Biased Genetic Screen Identifies Novel Genes Involved In Antiviral Defense, Tianyun Long

LSU Doctoral Dissertations

ABSTRACT

RNA interference (RNAi) mediates potent antiviral response across kingdoms. In Caenorhabditis elegans nematodes, antiviral RNAi requires a virus sensor that is conserved in mammals and is amplified by secondary small interfering RNAs that are produced in a Dicer-independent manner.

To better understand worm antiviral RNAi, I carried out a biased genetic screen, aiming to identify novel antiviral RNAi genes. To speed up the gene discovery process, the reporter worms used for this genetic screen were engineered to contain extra copies of 4 known antiviral RNAi genes. Therefore, genetic alleles derived from these 4 genes will be automatically rejected during …

Mechanism Elucidation And Inhibitor Discovery Against Serine And Metallo-Beta-Lactamases Involved In Bacterial Antibiotic Resistance, Orville A. Pemberton

USF Tampa Graduate Theses and Dissertations

The emergence and proliferation of Gram-negative bacteria expressing β-lactamases is a significant threat to human health. β-Lactamases are enzymes that degrade the β-lactam antibiotics (e.g., penicillins, cephalosporins, monobactams, and carbapenems) that we use to treat a diverse range of bacterial infections. Specifically, β-lactamases catalyze a hydrolysis reaction where the β-lactam ring common to all β-lactam antibiotics and responsible for their antibacterial activity, is opened, leaving an inactive drug. There are two groups of β-lactamases: serine enzymes that use an active site serine residue for β-lactam hydrolysis and metalloenzymes that use either one or two zinc ions for catalysis. Serine enzymes …

High-Throughput Single-Molecule Telomere Characterization, Jennifer Mccaffrey, Eleanor Young, Katy Lassahn, Justin Sibert, Steven Pastor, Harold Riethman, Ming Xiao

School of Medical Diagnostics & Translational Sciences Publications

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG) n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG) n tract length at the end of each …

Using Competition Assays To Quantitatively Model Cooperative Binding By Transcription Factors And Other Ligands., Jacob Peacock, James B Jaynes

Department of Biochemistry and Molecular Biology Faculty Papers

BACKGROUND: The affinities of DNA binding proteins for target sites can be used to model the regulation of gene expression. These proteins can bind to DNA cooperatively, strongly impacting their affinity and specificity. However, current methods for measuring cooperativity do not provide the means to accurately predict binding behavior over a wide range of concentrations.

METHODS: We use standard computational and mathematical methods, and develop novel methods as described in Results.

RESULTS: We explore some complexities of cooperative binding, and develop an improved method for relating in vitro measurements to in vivo function, based on ternary complex formation. We derive …

An Organismal Model For Gene Regulatory Networks In The Gut-Associated Immune Response, Katherine M. Buckley, J P. Rast

Biochemistry and Molecular Medicine Faculty Publications

The gut epithelium is an ancient site of complex communication between the animal immune system and the microbial world. While elements of self-non-self receptors and effector mechanisms differ greatly among animal phyla, some aspects of recognition, regulation, and response are broadly conserved. A gene regulatory network (GRN) approach provides a means to investigate the nature of this conservation and divergence even as more peripheral functional details remain incompletely understood. The sea urchin embryo is an unparalleled experimental model for detangling the GRNs that govern embryonic development. By applying this theoretical framework to the free swimming, feeding larval stage of the …

Cell Division Regulation In Staphylococcus Aureus, Catherine M. Spanoudis

USF Tampa Graduate Theses and Dissertations

Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it …

Functional Characteristics And Genomic Sequencing Of Antarctic Environmental Isolates, Amanda Kemper, Christina Aigner, Michelle Tigges, Sara Anderson

Annual Symposium on Biomathematics and Ecology Education and Research

No abstract provided.

Microfluidic Biopsy Trapping Device For The Real-Time Monitoring Of The Tumor Microenvironment, Angela Holton

USF Tampa Graduate Theses and Dissertations

The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent …

The Efficiency Of Dna Repair Processes In Proximity Of G 4 Dna Structures, Gloria Alvarado

Theses and Dissertations

Guanine-rich segments of genomes promote the formation of a four-stranded DNA structure called G-quadruplex (G4 DNA). While evolutionarily conserved, guanine repeats show increased rates of mutagenesis and instability, and this is likely associated with G4 formation. In this thesis, I sought to clarify the molecular sources of genetic instability by testing the repair, excision, and synthesis through guanine-rich DNA. Using E. coli UDG and human SMUG1, I found that uracil is inefficiently repaired in proximity to G4 DNA in vitro. Investigation of E. coli exonuclease III and exonuclease I activity indicated that G4 DNA is a block to activity and …

Activation Of The Phospholipid Scramblase Tmem16f By Nanosecond Pulsed Electric Field (Nspef) Facilitates Its Diverse Cytophysiological Effects, Claudia Muratori, Andrei G. Pakhomov, Elena Gianulis, Jade Meads, Maura Casciola, Peter A. Mollica, Olga N. Pakhomova

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEF) are emerging as a novel modality for cell stimulation and tissue ablation. However, the downstream protein effectors responsible for nsPEF bioeffects remain to be established. Here we demonstrate that nsPEF activate TMEM16F (or Anoctamin 6), a protein functioning as a Ca²⁺-dependent phospholipid scramblase and Ca²⁺-activated chloride channel. Using confocal microscopy and patch clamp recordings, we investigated the relevance of TMEM16F activation for several bioeffects triggered by nsPEF, including phosphatidylserine (PS) externalization, nanopore-conducted currents, membrane blebbing, and cell death. In HEK 293 cells treated with a single 300-ns pulse of 25.5 kV/cm, …

Marine Reserves Promote Coral Reef Resilience By Mitigating Human Impacts Through The Restoration Of Parrotfish Populations, Increasing Their Reproductive Output And Seeding Neighboring Overfished Reefs, Brian L. Stockwell

Biological Sciences Theses & Dissertations

Coral reefs are some of the most productive ecosystems on the planet, providing fisheries resources for over a billion people with billions of dollars in revenue from tourism for developing nations. Coral reefs are under threat from overfishing and water pollution, resulting in less productive algae dominated reefs. Marine reserves are widely expected to promote the resilience of reefs by protecting and increasing the abundance of herbivorous fishes that can graze on algae, therefore directly or indirectly preventing coral to algal phase shifts. However, the ability of marine reserves to mitigate human impacts, restore herbivorous fish populations and seed nearby …

Human Rickettsial Pathogen Modulates Arthropod Organic Anion Transporting Polypeptide And Tryptophan Pathway For Its Survival In Ticks, Vikas Taank, Shovan Dutta, Amrita Dasgupta, Durland Fish, John F. Anderson, Hameeda Sultana, Girish Neelakanta

Biological Sciences Faculty Publications

The black-legged tick Ixodes scapularis transmits the human anaplasmosis agent, Anaplasma phagocytophilum. In this study, we show that A. phagocytophilum specifically up-regulates I. scapularis organic anion transporting polypeptide, isoatp4056 and kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), for its survival in ticks. RNAi analysis revealed that knockdown of isoatp4056 expression had no effect on A. phagocytophilum acquisition from the murine host but affected the bacterial survival in tick cells. Knockdown of the expression of kat mRNA alone or in combination with isoatp4056 mRNA significantly affected A. phagocytophilum survival …

Effects Of Carboxylated Nanodiamonds On Macrophages During And After Differentiation, Maisoun E. Bani Hani

Biological Sciences Theses & Dissertations

Nanodiamonds (ND) are a carbon-based nanomaterial that are increasingly being proposed for developing novel imaging techniques, as carriers of biomolecules and therapeutic drugs, as coatings for implants, and for other biomedical applications. The exceptional chemical, mechanical, and optical properties of ND make this material suitable in a wide range of fields. The application of ND in the biomedical field is attractive but requires more in-depth investigation into the safety of ND and its interactions with different cells and systems. The effects of ND on the immune system are not fully understood or investigated and there are several controverting reports regarding …

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 …

Structural Basis For Earp-Mediated Arginine Glycosylation Of Translation Elongation Factor Ef-P, Ralph Krafczyk, Jakub Macošek, Pravin Kumar Ankush Jagtap, Daniel Gast, Swetlana Wunder, Prithiba Mitra, Amit Kumar Jha, Jürgen Rohr, Anja Hoffmann-Röder, Kirsten Jung, Janosch Hennig, Jürgen Lassak

Pharmaceutical Sciences Faculty Publications

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-β-L-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for …

Characterization Of Different Molecular Markers For Identification Of Salmonella Enterica Serovar Typhi In Pakistani Population, Faizan Muttiullah, Fida Muhammad Khan, Fakhar-I- Abbas, Sabiha Shamim

Journal of Bioresource Management

Typhoid is caused by Salmonella enterica serovar Typhi that is usually diagnosed by using serologic and immuno-chromatographic techniques in developing counties including Pakistan, which is thought to be an unreliable diagnostic method. For accurate diagnosis we used molecular techniques to amplify 204 bp StyR-36 and 498 bp flagellin gene for the identification of Salmonella enterica serovar Typhi. This study was done on 58 individuals diagnosed positive of typhoid via serologic tests and 50 healthy individuals as a control group. Success rate of amplification for flagellin gene was 77.58% while that for StyR-36 gene was 68.97% showing that flagellin gene primer …

Mutations Of Conserved Non-Coding Elements Of Pitx2 In Patients With Ocular Dysgenesis And Developmental Glaucoma., Meredith E. Protas, Eric Weh, Tim Footz, Jay Kasberger, Scott C. Baraban, Alex V. Levin, L. Jay Katz, Robert Ritch, Michael A. Walter, Elena V. Semina, Douglas B. Gould

Natural Sciences and Mathematics | Faculty Scholarship

Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of …

Exploring Biological Heterogeneity And Its Consequences At Tissue And Cellular Scales Through Mathematical And Computational Modeling, Romica Kerketta

Biomedical Sciences ETDs

This dissertation explores the effects of heterogeneity across different biological scales in cancer as well as normal cells. At the tissue scale, we investigated the variability present in the tumor microenvironment and its effect on patient chemotherapeutic outcomes using a mathematical model of drug transport. We found that parameters such as tumor blood perfusion and radius of blood vessel had an impact on the tumor cytotoxicity. This indicated that the physical microenvironment of the tumor is an important regulator of the tumor response to chemotherapy. At the cellular scale, we investigated the heterogeneity present on the membrane landscape of ErbB2 …

Changes In Alternative Splicing As Pharmacodynamic Markers For Sudemycin D6, Morgan Thurman, Jacob Van Doorn, Barbara Danzer, Thomas R. Webb, Stefan Stamm

Molecular and Cellular Biochemistry Faculty Publications

Objective:

The aim of the study was to define pharmacodynamic markers for sudemycin D6, an experimental cancer drug that changes alternative splicing in human blood.

Methods:

Blood samples from 12 donors were incubated with sudemycin D6 for up to 24 hours, and at several time points total RNA from lymphocytes was prepared and the pre-messenger RNA (mRNA) splicing patterns were analyzed with reverse transcription-polymerase chain reaction.

Results:

Similar to immortalized cells, blood lymphocytes change alternative splicing due to sudemycin D6 treatment. However, lymphocytes in blood respond slower than immortalized cultured cells.

Conclusions:

Exon skipping in the DUSP11 and SRRM1 pre-mRNAs …