Molecular Biology Commons^™

Genetic Basis For The Virulence Of Enterohemorrhagic Escherichia Coli Strain Tw14359, Jason Kyle Morgan

USF Tampa Graduate Theses and Dissertations

Enterohemorrhagic Escherichia coli (EHEC) is a virulent pathotype of E. coli that is associated with major outbreaks of hemorrhagic colitis and the life-threatening kidney disease hemolytic uremic syndrome. For successful host colonization and attachment to the intestinal mucosa, EHEC requires the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (TTSS) responsible for secreting and translocating effector proteins into host colonocytes. Regulation of the LEE is primarily directed through the first operon, LEE1, encoding the locus encoded regulator (Ler), and occurs through the direct and indirect action of several regulators. The 2006 U.S. spinach outbreak …

Functional Analysis Of Cytosolic Hsp70 Nucleotide Exchange Factor Networks In Yeast, Jennifer Lynn Abrams

Dissertations & Theses (Open Access)

The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the …

Structure-Based Design Of Novel Inhibitors And Ultra High Resolution Analysis Of Ctx-M Beta-Lactamase, Derek Allen Nichols

USF Tampa Graduate Theses and Dissertations

The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform …

Enzyme Activity, Maturation And Regulation Of Anaerobic Reductases In Shewanella Oneidensis Mr-1, Kenneth L. Brockman

Theses and Dissertations

Shewanella oneidensis MR-1 is a metal-reducing bacterium capable of using a wide range of terminal electron acceptors. These include oxygen, metal oxides and organic compounds such as dimethyl sulfoxide (DMSO) and fumarate. In addition, several nitrogen and sulfur based compounds can be used as terminal electron acceptors, including sulfite, for which the terminal reductase was recently identified as an octaheme c-type cytochrome that contains an atypical heme binding site. In this study, several additional components involved in sulfite reduction were identified. These include SirCD that form a membrane-bound electron-transferring complex with SirA, SirBI that appear to be involved in protein …

Small Rna, Cyclic-Di-Gmp And Phenolic Compounds Regulate The Type Iii Secretion System In Bacterial Phytopathogens, Devanshi Khokhani

Theses and Dissertations

Type III Secretion System (T3SS) is an essential virulence factor in many Gram-negative bacterial pathogens. Expression of T3SS consumes large amount of energy. Hence it is tightly regulated by bacteria through several mechanisms. In this work we screened a library of phenolic compounds and found several compounds that

dramatically downregulate T3SS in Erwinia amylovora 273. Additionally, the role of small RNA (sRNA) chaperone, Hfq, and a secondary messenger, cyclic-di-GMP in T3SS regulation in Dickeya dadantii 3937 was also examined. Chapter 1 provides a brief overview of the history and virulence mechanisms of two phytopathogens - Erwinia amylovora 273 and Dickeya …

The Role Of Antimicrobial Compounds In The Life Cycle Of The Symbiotic Bacterium, Xenorhabdus Nematophila, Swati Singh

Theses and Dissertations

The bacterium Xenorhabdus nematophila maintains a mutualistic relationship with the entomopathogenic nematode Steinernema carpocapsae and is also pathogenic towards insect larvae. X. nematophila possesses a large number of gene clusters potentially involved in antimicrobial production. Several antibiotics, including xenocoumacin (Xcn) produced at high levels in broth cultures, have been characterized. In this study I established that during nematode invasion of the insect body cavity (hemocoel) gut microbiota enter the hemocoel representing potential competitors for X. nematophila. As infection progressed some transient species, such as Staphylococcus saprophyticus disappeared early in infection, while other persistent species such as Enterococcus faecalis proliferated. S. …

Determining The Presence Of Carbapenem Antibiotic Resistance In Clinical Isolates, Fidelis Uzoma Enyinnaya

UNLV Theses, Dissertations, Professional Papers, and Capstones

Carbapenems are β-lactam antibiotics reserved for the treatment of severe microbial infections, especially those targeting the Enterobacteriaceae. Introduced in the 1980s, carbapenems have been used successfully in hospitals, and in the 1990s resistance was discovered. Carbapenem resistance is conferred through the production of carbapenemases. In the U.S., the most common carbapenemase is Klebsiella pneumoniae carbapenemase (KPC). In 2012, the National Healthcare Safety Network reported a carbapenem resistance rate of 13.0% among Klebsiella pneumoniae infections, and indicated that the mortality rate associated with carbapenem resistant Enterobacteriaceae (CRE) infections ranged from 48.0-71.9%. According to the Food and Drug Administration (FDA), carbapenem resistance …

Characterization Of Ftsa-Ftsn Interaction During Escherichia Coli Cell Division, Kimberly.Busiek@Gmail.Com K. Busiek

Dissertations & Theses (Open Access)

Division of a bacterial cell into two equal daughter cells requires precise assembly and constriction of the division machinery, or divisome. The Escherichia coli divisome includes nearly a dozen essential cell division proteins that assemble at midcell between segregating sister chromosomes. FtsZ, a homolog of eukaryotic tubulin, is the first essential cell division protein to localize at midcell where it polymerizes into a ring-shaped scaffold (Z ring). Establishment of the Z ring is required for recruitment of downstream cell division proteins including FtsA, a cytoplasmic protein that tethers the Z ring to the inner membrane. Following localization of FtsA and …

The Role Of Intestinal Sweet Taste Receptors (Strs) In The Regulation Of Glucose Absorption: Effects Of Short Term High Sucrose Diet (Hsd), Tania Hussain

HIM 1990-2015

Sweet taste receptors are primarily found in the oral cavity of the mammalian species. However, recent studies have shown that sweet taste receptors can be found in extraoral tissues such as the pancreas, intestines, and adipose tissue. Our lab has previously found that sweet taste receptors are down-regulated on the pancreas in the presence of high plasma glucose levels. In order to assess the possibility that sweet taste receptors respond to high levels of glucose by suppressing its expression, we wanted to see if they reacted similarly on the intestines. We found that intestinal sweet taste receptors are down regulated …

The Sumoylation Of The Non-Structural Protein 1 Of The Influenza A Virus Plays A Dual Role During Viral Infection, Jason Michael Chacon

Open Access Theses & Dissertations

The potential for a highly pathogenic influenza pandemic remains a concern for global health. The ability of the influenza virus to undergo antigenic shift and genetic drift give circulating strains of influenza the high probability of developing resistance to current antiviral therapies. Emerging strains of higher virulence, to which the infected person has not had any previous exposure to viral antigens, pose a greater threat of serious illness and death. Currently, there is no antiviral therapy that is effective against all strains of influenza, emphasizing the need to develop new strategies that target cellular systems required for viral replication to …

The Burden Of Avian Influenza Viruses In Community Ponds In California, Zin Htway

Walden Dissertations and Doctoral Studies

Emerging influenza viruses continue to challenge public health. The problem is public health science professionals have been battling emerging human influenza diseases with tactile and reactionary methods because there is a lack of knowledge and data at the human-animal interface. This research was a baseline study of the proportion of influenza A virus (IAV) in urban and rural communities in California. The population was artificial recirculating water ponds in the geographic locations of rural and urban Californian communities. Surface water samples [N = 182] were collected from artificial recirculating ponds in California. Positivity for IAV was verified by real time …

Sumoylation Affects Ns1'S Ability To Neutralize The Interferon Response, Jeanette Cecilia Gonzalez

Open Access Theses & Dissertations

It is of huge concern the possibility that at some point in the future we may face a new pandemic involving a highly pathogenic virus due to our current preventive and treatment options to fight this viral disease. The main reason for such limitations is the ability of the virus to go over constant antigenic shifts and drifts on its viral surface proteins HA and NA. The constant mutations that affect this virus cause the need of developing new vaccines for each influenza season. Periodically, the World Health Organization (WHO) reports that around 10% of the world population gets infected …

The Effects Of Lipoxin A4 (Lxa4) On Neutrophil Biology In Sepsis, Benedict Wu

Graduate School of Biomedical Sciences Theses and Dissertations

During sepsis, neutrophils are inappropriately activated and have prolonged lifespans, thus becoming dysfunctional. Excessive neutrophil activation can lead to tissue injury while neutrophil dysfunction can lead to decreased free radical production and reduced phagocytosis, preventing the host from clearing preexisting infections. Lipoxin A4 (LXA4) is a specialized pro-resolution mediator which reduces neutrophil migration and expression of proinflammatory mediators. Intact neutrophil functions are critical for the host to efficiently clear invading pathogens. The effects of LXA4 on neutrophil function in sepsis have not been established. Using the cecal ligation and puncture (CLP) model of sepsis, LXA4 administered 1 h after sepsis …

Nuclear Import And Interactions Of Potato Yellow Dwarf Virus Nucleocapsid, Matrix, And Phosphoprotein, Gavin Lloyd Franklin Anderson

Theses and Dissertations--Plant Pathology

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis and alanine-scanning mutagenesis identified two amino acid motifs, ⁴¹⁹QKR⁴²¹ and ⁴³²KR⁴³³, that were shown to be essential for nuclear import and interaction with …

Ether Bridge Formation And Chemical Diversification In Loline Alkaloid Biosynthesis, Juan Pan

Theses and Dissertations--Plant Pathology

Loline alkaloids, found in many grass-Epichloë symbiota, are toxic or feeding deterrent to invertebrates. The loline alkaloids all share a saturated pyrrolizidine ring with a 1-amine group and an ether bridge linking C2 and C7. The steps in biosynthesis of loline alkaloids are catalyzed by enzymes encoded by a gene cluster, designated LOL, in the Epichloë genome. This dissertation addresses the enzymatic, genetic and evolutionary basis for diversification of these alkaloids, focusing on ether bridge formation and the subsequent modifications of the 1-amine to form different loline alkaloids.

Through gene complementation of a natural lolO mutant and comparison …

Characterization Of Ns1 Sumoylation And Its Effect On Influenza A Viral Infection, Katherine Anne Meraz

Open Access Theses & Dissertations

Influenza virus is a contagious respiratory virus responsible for seasonal epidemics and several catastrophic pandemics in the last century. Its genome is comprised of negative sense, single-stranded RNA and, after entering the cell, it is capable of hijacking the host cellular machinery for reproducing its own genetic material. The activation of cellular defenses against influenza viral infection are triggered upon viral entry and help regulate the course of viral infection. This study focused on the interplay between the influenza A virus and the cellular SUMOylation system during viral infection. The first part of this Dissertation deals with the relevance of …

Characterizing Dsrna Production In Virus-Infected Fish Cells, Amal Brek Aloufi

Theses and Dissertations (Comprehensive)

Viral dsRNA is produced by almost all viruses sometime during their replicative cycle. These viral nucleic acids are potent inducers of both innate and adaptive immune responses, and are therefore considered important immuno-modulators. Previous studies have shown that viruses produce dsRNA when replicating in mammalian cells; however, to date no one has demonstrated viral dsRNA production in virus infected fish cells. Therefore, the goal of this study is to investigate dsRNA production by fish viruses in fish cells, verifying production and performing initial characterization of the dsRNA molecules being produced. Three different rainbow trout cell lines were used in this …

Characterizing The Human Vaginal Microbiome Using High-Throughput Sequencing, Jean Megan E. Macklaim

Electronic Thesis and Dissertation Repository

The human vaginal microbiome undoubtedly has a significant role in reproductive health and for protection from infectious organisms. Recent efforts to characterize the bacterial species of the vagina using molecular techniques have uncovered an unexpected diversity. Using high-throughput sequencing I sought to describe the structure and function of the vaginal microbiome under different physiological states including healthy, bacterial vaginosis (BV), post-menopausal vaginal atrophy, and acute vulvovaginal candidiasis (VVC).

Partial 16S rRNA gene sequencing revealed that healthy, asymptomatic women most often have vaginal biotas dominated by Lactobacillus iners or L. crispatus. In contrast, BV is a heterogeneous, highly diversified condition …

The Glycine And Proline Reductase Systems: An Evolutionary Perspective And Presence In Enterobacteriaceae, Joshua Witt

HIM 1990-2015

The Glycine and Proline Reduction systems are two of the best characterized selenoenzymes in bacteria and have been found to occur in a wide variety of clostridia [1-5]. These enzymes are utilized to reduce glycine or D-proline to obtain energy via substrate level phosporylation or membrane gradients, respectively [6, 7]. This includes the pathogens C. difficile and C. botulinum [5, 8]. Strains of C. difficile are activate toxigenic pathways whenever either of these pathways is active within the cell [5, 8]. Though evolutionary studies have been conducted on ammonia producing bacteria [9] none has been done to directly characterize these …

Structural Studies On The Rubella Virus Capsid Protein And Its Organization In The Virion, Vidya Mangala Prasad

Open Access Dissertations

Rubella virus is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, rubella infection leads to severe damage in fetuses. Despite its medical importance, very little is known about the structure of the pleomorphic rubella virus as compared to its alphavirus relatives. The rubella capsid protein is a critical structural component of virions as well as a key factor in virus-host interactions. Three crystal structures of the structural domain of the rubella capsid protein have been described here. The polypeptide fold of the capsid protomer has not been observed previously. The capsid protein structure, along …

The Effects Of Growth Hormone And Thyroxine Treatment On The Insulin Signaling Of Female Ames Dwarf Mouse Skeletal Muscle Tissue, Andrew Do

HIM 1990-2015

Ames dwarf (df/df) mice are deficient in anterior pituitary hormones: growth hormone (GH), thyroid stimulating hormone (TSH), and prolactin (PRL) due to a spontaneous, homozygous mutation of prop1[superscript df] gene. These dwarf mice exhibit characteristics such as delayed growth and development coupled with delayed aging, increased lifespan, overall increased insulin sensitivity, as well as resistance to certain diseases and cancers. The mutant mice possess low blood glucose, low serum insulin, and lower body temperature. Their enhanced longevity (about 40-60% longer lifespan than normal mice) is associated with their GH deficiency and disruption in the somatotropic axis (GH/IGF-1 hormonal pathway) as …

Human Adenovirus E1a Binds And Retasks Cellular Hbre1, Blocking Interferon Signalling And Activating Virus Early Gene Transcription, Gregory J. Fonseca

Electronic Thesis and Dissertation Repository

Upon infection, human adenovirus (HAdV) must block interferon signaling and activate the expression of its early genes to reprogram the cellular environment to support virus replication. During the initial phase of infection, these processes are orchestrated by the first HAdV gene expressed during infection, early region 1A (E1A). E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. We have identified hBre1/RNF20 as a novel target of E1A. hBre1 is an E3 ubiquitin ligase which acts with the Ube2b E2 conjugase and accessory factors RNF40 and WAC1 to monoubiquitinate …

Manipulating Aktivated Metabolism Via Mtorc1, Ivan Von Hack Prestinary

HIM 1990-2015

Although poorly understood, normal cells and cancerous cells of the same type exhibit different patterns of nutrient consumption, processing and utility of metabolic substrates. Differences in substrate uptake, preference, and alternately emphasized metabolic pathways offer opportunities for selective targeting of cancer versus stroma. This may be accomplished by using a sequential approach of nutrient deprivation and pharmaceutical perturbation of metabolic pathways to inhibit cellular proliferation. The purpose of this study was to investigate the effects of restricting glucose and glutamine concentrations, in vitro, to levels that resemble a potential human fasting state. The mammalian target of rapamycin (mTOR), a mediator …

Cell Surface Adhesins, Exopolysaccharides And The Por (Type Ix) Secretion System Of Flavobacterium Johnsoniae, Abhishek Shrivastava

Theses and Dissertations

Flavobacterium johnsoniae, a member of the phylum Bacteroidetes, crawls rapidly over surfaces. Cell movement is thought to result from the action of the gliding motor, composed of Gld proteins, on the cell-surface adhesin SprB. Cells lacking SprB are partially defective in motility. Transposon mutagenesis of an sprB mutant resulted in the identification of remA, which encodes a motility adhesin that is partially redundant with SprB. Cells lacking SprB and RemA had more severe motility defects than did cells lacking just SprB. RemA moves on the cell-surface with a speed of 1 to 2 micrometer per sec, similar to SprB. RemA …

Quorum Sensing In Vibrios And Cross-Species Activation Of Bioluminescence Lux Genes By Vibrio Harveyi Luxr In An Arabinose-Inducible Escherichia Coli Expression System, Anne Marie Wannamaker

Theses and Dissertations

Bacterial bioluminescence is observed in over twenty known species, primarily in the family Vibrionaceae. However, only Vibrio fischeri and Vibrio harveyi bioluminescence expression mechanisms are well studied. In V. harveyi, expression of the lux operon is activated by the transcription factor LuxR (LuxR_VH), resulting in bioluminescence. Homologs of LuxR_VH in other Vibrio species have been shown to regulate transcription of a variety of genes. Three parallel quorum sensing pathways co-regulate the expression of LuxR_VH. The first objective was to assess possible quorum sensing regulation of lux operon expression in V. cholerae, V. chagasii, …

Prolonged Glucose Deprivation Sensitizes Snf1 To Negative Regulation By Pka To Delay Entry Into Quiescence, Leah Bernadette Doughty

Theses and Dissertations

AMPK, the fuel gauge of the cell, and its upstream kinase, LKB1, have been implicated in cancer prevention and stress response associated with energy exhaustion. In the yeast Saccharomyces cerevisiae, Snf1 is the ortholog of mammalian AMPK. In S. cerevisiae, Snf1 is activated by phosphorylation of its T–loop at Thr210, primarily by its upstream kinase Sak1, in absence of the preferred carbon source, glucose, or during some other stress responses. Cyclic AMP–dependent protein kinase A, PKA, is involved in nutrient signaling largely antagonistically to Snf1. Using yeast strains of the Sigma 1278b genetic background, which have a high basal level …

Identification Of Potential Lead Antimalarial Compounds From Marine Microbial Extracts, Abigail Carbonell

HIM 1990-2015

Malaria, caused by the parasite Plasmodium falciparum, has a long history as a global health threat. The vector-borne disease causes millions of deaths yearly, especially in developing countries with tropical climates that facilitate transmission. Compounding the problem is the emergence of drug-resistant strains due to overuse of outdated treatments. New compounds with antiplasmodial activity are needed to be developed as effective drugs against malaria. The hypothesis for this project is that marine microorganisms have a high likelihood of yielding novel antiplasmodial chemotypes because of their high diversity, which has not yet been explored for antimalarial development. In this project, microbes …

Development Of An Alkaline Phosphatase Reporter System For Use In The Lyme Disease Spirochete Borrelia Burgdorferi, Selina Sutchu

HIM 1990-2015

The use of the periplasmic alkaline phosphatase (PhoA) reporter protein from E. coli has been critical for definition of the topology of transmembrane proteins of multiple bacterial species. This report demonstrates development of a PhoA reporter system in B. burgdorferi. Codon usage of the E. coli phoA in B. burgdorferi was analyzed and an optimized version of the gene was obtained. In order to assess the differential activity of the reporter system, two optimized PhoA-fusion construct using B. burgdorferi proteins were engineered: one using the periplasmic protein OppAIV and one using the cytoplasmic protein PncA. The activity of PhoA requires …

Expression Of An Epitope Tagged Tarp Effector In Chlamydia Trachomatis, Brenda Nguyen

HIM 1990-2015

Previous studies performed on Chlamydia trachomatis have demonstrated how these obligate intracellular microbes invade host cells through the utilization of secreted effector proteins. One secreted effector called Tarp (translocated actin recruiting protein) is implicated in cytoskeleton rearrangements that promote bacterial entry into the host cell. The focus of our study is to create a plasmid that carries the tarP gene that when transcribed and translated from within Chlamydia trachomatis will generate a c-Myc epitope tagged Tarp. The tag will be used in future studies to track the progression of the protein through the infectious process and will allow us to …

Understanding The Role Of Plasmodium Falciparum Vamp8 Snare Homologue, Katherine Ferreira

HIM 1990-2015

Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia [37]. According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of …