Molecular Biology Commons^™

Structure Based Drug Design Targeting Bacterial Antibiotic Resistance And Alzheimer's Disease, Eric Michael Lewandowski

USF Tampa Graduate Theses and Dissertations

Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, …

Tryptophan 207 Is Crucial To The Unique Properties Of The Human Voltage-Gated Proton Channel, Hhv1, Vladimir V. Cherny, Deri Morgan, Boris Musset, Susan M. E. Smith

Faculty Articles

Part of the “signature sequence” that defines the voltage-gated proton channel (HV1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence HV1 genes. Replacing Trp207 in human HV1 (hHV1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, Ea, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp207 …

Characterization And Diurnal Measurement Of Oral Inflammation In Association With Glycemic Control, Periodontal Status, & Glucose Stimulation, Melanie N. Kuehl

USF Tampa Graduate Theses and Dissertations

Diabetes has afflicted 8.3%, approximately 25.8 million, of the United States population and is the seventh leading cause of death [1]. Type I diabetes (T1D) accounts for 5 to 10% of all diagnosed cases of diabetes in the United States [2]. If present trends continue, the rate of T1D incidence among children under the age of 14 will increase by 3% globally [3]. T1D is an autoimmune disorder in which the β-cells of the pancreatic islets are destroyed, leading to high blood sugar. Hyperglycemia and loss of immunological tolerance to self-antigens are common associations of T1D [4]. Periodontal disease impacts …

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 …

Reactive Oxygen Species-Mediated Neurodegeneration Is Independent Of The Ryanodine Receptor In Caenorhabditis Elegans, Lyndsay E.A. Young, Daniel C. Williams

Journal of the South Carolina Academy of Science

Despite the significant impacts on human health caused by neurodegeneration, our understanding of the degeneration process is incomplete. The nematode Caenorhabditis elegans is emerging as a genetic model organism well suited for identification of conserved cellular mechanisms and molecular pathways of neurodegeneration. Studies in the worm have identified factors that contribute to neurodegeneration, including excitotoxicity and stress due to reactive oxygen species (ROS). Disruption of the gene unc-68, which encodes the ryanodine receptor, abolishes excitotoxic cell death, indicating a role for calcium (Ca²⁺) signaling in neurodegeneration. We tested the requirement for unc-68 in ROS-mediated neurodegeneration using the …

Dna Repair Deficiency In Huntington's Disease Fibroblasts And Induced Pluripotent Stem Cells, Peter Anthony Mollica

Biological Sciences Theses & Dissertations

Mutant huntingtin protein (mhtt)– the protein responsible for cellular dysfunction in Huntington’s disease (HD) –is a product of an expanded trinucleotide repeat (TNR) cytosine-adenine-guanine (CAG) sequence in exon 1 of the huntingtin (HTT) gene. The pathology of HD has been extensively researched; however, the mechanism by which the disease-causing TNR expansions occur in somatic cells remains elusive. Interestingly, HD has often been referred to a ‘DNA repair disease’, even though DNA repair dysfunction in situ has not been identified. We hypothesized that presence of the mhtt protein affects the expression of DNA repair genes used to address DNA repair, ultimately …

Cell Type–Dependent Mechanisms For Formin-Mediated Assembly Of Filopodia, Lorna E. Young, Ernest G. Heimsath, Henry N. Higgs

Dartmouth Scholarship

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in …

Mutations Of Adjacent Amino Acid Pairs Are Not Always Independent, Jyotsna Ramanan, Peter Revesz

School of Computing: Conference and Workshop Papers

Evolutionary studies usually assume that the genetic mutations are independent of each other. This paper tests the independence hypothesis for genetic mutations with regard to protein coding regions. According to the new experimental results the independence assumption generally holds, but there are certain exceptions. In particular, the coding regions that represent two adjacent amino acids seem to change in ways that sometimes deviate significantly from the expected theoretical probability under the independence assumption.

Biophysical Properties Of Cellular Membranes In Gram-Positive Bacterial Pathogens And Their Impact On Major Physiological Attributes And Virulence Determinants, Suranjana Sen

Theses and Dissertations

The cytoplasmic membrane of bacterial cells, forming an essential barrier from the surroundings, is a critical component of cellular physiology ensuring proper survival and maintenance of major cellular functions. The integrity of the membrane is an important feature that plays an essential role in the transport of solutes and nutrients through active and passive pathways, functions of membrane-associated proteins, electron transport and ATP synthesis, maintaining turgor pressure and combating environmental stresses, and thus is a crucial factor of a majority of cellular adaptations. The various biophysical properties affecting the integrity of this membrane are mainly determined by the composition and …

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 …

Regulation Of The Vacuolar Atpase Activity, Paulina Konarzewska

Dissertations, Theses, and Capstone Projects

In eukaryotes, V-ATPases play an essential role in cellular pH homeostasis as they transport hydrogen ions into the vacuoles. This assures an acidic vacuolar interior and normal physiological processes associated with this organelle. Studies show that mutations in any of the VMA genes encoding for subunits of the V-ATPase pump result in growth inhibition in the absence of inositol, suggesting that V-ATPases may play an important role in phospholipid biosynthesis or vice versa. It is not clear how VMA genes affect phospholipid biosynthesis and how the regulators of phospholipid biosynthetic genes may affect ATPase activity. Here, we employ biochemical and …

Incipient Speciation In Freshwater Fish Species From Two Isolated Watersheds, Paula Gore Miller

Dissertations, Theses, and Capstone Projects

The process of speciation occurs as a result of restricted gene flow between segments of an interbreeding population occupying different geographic areas. This separation may result in isolated populations which undergo genetic and phenotypic changes. The Wisconsin glacial period, which ended approximately 17,500 years ago, dramatically altered the geography of North America. The glacier covered almost the entire North America as it advanced. Areas that were not covered with ice provided suitable habitats (refugia) for relict species that were previously widespread in the northern section of the continent. As the ice sheet retreated, animals and plants were able to return …

Genomic Deoxyuracil: Targeting, Regulation And Repair, Nate Warren Holton

Theses and Dissertations

Maintaining genomic integrity is critical to avoid sequence mutations and genetic disease. However the immune system requires genomic rearrangements in order to produce novel antibodies and illicit a full immunological response. The expression of DNA modifying enzymes is essential for antibody production but the off target effects are not well understood. The work presented here was performed to elucidate molecular mechanisms responsible for the regulation and targeting of DNA editing enzymes, as well as characterize repair of these lesions at off target loci.

Elongation Factor-P At The Crossroads Of The Host-Endosymbiont Interface, Andrei Rajkovic, Anne Witzky, William Navarre, Andrew J. Darwin, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is an ancient bacterial translational factor that aids the ribosome in polymerizing oligo-prolines. EF-P structurally resembles tRNA and binds in-between the exit and peptidyl sites of the ribosome to accelerate the intrinsically slow reaction of peptidyl-prolyl bond formation. Recent studies have identified in separate organisms, two evolutionarily convergent EF-P post-translational modification systems (EPMS), split predominantly between gammaproteobacteria, and betaproteobacteria. In both cases EF-P receives a post-translational modification, critical for its function, on a highly conserved residue that protrudes into the peptidyl-transfer center of the ribosome. EPMSs are comprised of a gene(s) that synthesizes the precursor molecule …

A Forward Genetic Screen Identifies Factors Associated With Fever Pathogenesis In Plasmodium Falciparum, Phaedra J. Thomas

USF Tampa Graduate Theses and Dissertations

Infectious diseases that spread from person-to-person and continent-to-continent are a cause for concern for any health entity. One such disease is malaria, a mosquito-borne infection instigated by the protozoan parasite, Plasmodium falciparum. Hundreds of millions of people are affected annually and it is responsible for nearly 1 million deaths. It is the most fatal species causing malaria and proliferates in human red blood cells with a life cycle occurring every 48 hours. At this time, the parasite’s late stage form or schizont bursts from the erythrocyte releasing immune-inducing particles and infective forms (merozoites) into the bloodstream. The merozoites go …

Function Of Long Noncoding Rnas In Breast Cancer, Edward J. Richards

USF Tampa Graduate Theses and Dissertations

Breast cancer is a disease that will be diagnosed in about 1 in 10 women throughout their lifetime. The majority of breast cancers are originated from the epithelial cells of the mammary ducts, and this occurrence can be due to several factors including hereditary and acquired mutation. There are several major breast cancer subtypes, including estrogen receptor-α (ERα)-positive, HER2-enriched and triple-negative (TNBC). Patients diagnosed with ER+ tumors are generally treated with estrogen blockers (e.g., tamoxifen, letrozole and fulvestrant). Patients with HER2+ tumors are commonly administered with drugs that block HER2 signaling (e.g., trastuzumab) or inhibit HER2’s tyrosine kinase activity (e.g., …

Evaluation Of Reproductive Strategies In Captive California Yellowtail (Seriola Dorsalis) Using Genetic Parentage Analyses, Elizabeth K. Smith, Mark Drawbridge Ms, John Hyde Phd, Geoffrey Morse Phd

Theses

A good understanding of spawning dynamics of species in aquaculture is vital in order to maximize egg production and quality as well as efficient allocation of food and space resources. The breeding program of California yellowtail (CYT; Seriola dorsalis previously Seriola lalandi) at Hubbs-SeaWorld Research Institute (HSWRI) is set up such that up to 30 wild caught brood fish can broadcast spawn in a group setting, just as CYT reproduce in the wild. The population of brood fish were originally caught offshore from Southern California, and are maintained under ambient sea water and natural lighting conditions. The spawning season …

Characterizing The Rogfp2-Orp1 Fluorescent Biosensor For Detecting Oxidative Stress In Mammalian Cells, Sara A. Doan, Stevie Norcross, Mathew Tantama

The Summer Undergraduate Research Fellowship (SURF) Symposium

Parkinson’s disease is a neurodegenerative disease involving the death of neurons in the substantia nigra and loss of the neurotransmitter, dopamine. The disease leads to progressive loss of motor control. Exact causes and mechanisms by which Parkinson’s disease proceeds are unknown, however, previous experiments determine oxidative stress in mitochondria as a factor that results in cell death. Strategies have been implemented to generate fluorescent biosensors to monitor reactive oxygen species (ROS) concentrations while simultaneously measuring the spatiotemporal distribution and correlation between the ROS, cellular function and organelle. Orp1, an enzyme found in yeast, is a sensitive oxidizing species and when …

Precise Repair Of Mping Excision Sites Is Facilitated By Target Site Duplication Derived Microhomology, David M. Gilbert, M. Catherine Bridges, Ashley E. Strother, Courtney E. Burckhalter, James M. Burnette Iii, C. Nathan Hancock

Faculty Publications

A key difference between the Tourist and Stowaway families of miniature inverted repeat transposable elements (MITEs) is the manner in which their excision alters the genome. Upon excision, Stowaway-like MITEs and the associated Mariner elements usually leave behind a small duplication and short sequences from the end of the element. These small insertions or deletions known as “footprints” can potentially disrupt coding or regulatory sequences. In contrast, Tourist-like MITEs and the associated PIF/Pong/Harbinger elements generally excise precisely, returning the genome to its original state. The purpose of this study was to determine the mechanisms underlying these …

Wintering Bald Eagle Count Trends In The Conterminous United States, 1986-2010, Wade L. Eakle, Laura Bond, Mark R. Fuller, Richard A. Fischer, Karen Steenhof

Biomedical Research Institute Publications and Presentations

We analyzed counts from the annual Midwinter Bald Eagle Survey to examine state, regional, and national trends in counts of wintering Bald Eagles (Haliaeetus leucocephalus) within the conterminous 48 United States from 1986 to 2010. Using hierarchical mixed model methods, we report trends in counts from 11 729 surveys along 844 routes in 44 states. Nationwide Bald Eagle counts increased 0.6% per yr over the 25-yr period, compared to an estimate of 1.9% per yr from 1986 to 2000. Trend estimates for Bald Eagles were significant (P ≤ 0.05) and positive in the northeastern and northwestern U.S. …

An Exploration Of The Phylogenetic Placement Of Recently Discovered Ultrasmall Archaeal Lineages, Jeffrey M. O'Brien

Honors Scholar Theses

In recent years, several new clades within the domain Achaea have been discovered. This is due in part to microbiological sampling of novel environments, and the increasing ability to detect and sequence uncultivable organisms through metagenomic analysis. These organisms share certain features, such as small cell size and streamlined genomes. Reduction in genome size can present difficulties to phylogenetic reconstruction programs. Since there is less genetic data to work with, these organisms often have missing genes in concatenated multiple sequence alignments. Evolutionary Biologists have not reached a consensus on the placement of these lineages in the archaeal evolutionary tree. There …

Intracranial Glioblastomas: New Hope For An Effective Treatment, Arthur J L Cooper

NYMC Faculty Publications

Comment on Successful Treatment of Intracranial Glioblastoma Xenografts With a Monoamine Oxidase B-Activated Pro-Drug. [EBioMedicine. 2015]

Transcriptional And Post-Transcriptional Regulation Of Hepcidin And Iron Metabolism By Lipid Signaling In The Liver, Sizhao Lu

Theses & Dissertations

Although iron is required for essential biological processes, excess iron is detrimental due to oxidative damage induced by iron-mediated Fenton reactions, which promote tissue injury. Cellular iron uptake, transport and storage must therefore be tightly regulated. This task is accomplished mainly through hepcidin, the key iron-regulatory hormone. Hepcidin is synthesized primarily in hepatocytes as a circulatory antimicrobial peptide. It controls iron metabolism by inhibiting iron absorption from the duodenum and iron release from reticuloendothelial macrophages. Besides synthesizing hepcidin, the liver plays an important role in maintaining iron homeostasis by serving as the main storage organ for excess iron. Patients with …

Role Of Ime4 Protein In Pho Regulon Of S.Cerevisiae., Jenisha Ghimire

LSU New Orleans Theses and Dissertations

In the yeast Saccharomyces cerevisiae, the IME4 methyltransferase, interacts genetically with methyl binding protein, Pho92, to affect the expression of PHO regulon target genes. Cells mutant in IME4 or PHO92 show increases in the RNA abundance of PHO regulon target genes. The increase in the RNA abundance of the PHO regulon target genes is not additive in the cells double mutant in IME4 and PHO92. Hence, Ime4 and Pho92 interact in a single pathway in PHO regulon. Surprisingly, cells overexpressing IME4 and MUM2 shows increase in some PHO regulon target genes, indicating that IME4 affects the PHO regulon target …

A Novel Synthetic Yeast For Enzymatic Biodigester Pretreatment, Tianyu Tan, Mark S. Aronson, Arren Liu, Jill H. Osterhus, Melissa Robins, Suraj Mohan, Erich Leazer, Bowman Clark, Alexa Petrucciani, Katherine Lowery, James Welch, Casey Martin, Helena Lysandrou, Michael E. Scharf, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Lignin, a complex organic polymer, is a major roadblock to the efficiency of biofuel conversion as it both physically blocks carbohydrate substrates and poisons biomass degrading enzymes, even if broken down to monomer units. A pretreatment process is often applied to separate the lignin from biomass prior to biofuel conversion. However, contemporary methods of pretreatment require large amounts of energy, which may be economically uncompelling or unfeasible. Taking inspiration from several genes that have been isolated from termites and fungi which translate to enzymes that degrade lignin, we want to establish a novel “enzymatic pretreatment” system where microbes secrete these …

Elucidating The Role Of Hausp Ubiquitin Like Domains In The Catalytic Function Of Usp7, Anuj Patel, Nicole Davis, Andrew Mesecar

The Summer Undergraduate Research Fellowship (SURF) Symposium

Ubiquitin specific proteases (USPs) are a class of enzymes involved in myriad cellular processes. One USP of great interest due to its oncogenic properties is USP7. In normal conditions USP7 is closely regulated due to its responsibility for destabilizing the tumor suppressor, p53, through the deubiquitination of MDM2. In multiple myeloma cases, it appears the regulation of USP7 subsides, as it is largely overexpressed, leading to the inappropriate degradation of p53. Inhibition of USP7 could, therefore, prove a viable target for cancer therapy. A greater understanding of USP7’s function and structure can lead to more insight into how this enzyme …

How Many Particles Are Present In The Air? Bioaerosol Detection Using An Air Particle Counter, Angie Pamela Rivera, Parag Vaishampayan

STAR Program Research Presentations

Relative cleanliness in terms of particle abundance in spacecraft assembly facilities is determined by particle counts carried out in clean rooms during resting conditions. Particle counters assess total particles and particle size distribution, but do not distinguish inert particles from biological particles, which may include bacterial spores that are resistant to standard cleanroom sterilization procedures. Current cleanroom certifications do not fully assess the effects of human presence on spacecraft contamination since humans are known symbionts to enumerate microorganisms and assessments are performed at rest when there is no human presence. In this study, contamination risks and bioburden in spacecraft assembly …

Conformational Dynamics Of Dnaa Protein Of Bacillus Anthracis In The Initiation Of Dna Replication: A Fluorescence Resonance Energy Transfer Study, Meera J. Patel

Graduate School of Biomedical Sciences Theses and Dissertations

A precursor of the origin recognition complex proteins in eukaryotes, the DnaA protein, a single-subunit protein found in all prokaryotes, p1ays a significant role in the initiation of DNA replication. The DnaA protein in Gram-negative Escherichia coli (DnaA_EC) identifies, binds and activates specific DNA sequences at the origin of replication, oriC, initiating the duplication of the prokaryotic genome. Using DnaA_EC as a guide, we selected the full-length form of DnaA from Gram-positive Bacillus anthracis (DnaA_BA) as our model for studying. We have cloned, mutated, expressed and purified large quantities of DnaA_BA protein due to …