipase D (PLD) activity relative to the androgen-sensitive prostate cancer cells. PLD …

Wnt Secretion Proteins Modulate Rankl-Induced Expression Of Aire In Thymic Epithelial Cells, Daniel Pollack

Dissertations and Theses

Thymic epithelial cells (TEC) are essential for a proper adaptive immune response by regulating thymocyte development and establishing central tolerance. In the thymus, TECs differentially express Wnt proteins, which activate canonical and non-canonical Wnt signaling pathways. Wnt signaling is thought to regulate cell survival, proliferation, and development although the direct molecular mechanisms in TECs have yet to be elucidated. The inducible inhibition of canonical Wnt signaling with Dkk1 leads to a rapid loss of TEC progenitors as well as a decline in mature Aire-expressing mTECs. Therefore, we explore the role of Wnt ligands potentially responsible for stimulating and/or regulating Wnt …

Pharmacological Antagonism And The Olfactory Code, Mihwa Na

Dissertations, Theses, and Capstone Projects

Mammals can detect and discriminate uncountable odors through their odorant receptors. To accommodate the countless and diverse odors, exceptionally large numbers of odorant receptor (OR) genes are expressed in mammals. In addition, the mammals utilize a combinatorial code, where an odorant molecule can activate multiple ORs; an OR also responds to a set of multiple odorants. In nature, an odor is often a complex mixture of multiple odorant molecules. The combination of the ORs activated by each constituent generates the unique olfactory code for the particular odor.

Some odorants can antagonize select ORs, as discussed in Chapter 1. An antagonist …

Structural And Biochemical Studies Of The Dna Replication Initiation Mechanism Of The Second Chromosome Of Vibrio Cholerae, Natalia Orlova

Dissertations, Theses, and Capstone Projects

Transmission of genetic information through DNA replication is one of the key processes for any living organism. Despite the extensive effort put into studies of the mechanism of DNA replication, the understanding of the process on the molecular level is still incomplete. Specifically the molecular details of the very first events of DNA replication initiation are not sufficiently understood.

The majority of bacteria possess a single circular chromosome, and in order to initiate DNA replication these organisms utilize a conserved system, consisting of a specific DNA sequence - replication origin, called oriC, and replication initiator protein DnaA. However, bacteria …

Mutagenesis Of Human Alpha-Galactosidase A For The Treatment Of Fabry Disease, Erin Stokes

Dissertations, Theses, and Capstone Projects

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40-60 years of age. There is currently only one FDA approved treatment (Fabrazyme) classified as an enzyme replacement therapy. However, approximately 88% of patients experience a severe immune response that, rarely, can be fatal and is a huge cost burden at average $250,000 a year per patient. The structure of α-galactosidase A has been previously determined to be a …

A Combined Computational Strategy Of Sequence And Structural Analysis Predicts The Existence Of A Functional Eicosanoid Pathway In Drosophila Melanogaster, Michael Scarpati

Dissertations, Theses, and Capstone Projects

With increased understanding of their roles in signal transduction and metabolism, eicosanoids have emerged as important players in human health and disease. Mammalian prostanoids and related lipid mediators perform varied functions in different tissues and organs. Synthesized through the oxygenation of C20 polyunsaturated fatty acids, mammalian eicosanoids are both pro- and anti-inflammatory. The physiological contexts in which eicosanoid family members act at the cellular level are not well understood. In this study, we examined whether the genome of Drosophila melanogaster, a powerful model for innate immunity and inflammation, codes for the enzymes required for eicosanoid biosynthesis. We report the …

Insight Into The Interaction Between The Peroxisome Proliferator-Activated Receptor Gamma (Pparγ) And Adipocyte Fatty Acid-Binding Protein (A-Fabp), Qian Wang

Dissertations, Theses, and Capstone Projects

The Adipocyte Fatty Acid-Binding Protein (AFABP) is mainly expressed in fat cells. It can bind fatty acids and other lipophilic substances such as eicosanoids and retinoids. The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor protein that requires ligand binding to regulate the specific gene transcription. PPARγ is expressed at extremely high levels in adipose tissue, macrophages, and the large intestine, where it controls lipid adipogenesis and energy conversion. Moreover, it has been found that AFABP and PPARγ can form a complex in vivo. It is proposed that AFABP carries the ligand and enters into the nucleus where it …

Engineered Aptamers To Probe Molecular Interactions On The Cell Surface, Sana Batool, Sanam Bhandari, Shanell George, Precious Okeoma, Nabeela Van, Hazan E. Zümrüt, Prabodhika Mallikaratchy

Publications and Research

Significant progress has been made in understanding the nature of molecular interactions on the cell membrane. To decipher such interactions, molecular scaffolds can be engineered as a tool to modulate these events as they occur on the cell membrane. To guarantee reliability, scaffolds that function as modulators of cell membrane events must be coupled to a targeting moiety with superior chemical versatility. In this regard, nucleic acid aptamers are a suitable class of targeting moieties. Aptamers are inherently chemical in nature, allowing extensive site-specific chemical modification to engineer sensing molecules. Aptamers can be easily selected using a simple laboratory-based in …

Thermodynamic And Kinetic Analyses Of Iron Response Element (Ire)-Mrna Binding To Iron Regulatory Protein, Irp1, Mateen A. Khan, William E. Walden, Elizabeth C. Theil, Dixie J. Goss

Publications and Research

Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRERNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 μM, 25 °C) increased IRE-RNA/IRP1 …

Bow-Tie Signaling In C-Di-Gmp: Machine Learning In A Simple Biochemical Network, Jinyuan Yan, Maxime Deforet, Kerry E. Boyle, Rayees Rahman, Raymond Liang, Chinweike Okegbe, Lars E. P. Dietrich, Weigang Qiu, Joao B. Xavier

Publications and Research

Bacteria of many species rely on a simple molecule, the intracellular secondary messenger c-di-GMP (Bis-(3'-5')-cyclic dimeric guanosine monophosphate), to make a vital choice: whether to stay in one place and form a biofilm, or to leave it in search of better conditions. The c-di-GMP network has a bow-tie shaped architecture that integrates many signals from the outside worldÐthe input stimuliÐinto intracellular c-di-GMP levels that then regulate genes for biofilm formation or for swarming motilityÐthe output phenotypes. How does the `uninformed' process of evolution produce a network with the right input/output association and enable bacteria to make the right choice? Inspired …

Range Of Detection For Proteins And Dna From Fingerprints On Fired And Unfired Cartridge Casings, Stacey-Ann R. Sterling

Student Theses

Cartridges and spent cartridge casings can be probative pieces of evidence. Unfortunately due to a combination of factors such as exposure to high temperatures and initially low amounts of biological material on the surface, DNA testing so far has been mostly unsuccessful for these items. Typing other marker systems, such as protein polymorphisms, on the same biological evidence would add power of discrimination. To explore this option we developed a DNA-protein trypsin-based co-extraction method that was optimized for unfired and fired cartridges. Various sample wet and dry collection methods and multiple metal casings, such as aluminum, nickel, steel, and brass …

Transcriptional And Post-Transcriptional Regulation Of Histone Variant H2a.Z During Sea Urchin Development, Mihai Hajdu

Dissertations, Theses, and Capstone Projects

Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3’ UTR sequences stabilize GFP mRNAs relative to those with SV40 3’ UTR sequences, although the 3’UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We …

Evolution Of Complex Target Selex To Identify Aptamers Against Mammalian Cell-Surface Antigens, Prabodhika R. Mallikaratchy

Publications and Research

The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs) represent a class of molecules able to meet this demand. In particular, aptamers, a class of small nucleic acid ligands that are composed of single-stranded modified/unmodified RNA/DNA molecules, can be evolved from a complex library using Systematic Evolution of Ligands by EXponential enrichment (SELEX) against almost any molecule. Since its introduction in 1990, in stages, SELEX technology has itself undergone several modifications, improving selection and broadening the repertoire of targets. This …

Specific Binding Affinity Of The Non-Catalytic Domain Of Eukaryotic Like Type Ib Topoisomerase Of Vaccinia Virus, Benjamin R. Reed

Dissertations, Theses, and Capstone Projects

Topoisomerases are ubiquitous proteins that alter supercoiling in double stranded DNA (dsDNA) during transcription and replication and. vaccinia and the closely related poxvirus variola virus, at 314 amino acids in length, encode the smallest of the type I topoisomerases(TopIB). TopIB is a two domain protein that recognizes the sequence 5’-T/CCCTT, cleaves at the 3’-end and relaxes supercoiling through rotation. The C-terminal domain (CTD) alone contains the catalytic activity and specificity. Deletion of the N-terminal domain results in a greatly reduced rate of relaxation and rapid dissociation. Biochemical data suggests that the N-terminal domain (NTD) is important for pre-cleavage binding and …

Molecular Analysis Of Ftsz-Ring Assembly In E. Coli Cytokinesis, Kuo-Hsiang Huang

Dissertations, Theses, and Capstone Projects

An essential first step in bacterial division is the assembly of a cytokinetic ring (Z-ring) formed by the tubulin-like FtsZ at midcell. The highly conserved core domain of FtsZ has been reported to mediate assembly of FtsZ polymers in vivo and in vitro. Species-specific differences in the FtsZ C-terminal domain such as the FtsZ CTV region and interactions with several modulatory proteins such as ZapC and ZapD, restricted to certain bacterial classes, also serve as key determinants of FtsZ protofilament bundling. Here, we characterize (i) the roles of the FtsZ CTV region in mediating both longitudinal and lateral interactions …

Tuning Into Toxins And Channels: The Characterizations Of Tv1 And A Human Cardiac Sodium Channel Voltage-Sensor Domain, Mohammed H. Bhuiyan

Dissertations, Theses, and Capstone Projects

In nature, peptide toxins are an abundant resource, produced both by marine and terrestrial organisms. A major target of these peptide toxins is the group of the highly important voltage-gated ion channels. Due to their high specificity and affinity, peptide toxins have been used for over a decade in discovery and characterization of voltage-gated ion channels. Although peptide toxins have been extensively characterized structurally, the structural characterization of eukaryotic voltage-gated sodium channels has seen much less progress, due to their large size and high hydrophobicity. Voltage-gated sodium channels play crucial roles in many physiological processes, and when these processes are …

Insights Into The Molecular Roles Of Heparan Sulfate Proteoglycans (Hspgs-Syndecans) In Autocrine And Paracrine Growth Factor Signaling In The Pathogenesis Of Hodgkin's Lymphoma, Rajendra Ghardbaran

Publications and Research

Syndecans (SDC, SYND) comprise a group of four structurally related type 1 transmembrane heparan sulfate proteoglycans (HSPGs) that play important roles in tumorigenic processes. SDCs exert signaling via their protein cores and their conserved transmembrane and cytoplasmic domains or by forming complexes with growth factors (GFs). In classical Hodgkin's lymphoma (cHL), a lymphoid neoplasm of predominantly B cell origin, SDC1 and SDC4 are the active SDCs, and a number of GF (vascular endothelial growth factor, fibroblast growth factor, etc.) signaling pathways have been studied. However, despite extensive pre-clinical and clinical research on SDC-mediated GF signaling in many cancer types, there …

Circular Oligodeoxynucleotides (Coligos) As Chemically Synthesized, Promoter-Independent Small Rna Expression Vectors, Lodoe Lama

Dissertations, Theses, and Capstone Projects

Small RNAs (sRNA) such as microRNA (miRNA), small interfering RNA (siRNA), and other types of RNA have been found to play many important biological roles in regulation of different cellular process leading to their possible use as therapeutics. However, one of the biggest hurdles in exploiting these sRNAs in therapeutics has been the difficulty in delivering them safely and stably into human tissues. Many delivery approaches have been undertaken to deliver these sRNA, albeit with many shortcomings in terms of cost, stability, and side effects, necessitating the need for alternative approaches. We are exploring an entirely different approach for conveying …

Antioxidant And Hepatoprotective Effects Of Procyanidins Fromwild Grape (Vitis Amurensis) Seeds In Ethanol-Induced Cells And Rats, Min Ji Bak, Van-Long Truong, Se-Yeon Ko, Xuan Ngan Giang Nguyen, Pajaree Ingkasupart, Mira Jun, Jin Young Shin, Woo-Sik Jeong

Publications and Research

In the present study, we characterized the antioxidant and hepatoprotective mechanisms underlying of wild grape seed procyanidins (WGP) against oxidative stress damage in ethanol-treated HepG2 cell and Sprague-Dawley (SD)-rat models. In HepG2 cells, WGP not only diminished the ethanol (EtOH, 100 mM)-induced reactive oxygen species (ROS) formation and cytochrome P450 2E1 (CYP2E1) expression, but also renovated both the activity and expression of antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase. Additionally, to investigate the hepatoprotective effect of WGP, rats were orally administered 10 or 50 mg/kg WGP once daily for seven days prior to the single oral administration of …

The Interaction Between Eukaryotic Translation Initiation Factor Eif4g And 3’ Cap Independent Translation Element Of Barley Yellow Dwarf Virus Is Affected By Multiple Initiation Factors, Pei Zhao

Dissertations, Theses, and Capstone Projects

Barley Yellow Dwarf Virus (BYDV) lacks a 5’ (7-methyl guanosine) cap as well as a 3’poly A tail. Like many plant viruses, BYDV contains a cap independent translation element (CITE) in the 3’ untranslated region of the viral mRNA. BTE (Barley Yellow Dwarf Virus like cap-independent translation element) is one of the well characterized CITEs. BTE mediated translation primarily depends on eukaryotic initiation factor eIF4G. BTE binds to eIF4G; however, the details of BTE initiated translation are still unclear. Three eIF4G deletion mutants with different domain organization were used to investigate BTE interaction with eIF4G: eIF4G601-1196 is the eIF4G fragment …

The Evolution Of The Viral Rna Sensor Oas1 In Old World Monkeys And Cetartiodactyls, Ian Fish

Dissertations, Theses, and Capstone Projects

Animals produce an array of sensors patrolling the intracellular environment poised to detect and respond to viral infection. The oligoadenylate synthetase family of enzymes comprises a crucial part of this innate immune response, directly signaling endonuclease activity responsible for inhibiting viral replication. Oligoadenylate synthetase 1 plays a vital role in animal susceptibility to pathogens including flaviviruses such as dengue, West Nile, and hepatitis c virus. This thesis includes a population level analysis of OAS1 diversity within macaque and baboon species followed by a broader survey of the gene in nineteen Old World monkeys. My research found that at the species …

Chamber-Specific Patterns Of Epicardium Formation In Zebrafish, Sana Khan

Dissertations, Theses, and Capstone Projects

The outer cardiac layer, the epicardium, coordinates the final steps of vertebrate heart development. This cardiac tissue arises from cells in the proepicardial organ (PEO) that forms around the base of the inflow tract. Its general location is conserved across species despite morphological differences. Cellular mechanisms of migration differ across species. Three strategies of PEO migration are described: 1) The floating cyst model - PEO cells released into the pericardial cavity are directed by fluid movements to migrate onto the myocardium; 2) Villi transfer - cardiac contractions may mediate multicellular PEO villi contact to the myocardium; and 3) Tissue bridge-mediated …

Messenger Rna Transport And Translation Regulated By The 3' Utrs Of Dendritic Mrnas And Abnormal Alternative Splicing Of Neuroligin1 In The Fmr1 Ko Mouse Hippocampus, Tianhui Zhu

Dissertations, Theses, and Capstone Projects

Fragile X Syndrome (FXS) is one of the most commonly inherited mental retardations. It is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of functional FMRP is the most widespread single-gene cause of autism. The most prominent phenotype of FXS patients is an IQ ranging from 20 to 70. FMRP is an RNA binding protein, widely expressed in almost all tissues and highly expressed in brain. As a RNA binding protein, 85-90 % of FMRP in the brain is associated with polyribosomes. Approximately 4 % of total mRNA is associated with FMRP, which functions in …

Cladribine Analogues Via O6-(Benzotriazolyl) Derivatives Of Guanine Nucleosides, Sakilam Satishkumar, Prasanna K. Vuram, Siva Subrahmanyam Relangi, Venkateshwarlu Gurram, Hong Zhou, Robert J. Kreitman, Michelle M. Martínez Montemayor, Lijia Yang, Muralidharan Kaliyaperumal, Somesh Sharma, Narender Pottabathini, Mahesh K. Lakshman

Publications and Research

Cladribine, 2-chloro-2′-deoxyadenosine, is a highly efficacious, clinically used nucleoside for the treatment of hairy cell leukemia. It is also being evaluated against other lymphoid malignancies and has been a molecule of interest for well over half a century. In continuation of our interest in the amide bond-activation in purine nucleosides via the use of (benzotriazol-1yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate, we have evaluated the use of O6-(benzotriazol-1-yl)-2′-deoxyguanosine as a potential precursor to cladribine and its analogues. These compounds, after appropriate deprotection, were assessed for their biological activities, and the data are presented herein. Against hairy cell leukemia (HCL), T-cell lymphoma (TCL) and chronic lymphocytic …

Exploiting Cancer Cell Signaling And Metabolism: Implications For Therapeutic Approach, Suman Mukhopadhyay

Dissertations, Theses, and Capstone Projects

Over the last decade, metabolic dysregulation in cancer cells has stimulated a significant amount of interest in basic research. It has been established that cancer cells increase glucose uptake and alter the fate of glycolytic and tricarboxylic acid (TCA) cycle intermediates for the synthesis of biological molecules to accommodate high rates of cellular growth and proliferation. Moreover, it is more prominent that some metabolic dysregulations are specific to particular oncogenes. Exploiting the dysregulated metabolic dependency of cancer cells with therapeutic means could represent a novel approach for clinical aspect.

To meet the need of increased anabolic metabolism cancer cells engage …

Exploring Chromatin-Bound Mdm2 Functions In Compromised Transcriptional Regulation Of P53 Target Genes, Melissa Rosso

Dissertations, Theses, and Capstone Projects

MDM2 overexpression is a common occurrence in many types of cancer. A single nucleotide polymorphism (T to G) near the mdm2 promoter, termed mdm2 SNP309, leads to MDM2 overexpression. This polymorphism is associated with accelerated tumor formation, decreased sensitivity to DNA damage treatment and compromised p53 transcriptional activity. Two G/G SNP309 cancer cell lines MANCA and A875, a Burkitts' lymphoma and melanoma respectively, express a stable wild-type p53 protein. We previously reported these cells have DNA damage resistant MDM2-p53 chromatin complexes and hypothesized that MDM2 is the contributing factor for the compromised p53 transcriptional activity. We created constitutive mdm2 shRNA …

The Role Of Atp And Cleavage Factor Phosphorylation In Pre-Mrna 3' Cleavage Reaction, Asya Khleborodova

Dissertations, Theses, and Capstone Projects

The 3' end cleavage/polyadenylation (3' processing) is important in generating a functional messenger RNA (mRNA) transcript. It is long-known that ATP can significantly stimulate the in vitro cleavage of adenovirus type 2 L3 (Ad2L3) RNA substrate. Here, we used ATP analogs in structure-activity assays to show that the structural features of ATP and its analogs determine in vitro 3' cleavage efficiency. More specifically, we demonstrate that the structure of the nitrogenous base, the nucleotide sugar and the triphosphate group contribute to the efficiency of the nucleotide-stimulated in vitro 3' cleavage reaction.

It was found that AppNp, an analog of ATP …