Molecular Biology Commons^™

Innate Immunity In Chickens: In Vivo Responses To Different Pathogen Associated Molecular Patterns, Kristen Alicia Byrne

Graduate Theses and Dissertations

Pattern recognition receptors (PRRs) on host cells recognize motifs known as pathogen associated molecular patterns (PAMPs) that are common to groups of microbes. Examples include LPS on Gram-negative bacteria, the structural motif PGN common to all bacteria, MDP the smallest immunostimulatory unit of PGN, and poly I:C the dsRNA analog. PAMP recognition by and stimulation of the innate immune system is crucial to an individual’s ability to quickly limit microbial growth and stimulate the adaptive immune system. Characterization of the in vivo immune responses initiated by PAMPs has not been directly addressed. Using growing feathers (GF) as a novel intradermal …

Applications Of The Gst- Affinity Tag In The Purification And Characterization Of Proteins, Wibke Beatrice Kachel

Graduate Theses and Dissertations

With the latest innovations in biological sciences, large quantities of biologically active polypeptides as well as high throughput screening methods to quickly evaluate if these biomolecules potentially have therapeutic, diagnostic, or industrial purposes are required. The synthesis and purification of peptides and small proteins continue to be demanding as the production of high yields through chemical synthesis can involve large costs. On the other hand, there are only few examples of acquiring those biomolecules through cloning and expression in bacterial systems in form of recombinant fusion proteins. Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to …

Computational Modeling Of Allosteric Stimulation Of Nipah Virus Host Binding Protein, Priyanka Dutta

USF Tampa Graduate Theses and Dissertations

Nipah belongs to the family of paramyxoviruses that cause numerous fatal diseases in humans and farm animals. There are no FDA approved drugs for Nipah or any of the paramyxoviruses. Designing antiviral therapies that are more resistant to viral mutations require understanding of molecular details underlying infection. This dissertation focuses on obtaining molecular insights into the very first step of infection by Nipah. Such details, in fact, remain unknown for all paramyxoviruses. Infection begins with the allosteric stimulation of Nipah virus host binding protein by host cell receptors. Understanding molecular details of this stimulation process have been challenging mainly because, …

A Caenorhabditis Elegans Model To Detect Developmental Teratogens With Adverse Reproductive And Metabolic Effects, Alexis Ann Killeen

USF Tampa Graduate Theses and Dissertations

Obesity is a current epidemic in the United States. Greater than one third of adults and nearly one fifth of children are classified as obese (Ogden, 2014). Increased awareness of this problem has triggered interest into the potentially causal agents in our environment. These agents, termed obesogens, are a type of teratogen hypothesized to work during embryonic development by programming offspring to store excess fat. A type of teratogen, obesogens are also typically reprotoxic, affecting germline development and embryonic viability. These noxious teratogens span a variety of different chemical classes including estrogen mimics, metals and metalloids, biocides, and even voluntary …

Trimethylated Lysine 4 At Histone 3 Shows The Same Circadian Rhythm At Promoters Of Diversely-Expressed Genes In Chlamydomonas Reinhardtii, Robyn M. Wilson

Masters Theses & Specialist Projects

Circadian clocks are biochemical mechanisms that allow eukaryotic and some prokaryotic organisms to coordinate their physiology with daily environmental changes. It enables organisms to increase their fitness by taking advantage of beneficial environmental conditions while also avoiding or restricting certain sensitive processes during harsh conditions. Similarly, post-translational histone modifications allow eukaryotic organisms to regulate gene expression in response to environmental or developmental factors. Some post-translational modifications of histones are associated with active transcription while others are associated with repressed transcription depending upon the location, type and degree of modification. Trimethylation of lysine 4 on the N-terminal tail of histone H3 …

Multiplexed Detection Of Double-Stranded Pathogenic Dna With Engineered Zinc Finger Proteins, Juhwa Kim

Masters Theses & Specialist Projects

The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to …

Identification Of Collagen Iv Associated Proteins In Drsophila Using Genetics And Mass Spectrometry, Mayank S. Kapadia

Masters Theses & Specialist Projects

Metastatic cancer cells invade and spread to other locations by disrupting the basement membrane (BM). The membrane plays a major role during the normal development of an organism as well. In order to understand the invasion mechanism it is important to know about the interactions occurring between the proteins of the BM during normal development. This study concentrates on isolating and identifying the major factors associated with collagen IV, a major component of BM, during the third instar larval development of Drosophila. Western blot and mass spectrometry analysis revealed that collagen IV associates with various growth factors, signaling molecules, and …

Functional And Expression Analysis Of A Novel Basement Membrane Degrader In Drosophila Melanogaster, Christopher J. Fields

Masters Theses & Specialist Projects

The Srivastava Lab is focused on the identification and characterization of genes that play a role in basement membrane remodeling. Previously, we identified putative basement membrane degraders through a genetic screen. One such gene has been suggested to play a role in the maintenance of the stem cell niche in Drosophila melanogaster, but no other information about the role this gene plays in development or disease has been published. Here, data are presented from experiments utilizing Drosophila genetics and immunohistochemistry that provide important insights on the biological role of this gene.

Collagenase activity was up-regulated upon overexpression of this gene, …

Heterologous Expression Of Pantoea Agglomerans Phytase Gene Optimized For Plant-Host Expression, N. N. Khabipova, L. R. Valeeva, I. B. Chastukhina, M. R. Sharipova, Eugene V. Shakirov

Biological Sciences Faculty Research

Here we report expression and characterization of recombinant bacterial phytase PaPhyC from Pantoea sp. Codon-optimized phytase gene was expressed E.coli BL21 pLysS and protein expression was confirmed by Western blotting. Recombinant protein expressed in E.coli has high phytase activity. We show that PaPhyC recombinant phytase has different molecular masses when expressed in bacteria and plants, suggesting that possible protein glycosylation in plants may influence its overall size.

Cell Cycle Arrest By Tgfß1 Is Dependent On The Inhibition Of Cmg Helicase Assembly And Activation, Brook Samuel Nepon-Sixt

USF Tampa Graduate Theses and Dissertations

Tumorigenesis is a multifaceted set of events consisting of the deregulation of several cell-autonomous and tissue microenvironmental processes that ultimately leads to the acquisition of malignant disease. Transforming growth factor beta (TGFß) and its family members are regulatory cytokines that function to ensure proper organismal development and the maintenance of homeostasis by controlling cellular differentiation, proliferation, adhesion, and survival, as well as by modulating components of the cellular microenvironment and immune system. The pleiotropic control by TGFß of these cell intrinsic and extrinsic factors is intimately linked to the prevention of tumor formation, the specifics of which are dependent on …

Anticancer Activities Of Resveratrol In Colorectal Cancer, Evelien Schaafsma, Tze-Chen Hsieh, Barbara B. Doonan, John T. Pinto, Joseph M. Wu

NYMC Faculty Publications

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a dietary polyphenolic phytochemical that has demonstrated health benefits such as cardioprotection, the prevention of neurodegeneration and chemoprevention. Resveratrol has shown great potential in the prevention and treatment of carcinomas and clinical trials support resveratrol as anticancer compound in colorectal carcinoma. Colorectal cancer remains a major cause of cancer-related deaths for both men and women in industrialized countries. Because of this widespread prevalence, identifying major risk factors and initiating colorectal screening procedures provide the distinct advantage for recognizing early disease and addressing treatable forms of CRC. Epidemiological studies of fruit and vegetable consumption in relationship to developing …

Intrinsic Disorder Where You Least Expect It: The Incidence And Functional Relevance Of Intrinsic Disorder In Enzymes And The Protein Data Bank, Shelly Deforte

USF Tampa Graduate Theses and Dissertations

Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) exist as interconverting conformational ensembles, without a single fixed three-dimensional structure in vivo. The focus in the literature up to this point has been primarily on IDPs that are mostly or entirely disordered. Therefore, we have an incomplete understanding of the incidence and functional relevance of IDPRs in proteins that have regions of both order and disorder. This work explores these populations, by examining IDPRs in the Protein Data Bank (PDB) and in enzymes. By applying disorder prediction methods combined with an analysis of missing regions in crystal structure data, …

Functional/Activity Network (Fan) Analysis Of Gene-Phenotype Connectivity Liaised By Grape Polyphenol Resveratrol, Tze-Chen Hsieh, Sheng-Tang Wu, Dylan J. Bennett, Barbara B. Doonan, Erxi Wu, Joseph M. Wu

NYMC Faculty Publications

Resveratrol is a polyphenol that has witnessed an unprecedented yearly growth in PubMed citations since the late 1990s. Based on the diversity of cellular processes and diseases resveratrol reportedly affects and benefits, it is likely that the interest in resveratrol will continue, although uncertainty regarding its mechanism in different biological systems remains.We hypothesize that insights on disease-modulatory activities of resveratrol might be gleaned by systematically dissecting the publicly available published data on chemicals and drugs. In this study, we tested our hypothesis by querying DTome (Drug-Target Interactome), a web-based tool containing data compiled from open-source databases including DrugBank, PharmGSK, and …

Inhibiting The Interaction Between Grp94 And Myocilin To Treat Primary Open-Angle Glaucoma, Andrew Stothert

USF Tampa Graduate Theses and Dissertations

Glaucoma is a neurodegenerative protein misfolding disorder classified by increases in IOP, damage to retinal ganglion cells (RGCs), optic nerve (ON) head damage, and progressive irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form of glaucoma, constituting over 90% of clinical cases. POAG is observed in patients where normal outflow channels, mainly the trabecular meshwork (TM), are exposed at the angle formed by the iris and cornea. However, due to TM cellular dysfunction, aqueous outflow resistance is increased preventing normal circulation of aqueous humor. Recent studies have shown that in 2-4% of POAG cases, increased intracellular levels of …

Splice-Switching Antisense Oligonucleotides As Therapeutic Drugs, Mallory A. Havens, Michelle L. Hastings

Biology Department Faculty Articles

Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal …

Population Dynamics And Community Composition Of Ammonia Oxidizers In Salt Marshes After The Deepwater Horizon Oil Spill, Anne E. Bernhard, Roberta Sheffer, Anne E. Giblin, John M. Marton, Brian J. Roberts

Biology Faculty Publications

The recent oil spill in the Gulf of Mexico had significant effects on microbial communities in the Gulf, but impacts on nitrifying communities in adjacent salt marshes have not been investigated. We studied persistent effects of oil on ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities and their relationship to nitrification rates and soil properties in Louisiana marshes impacted by the Deepwater Horizon oil spill. Soils were collected at oiled and unoiled sites from Louisiana coastal marshes in July 2012, 2 years after the spill, and analyzed for community differences based on ammonia monooxygenase genes (amoA). Terminal Restriction Fragment …

Cmg Helicase Assembly And Activation: Regulation By C-Myc Through Chromatin Decondensation And Novel Therapeutic Avenues For Cancer Treatment, Victoria Bryant

USF Tampa Graduate Theses and Dissertations

The CMG (Cdc45, MCM, GINS) helicase is required for cellular proliferation and functions to unwind double-stranded DNA to allow the replication machinery to duplicate the genome. Cancer cells mismanage helicase activation through a variety of mechanisms, leading to the potential for the development of novel anti-cancer treatments. Mammalian cells load an excess of MCM complexes that act as reserves for new replication origins to be created when replication forks stall due to stress conditions, such as drug treatment. Targeting the helicase through inhibition of the MCM complex has sensitized cancer cells to drugs that inhibit DNA replication, such as aphidicolin …

Mof Acetylates The Histone Demethylase Lsd1 To Suppress Epithelial-To-Mesenchymal Transition., Huacheng Luo, Anitha K Shenoy, Xuehui Li, Yue Jin, Lihua Jin, Edward Seto, +10 Additional Authors

Biochemistry and Molecular Medicine Faculty Publications

The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby …

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 …

4,4’-Dichlorodiphenyltrichloroethane (Ddt) And 4,4’-Dichlorodiphenyldichloroethylene (Dde) Promote Adipogenesis In 3tl1 Adipocyte Cell Culture, J Kim, Q Sun, Y Yue, Kyong-Sup Yoon, K -Y Whang, J M. Clark, Y Park

SIUE Faculty Research, Scholarship, and Creative Activity

4,4’-Dichlorodiphenyltrichloroethane (DDT), a chlorinated hydrocarbon insecticide, was extensively used in the 1940s and 1950s. DDT is mainly metabolically converted into 4,4’- dichlorodiphenyldichloroethylene (DDE). Even though most countries banned DDT in the 1970s, due to the highly lipophilic nature and very stable characteristics, DDT and its metabolites are present ubiquitously in the environment, including food. Recently, there are publications on relationships between exposure to insecticides, including DDT and DDE, and weight gain and altered glucose homeostasis. However, there are limited reports regarding DDT or DDE and adipogenesis, thus we investigated effects of DDT and DDE on adipogenesis using 3T3-L1 preadipocytes. Treatment …

Identification Of Genes That Are Essential To Restrict Genome Duplication To Once Per Cell Division., Alex Vassilev, Chrissie Y. Lee, Boris Vassilev, Wenge Zhu, Pinar Ormanoglu, Scott E. Martin, Melvin L. Depamphilis

Biochemistry and Molecular Medicine Faculty Publications

Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor …

Combined Metformin And Resveratrol Confers Protection Against Uvc-Induced Dna Damage In A549 Lung Cancer Cells Via Modulation Of Cell Cycle Checkpoints And Dna Repair, Yong-Syu Lee, Barbara B. Doonan, Joseph M. Wu, Tze-Chen Hsieh

NYMC Faculty Publications

Aging in humans is a multi-factorial cellular process that is associated with an increase in the risk of numerous diseases including diabetes, coronary heart disease and cancer. Aging is linked to DNA damage, and a persistent source of DNA damage is exposure to ultraviolet (UV) radiation. As such, identifying agents that confer protection against DNA damage is an approach that could reduce the public health burden of age-related disorders. Metformin and resveratrol have both shown effectiveness in preventing several age-related diseases; using human A549 cells, we investigated whether metformin or resveratrol, alone or combined, prevent UVC-induced DNA damage. We found …

The Role Of Daf-19 In Non-Ciliated Neurons: How Is Neural Development Regulated By Different Daf-19 Isoforms?, Zabdiel Ek Vazquez

Lawrence University Honors Projects

A degenerative disease-like phenotype, specifically reduction in synaptic protein levels in adult worms, is correlated with loss-of-function of the only RFX transcription factor gene, daf-19, in C. elegans. This gene encodes four known transcription factor isoforms, two of which are correlated with particular functions. The DAF-19C isoform activates genes responsible for cilia development, while DAF-19M is needed for cilia specification in males. A comparison of the transcriptome of daf-19 null and isogenic wild type adult worms suggests both positive and negative regulation of gene expression is correlated with the presence of DAF-19 proteins. We have assessed DAF-19 regulation …

Influencing Pathways That Cause Metastasis And Stemness In Epithelial Ovarian Cancer, Alyse Lynn Huisken-Hill

Electronic Theses, Projects, and Dissertations

Ovarian cancer is the fifth leading cause of cancer death in women between the ages of 35 and 74. With 22 thousand new cases and 15 thousand deaths annually ovarian cancer is among the most deadly cancers with a death to incidence ratio of 68%. With 70% of cases High Grade Serous Ovarian Carcinoma (HGSOC) is the most common type of ovarian cancer and causes 90% of ovarian cancer deaths. 80% of patients have reoccurrence within five years and only 15-30% of patients with recurrent metastatic ovarian cancer respond to current therapies, chemotherapy and surgery. One reason for the high …

Purification, Optimization, And Growth Of New Delhi Metallo-Β-Lactamase-1 Protein Crystals Mixed With Nz218 Inhibitor, Brandon M. Wills

Celebration of Learning

New Delhi metallo-β-lactamase-1 is a problematic gene found in certain strains of bacteria that cause them to become antibiotic resistant to nearly all known antibiotics. While some antibiotics are available to treat patients with a bacterial infection, most are toxic or do not have 100% success rates. With that being said, it is imperative that we search for a molecule that is successfully able to inhibit the effects of this gene every time. Such a discovery would help tremendously with new antibiotic drug development and also prevent further damage by these dangerous bacteria. In this presentation, I will describe the …

Hiv Vaccines: Progress, Limitations And A Crispr/Cas9 Vaccine, Omar A. Garcia Martinez

Biology: Student Scholarship & Creative Works

ABSTRACT: The HIV-1 pandemic continues to thrive due to ineffective HIV-1 vaccines. Historically, the world’s most infectious diseases, such as polio and smallpox, have been eradicated or have come close to eradication due to the advent of effective vaccines. Highly active antiretroviral therapy is able to delay the onset of AIDS but can neither rid the body of HIV-1 proviral DNA nor prevent further transmission. A prophylactic vaccine that prevents the various mechanisms HIV-1 has to evade and attack our immune system is needed to end the HIV-1 pandemic. Recent advances in engineered nuclease systems, like the CRISPR/Cas9 system, have …

Translation Control Of Swarming Proficiency In Bacillus Subtilis By 5-Amino-Pentanolylated Elongation Factor P, Andrei Rajkovic, Katherine R. Hummels, Anne Witzky, Sarah Erickson, Philip R. Gafken, Julian P. Whitelegge, Kym F. Faull, Daniel B. Kearns, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys³² of B. subtilis EF-P that is …

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 …

Flow-Dependent Myosin Recruitment During Drosophila Cellularization Requires Zygotic Dunk Activity, Bing He, Adam Martin, Eric Wieschaus

Dartmouth Scholarship

Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts …

Multiple Quality Control Pathways Limit Non-Protein Amino Acid Use By Yeast Cytoplasmic Phenylalanyl-Trna Synthetase, Adil Moghal, Lin Hwang, Kym F. Faull, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA^Phe. We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA^Phe with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic …