Molecular Biology Commons^™

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 …

Real-Time Qpcr Assay Development For Detection Of Bacillus Thuringiensis And Serratia Marcescens Dna And The Influence Of Complex Microbial Community Dna On Assay Sensitivity, Jonathan Segal

FIU Electronic Theses and Dissertations

Real-time quantitative polymerase chain reaction (real-time qPCR) assays are an effective technique to detect biological warfare agents and surrogate organisms. In my study, primers were designed to detect chromosomal DNA of biological warfare agent surrogates B. thuringiensis and S. marcescens (representing B. anthracis and Y. pestis, respectively) via real-time qPCR. Species-level specificity of the primers was demonstrated through comparisons with a bacterial strain panel and corroborated by qPCR data. Additionally, the primer efficacy was tested when template DNA was spiked into metagenomic DNA extracted from clinical lung microbiome samples. The results showed that while detection of B. thuringiensis or …

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 …

Divergent Mechanisms Of Interaction Of Helicobacter Pylori And Campylobacter Jejuni With Mucus And Mucins, Julie Ann Naughton, Karina Mariño, Brendan Dolan, Colm Reid, Ronan Gough, Mary Gallagher, Michelle Kilcoyne, Jared Gerlachscience,, Lokesh Joshi, Pauline Rudd, Stephen Carrington, Billy Bourke, Marguerite Clyne

Articles

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but …

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 …

Section Abstracts: Biology With Microbiology And Molecular Biology

Virginia Journal of Science

Abstracts of the Biology with Microbiology and Molecular Biology Section for the 91st Annual Virginia Journal of Science Meeting, May 2013

Analysis Of The Chondroitinase Operon Of Flavobacterium Columnare, Erin L. Sorlien

Senior Honors Projects

Analysis of the chondroitinase operon of Flavobacterium columnare

Erin Sorlien

Major

Cell and Molecular Biology, Chemistry

Advisor

Dr. David R. Nelson

Date

May 2, 2013

Keywords

Flavobacterium columnare, columnaris disease, chondroitin AC lyase, complementation, csl operon

Abstract
Flavobacterium columnare, an opportunistic bacterial pathogen of fish, is the causative agent of columnaris disease (CD). The bacterium is a Gram-negative rod that exhibits gliding motility and avidly forms biofilms. CD affects both wild and cultured freshwater fish, and continues to cause large economic losses to the fish farming industry. According to an investigation conducted by the National Animal Health Monitoring System, CD …

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 …

Evolutionary And Molecular Analysis Of Conserved Vertebrate Immunity To Fungi, Erin Carter

Honors College

The innate immune system is highly conserved amongst all multicellular organisms. Yet a constant battle exists between host cells and pathogens due to the rapid evolution of immune system components. Functional genomics and in silico methods can be employed to elucidate the evolutionary patterns of vertebrate immunity to pathogenic fungi such as Candida albicans, an opportunistic fungal pathogen that can cause lethal candidiasis in the immunocompromised. Mammals such as humans and mice possess conserved C-type lectin receptors that recognize the C. albicans cell wall. However, these receptors have not been identified in fish. Here I describe how we identified potential …

Modifying Commercially Available Vectors For Expression Of Standardized Igem Biobricks, Merhawi T. Yigzaw, Dafna Lapido, Holly M. Bowman

Georgia State Undergraduate Research Conference

No abstract provided.

Characterization Of Myxococcus Xanthus Mazf And Implications For A New Point Of Regulation, Tye O. Boynton, Jonathan L. Mcmurry, Lawrence J. Shimkets

Jonathan McMurry

During development, Myxococcus xanthus cells undergo programmed cell death (PCD) whereby 80% of vegetative cells die. Previously, the MazF RNA interferase has been implicated in this role. Recently, it was shown that deletion of the mazF gene does not eliminate PCD in wild-type strain DK1622 as originally seen in DZF1. To clarify the role of MazF, recombinant enzyme was characterized using a highly sensitive assay in the presence and absence of the proposed antitoxin MrpC. In contrast to previous reports that MrpC inhibits MazF activity, the hydrolysis rate was enhanced in a concentration-dependent manner with MrpC or MrpC2, an N-terminally …

Characterization Of Myxococcus Xanthus Mazf And Implications For A New Point Of Regulation, Tye O. Boynton, Jonathan L. Mcmurry, Lawrence J. Shimkets

Faculty and Research Publications

During development, Myxococcus xanthus cells undergo programmed cell death (PCD) whereby 80% of vegetative cells die. Previously, the MazF RNA interferase has been implicated in this role. Recently, it was shown that deletion of the mazF gene does not eliminate PCD in wild-type strain DK1622 as originally seen in DZF1. To clarify the role of MazF, recombinant enzyme was characterized using a highly sensitive assay in the presence and absence of the proposed antitoxin MrpC. In contrast to previous reports that MrpC inhibits MazF activity, the hydrolysis rate was enhanced in a concentration-dependent manner with MrpC or MrpC2, an N-terminally …

Phi X 174, David J. Hall

Infectious Pathogens

Phi X 174 database number # 1CD3. Phi X 174 is a virus that infects bacteria and was the first DNA-based genome to be sequenced in 1977. This bacteriophage has a [+] circular single-stranded DNA genome. The assembly of the capsid has been successfully done in vitro and is the focus of many studies on self-assembly, drug delivery and nanotechnology.

Human Papilloma Virus 16, David J. Hall

Infectious Pathogens

Human Papilloma virus 16, database# 1DZL Infection by most papillomaviruses is either asymptomatic or causes small benign tumors, known as papillomas or warts (e.g. human papillomavirus HPV6 or HPV11). Papillomas caused by some types, however, such as human papillomaviruses 16 and 18, carry a risk of becoming cancerous. Human papilloma virus is a non-enveloped, double stranded, circular DNA virus.

Dengue Virus, David J. Hall

Infectious Pathogens

Dengue virus, database # 1THD Dengue fever, is an infectious tropical disease caused by the dengue virus and transmitted by mosquito. Symptoms include fever, headache, muscle and joint pains, and a characteristic skin rash that is similar to measles. In a small proportion of cases the disease develops into the life-threatening dengue hemorrhagic fever. Dengue is a positive strand RNA virus that is part of the flavivirus family. Dengue is an enveloped virus meaning it has a nucleocapsid protecting the RNA and the nucleocapsid is surrounded by lipids (hence the name enveloped).

Hepatitis B, David J. Hall

Infectious Pathogens

Hepatitis B, database # 2g33. Hepatitis B is an infectious inflammatory illness of the liver caused by the hepatitis B virus (HBV). Hepatitis B virus (HBV) is a member of the Hepadnavirus family.The virus particle, (virion) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. These virions are 42 nM in diameter. The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity.

Inhibition Of Bacillus Cereus Growth By Bacteriocin Producing Bacillus Subtilis Isolated From Fermented Baobab Seeds (Maari) Is Substrate Dependent, Donatien Kaboré, Dennis S. Nielsen, Hagrétoui Sawadogo-Lingan, Bréhima Diawara, Mamoudou H. Dicko Prof., Mogens Jakobsen, Line Thorsen

Pr. Mamoudou H. DICKO, PhD

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 …

The Characterization Of The Interplay Between Sumoylation And Influenza A Virus, Andres Santos

Open Access Theses & Dissertations

Influenza A virus is a negative sense single stranded RNA virus responsible for multiple devastating pandemics in the past. This virus is capable of entering multiple eukaryotic hosts, sequestering, and using their cellular machinery for its own replicative purposes. This plethora of molecular events is all orchestrated through a shockingly limited repertoire of only 11 viral proteins. All of which are all strictly dependent on cellular processes such as post-transcriptional regulation, protein translation, post-translational modifications, etc. This study is focused on the characterization of the interplay between Influenza A virus and the cellular SUMOylation system. The first part of this …

Sumoylation Exerts Minimal Effects On The Splicing Regulatory Activity Of The Influenza A Virus Non-Structural Protein Ns1, Griselda Melendez

Open Access Theses & Dissertations

One of the greatest challenges facing the development of a universal treatment for influenza infection is the virus's ability to mutate rapidly and produce new antigenic properties. Targeting the viral components of one strain does not ensure that the treatment will be effective with a newly developed strain. One alternative would be to develop therapies that target cellular factors the virus depends on for survivability. One of these factors is the cellular SUMOylation system. We have previously demonstrated that the Small Ubiquitin-like MOdifier (SUMO) interacts with the influenza virus by SUMOylating several viral proteins and by affecting the interferon blocking …

Detection Of Viable Microorganisms Using Propidium Monoazide, Erik J. Mcfarland, Adrian Ponce Dr.

STAR Program Research Presentations

Propidium monoazide (PMA) is a molecular tool used to assess viability of microorganisms. Currently, PMA is thought to discern viability through membrane permeability; PMA enters only membrane compromised cells, irreversibly crosslinks to theirDNAand precipitates theDNAout of solution, preventing it from being amplified during polymerase chain reaction (PCR). Using PMA on a sample of live and dead microorganisms results in only theDNAof living organisms being amplified and identified. Therefore, a comparison ofPCRresults with and without PMA allows one to determine the live fraction and total population, respectively.

Current literature provides conflicting evidence as to the effectiveness of the technique. Our research …

Extracellular Proteases Are Key Mediators Of Staphylococcus Aureus Virulence Via The Global Modulation Of Virulence-Determinant Stability, Stacey L. Kolar, J. Antonio Ibarra, Frances E. Rivera, Joe M. Mootz, Jessica E. Davenport, Stanley M. Stevens Jr., Alexander R. Horswill, Lindsey N. Shaw

Molecular Biosciences Faculty Publications

Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community-associated methicillin-resistant S. aureus (CA-MRSA). The reason for enhanced CA-MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr-regulated toxins, including secreted proteases. In this study, we explore the contribution of exo-proteases to CA-MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide-rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. …