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 Role Of Histone H3 And H4 In Centromere Function And Genome Integrity, Payel Chaudhuri

Graduate Theses and Dissertations

Histone H2A plays an important role in chromosomal segregation among parent and daughter cells during mitosis. While it is established that this histone is important in maintaining chromosome number in cell, further work is carried out to explore the role of other histones like H3 and H4 for similar effects. A systematic study is initiated by screening a library based on mutation of different amino acid residues in these histones. This detailed screening identified specific regions within H3 and H4, which are critically important for centromeric function. These histones residing near the DNA entry/exit region of nucleosome effects the functionality …

Evaluating The Influence Of Environmental Factors On The Rate Of Extra-Pair Matings In Tropical And Temperate Populations Of The House Wren (Troglodytes Aedon), Kaitlin Claire Mckenney

Theses and Dissertations

Considerable variation exists in the rate of extra-pair matings (EPMs) in birds. Environmental variability likely influences EPM rates within species, but the effects of local environmental factors on EPM rates are largely unpredictable. To determine whether broad-scale environmental factors might be better predictors of EPM rates within species, we quantified levels of extra-pair paternity in the house wren (Troglodytes aedon) in four populations spanning a range of latitude, elevation, and primary productivity (measured by actual evapotranspiration rates). Our results indicated an intermediate and variable level of EPM among populations (6 -31% extra-pair young) that was not significantly affected by 3 …

Homeotic Gene Teashirt (Tsh) Has A Neuroprotective Function In Amyloid-Beta 42 Mediated Neurodegeneration, Michael T. Moran, Meghana Tare, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Background: Alzheimer's disease (AD) is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42) polypeptides formed by the improper cleavage of amyloid precursor protein (APP) in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s) responsible for this neurodegeneration still remain elusive.

Methodology/Principal Findings: We have generated a transgenic Drosophila eye model where …

A Study On The Regulation Of Amino Acids And Glucose Sensing Pathways In Saccharomyces Cerevisiae, Mengying Chiang

University of New Orleans Theses and Dissertations

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to …

Chromatin Insulators: Master Regulators Of The Eukaryotic Genome, Todd Andrew Schoborg

Doctoral Dissertations

Proper organization of the chromatin fiber within the three dimensional space of the eukaryotic nucleus relies on a number of DNA elements and their interacting proteins whose structural and functional consequences exert significant influence on genome behavior. Chromatin insulators are one such example, where it is thought that these elements assist in the formation of higher order chromatin loop structures by mediating long-range contacts between distant sites scattered throughout the genome. Such looping serves a dual role, helping to satisfy both the physical constraints needed to package the linear DNA polymer within the small volume of the nucleus while simultaneously …

Functional Analysis Of Corazonin And Its Receptor In Drosophila Melanogaster, Kai Sha

Doctoral Dissertations

Corazonin (Crz) is an amidated undecapeptide originally isolated from the American cockroach. It has been shown to affect diverse physiological functions in a species-specific manner. However, the functionality of Crz in Drosophila melanogaster has not yet been determined. To gain insight into the role of Crz signaling in vivo, Crz and CrzR null alleles were obtained by transposable element mobilization. Flies carrying a deficiency uncovering Crz and pr-set7 loci were generated via P-element excision, and the latter was rescued by wild-type pr-set7 transgene. A mutation of Crz receptor (CrzR) was generated by Minos-element mobilization from …

Med13p Prevents Stress-Independent Mitochondrial Hyperfragmentation And Aberrant Apoptosis Activation In Saccharomyces Cerevisiae By Controlling Cyclin C Nuclear Localization, Svetlana Khakhina

Graduate School of Biomedical Sciences Theses and Dissertations

During aging, and as a result of environmental changes, cells are exposed to elevated levels of reactive oxygen species (ROS). High ROS levels induce lipid oxidation, protein aggregation, mitochondrial hyperfragmentation, DNA damage and programmed cell death (PCD), also called apoptosis. PCD is a highly regulated process and its misregulation has been linked to neurodegenerative diseases and cancer development.

Our hypothesis is that cyclin C plays a role in the initiation of apoptosis. During normal conditions, cyclin C represses the transcription of stress response genes (SRG). In response to stress, cyclin C translocates to the cytoplasm where it facilitates mitochondrial hyperfragmentation …

Single-Nucleotide Polymorphisms Associated With Performance Traits In Beef Cattle Grazing Endophyte-Infected Tall Fescue, Bryan Christopher Bastin

Masters Theses

Tall fescue (Lolium arundinaceum Schreb.) is the most prevalent forage in the Midsouth United States due in part to the presence of the endophytic fungus Neotyphodium coenophialum. The fungus, while conferring hardiness to tall fescue, contributes to decreased production efficiency in cow-calf operations. A previous genome-wide association study was performed using the Illumina 50k bovine SNP chip. Twenty-four SNP were found to be associated (P < 0.05) with adjusted birth weight and adjusted 205-day weights of calves from 48 beef cows at Ames Plantation. The first objective was to validate each SNP by testing associations with several additional phenotypes. Custom Taqman genotyping assays (Applied Biosystems, Foster City, CA) were subsequently designed to genotype each SNP in beef cattle located at Tennessee Tech University (n = 654), to validate associations in a large, independent herd. The results yielded 15 associations that were significant (P < 0.05) with 6 phenotypes linked to those affected by fescue toxicosis. The second objective investigated the link between fescue toxicosis and the XK, Kell blood group complex subunit-related, member 4 …

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 …

Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham

Doctoral Dissertations

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as …

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 …

Reverse Genetic Analysis Of A Cysteine Protease-Encoding Gene (Rd19a) Of Arabidopsis Thaliana In Relation To The Mechanism Of Resistance To The Piercing/Sucking Insect Myzus Persicae, Siobhan A. Cusack

Honors College

A recent study in Solanum bulbocastanum (a wild relative of the cultivated potato) aiming to identify potential genes involved in aphid and pathogen resistance mechanisms found that a homolog of the Arabidopsis thaliana cysteine protease gene RD19a is upregulated during aphid infestation. RD19a is upregulated in response to abiotic stresses such as drought and high salinity, and rd19a mutants show increased susceptibility to bacterial infection. In this study, Arabidopsis rd19a mutants and wild-type plants were subjected to aphid feeding to observe and compare the molecular, physiological and phenotypic responses. The aim was to further establish the proof of concept regarding …

Genetic Parameters For Concentrations Of Minerals In Longissimus Muscle And Their Associations With Palatability Traits In Angus Cattle, R. G. Mateescu, A. J. Garmyn, Richard G. Tait Jr., Qing Duan, Q. Liu, Mary S. Mayes, Dorian J. Garrick, A. L. Van Eenennaam, D. L. Vanoverbeke, G. G. Hilton, Donald C. Beitz, James M. Reecy

Richard G. Tait Jr.

The objective of this study was to estimate genetic parameters for concentrations of minerals in LM and to evaluate their associations with beef palatability traits. Samples of LM from 2,285 Angus cattle were obtained and fabricated into steaks for analysis of mineral concentrations and for trained sensory panel assessments. Nine minerals, including calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc, were quantified. Restricted maximum likelihood procedures were used to obtain estimates of variance and covariance components under a multiple-trait animal model. Estimates of heritability for mineral concentrations in LM varied from 0.01 to 0.54. Iron and sodium were …

Genetic Parameters For Carnitine, Creatine, Creatinine, Carnosine, And Anserine Concentration In Longissimus Muscle And Their Association With Palatability Traits In Angus Cattle, R. G. Mateescu, A. J. Garmyn, M. A. O'Neil, Richard G. Tait Jr., Almass A. Abuzaid, Mary S. Mayes, Dorian J. Garrick, A. L. Van Eenennaam, D. L. Vanoverbeke, G. G. Hilton, Donald C. Beitz, James M. Reecy

Richard G. Tait Jr.

The objective of this study was to estimate genetic parameters for carnitine, creatine, creatinine, carnosine, and anserine concentration in LM and to evaluate their associations with Warner-Bratzler shear force (WBSF) and beef palatability traits. Longissimus muscle samples from 2,285 Angus cattle were obtained and fabricated into steaks for analysis of carnitine, creatine, creatinine, carnosine, anserine, and other nutrients, and for trained sensory panel and WBSF assessments. Restricted maximum likelihood procedures were used to obtain estimates of variance and covariance components under a multiple-trait animal model. Estimates of heritability for carnitine, creatine, creatinine, carnosine, and anserine concentrations in LM from Angus …

Cellular Adaptation Of Macrophages To Anthrax Lethal Toxin-Induced Pyroptosis Via Epigenetic Mechanisms, Chae Young Han

Electronic Thesis and Dissertation Repository

Cellular adaptation to microbial stresses has been demonstrated in several cell types. Macrophages (MФ) are sentinel immune cells fending off invading microbes. Anthrax lethal toxin (LeTx) is a key virulence factor released by Bacillus anthracis that causes rapid cell death, pyroptosis. A small number of RAW246.7 macrophages (~4%) exposed to a non-lethal dose of LeTx become resistant to LeTx-induced pyroptosis for ~ 4 weeks, termed “toxin-induced resistance (TIR)”. Here, I showed that high levels of DNA methyl transferase1 (DNMT1) expression were maintained although global genomic methylation levels were not high in TIR. TIR cells treated with the DNMT inhibitor 5-azacitidine …

Bed Bugs Evolved Unique Adaptive Strategy To Resist Pyrethroid Insecticides, Fang Zhu, Hemant Gujar, Jennifer R. Gordon, Kenneth F. Haynes, Michael F. Potter, Subba R. Palli

Entomology Faculty Publications

Recent advances in genomic and post-genomic technologies have facilitated a genome-wide analysis of the insecticide resistance-associated genes in insects. Through bed bug, Cimex lectularius transcriptome analysis, we identified 14 molecular markers associated with pyrethroid resistance. Our studies revealed that most of the resistance-associated genes functioning in diverse mechanisms are expressed in the epidermal layer of the integument, which could prevent or slow down the toxin from reaching the target sites on nerve cells, where an additional layer of resistance (kdr) is possible. This strategy evolved in bed bugs is based on their unique morphological, physiological and behavioral characteristics …

The Drosophila Interactions Database: Integrating The Interactome And Transcriptome, Thilakam Murali

Wayne State University Dissertations

In this thesis I describe the integration of heterogeneous interaction data for Drosophila into DroID, the Drosophilainteractions database, making it a one-stop public resource for interaction data. I have also made it possible to filter the interaction data using gene expression data to generate context-relevant networks making DroID a one-of-a kind resource for biologists. In the two years since the upgraded DroID has been available, several studies have used the heterogeneous interaction data in DroID to advance our understanding of Drosophila biology thus validating the need for such a resource for biologists. In addition to this, I have identified …

Aβ Alters The Dna Methylation Status Of Cell-Fate Genes In An Alzheimer’S Disease Model, Gary D. Isaacs, Noor Taher, Courtney Mckenzie, Rebecca Garrett, Matthew Baker, Nena Fox

Faculty Publications and Presentations

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-β plaques (Aβ). Despite ongoing research, some ambiguity remains surrounding the role of Aβ in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aβ treatment. We isolated the DNA from neurons treated with Aβ or vehicle, and digested the two samples with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. …

Overexpression/Silencing Of Selected Soybean Genes Alters Resistance To Pathogens, Mohamed H. El-Habbak

Theses and Dissertations--Plant Pathology

Plant diseases remain a major obstruction to meeting the world’s increased demand for soybean oil and protein. Reducing the losses caused by diseases in order to improve crop production is a high priority for agricultural research. The need for novel strategies for plant disease control cannot be overstated. In the present study, selected defense-related genes were silenced and/or overexpressed in soybean using a virus-based vector and the resultant plants were tested for their responses to pathogens. The first part of the study focused on Rps1k (Resistance to Phytophthora sojae) gene. The two conserved domains encoding ‘P-Loop NTPase’ and ‘PLN03210’ …

Investigation Of X Chromosome Recognition: The Role Of Small Rna In Drosophila Dosage Compensation, Debashish Unnikrishnan Menon

Wayne State University Dissertations

In humans and flies, females have two X chromosomes but males have one X chromosome and one Y chromosome. This leads to a fatal imbalance in X-linked gene expression in one sex. In mammals and in the fruit fly Drosophila, modulation of X chromosome expression is critical for survival. This process is termed dosage compensation. Flies increase expression from the male X chromosome two-fold. This is achieved by the Male Specific Lethal (MSL) complex, which consists of two large, non-coding RNA on the X transcripts (roX1 and roX2) and five proteins. The roX RNAs have a critical …

Investigation Of Bacterial Rna-Directed Dna Methylation Via Dcm And Hfq, Dandan Li

Wayne State University Theses

Bacterial small RNAs and the RNA chaperone Hfq play crucial roles in post-transcriptional gene regulation, often as parts of stress-response pathways, but little is known about their roles in regulation of gene transcription. A recent report showed that changes in methylation patterns caused by DNA cytosine methyltransferase (Dcm) were linked to gene regulation occurring during the transition to stationary phase. Here, we show that Dcm involves in the stress responses under nutrient starvation and cold stress. Dcm and Hfq together mediate gene expression under cold stress. Hfq promotes Dcm-catalyzed cytosine methylation at specific sites near the rpoS promoter, which is …

Mitochondrial Dna Instability In Cells Lacking Aconitase Correlates With Iron Citrate Toxicity, Muhammad A. Farooq, Tammy M. Pracheil, Zhejun Dong, Fei Xiao, Zhengchang Liu

Biological Sciences Faculty Publications

Aconitase, the second enzyme of the tricarboxylic acid cycle encoded by ACO1 in the budding yeast Saccharomyces cerevisiae, catalyzes the conversion of citrate to isocitrate. aco1 Delta results in mitochondrial DNA (mtDNA) instability. It has been proposed that Aco1 binds to mtDNA and mediates its maintenance. Here we propose an alternative mechanism to account for mtDNA loss in aco1 Delta mutant cells. We found that aco1 Delta activated the RTG pathway, resulting in increased expression of genes encoding citrate synthase. By deleting RTG1, RTG3, or genes encoding citrate synthase, mtDNA instability was prevented in aco1 Delta mutant …

Sirt1 Regulation Of The Heat Shock Response In An Hsf1-Dependent Manner And The Impact Of Caloric Restriction, Rachel Rene Raynes

USF Tampa Graduate Theses and Dissertations

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a …

Malnutrition In Sickle Cell Anemia: Implications For Infection, Growth And Maturation, Hyacinth I. Hyacinth, Oluwatoyosi A. Adekeye, Christopher S. Yilgwan

Journal of Social, Behavioral, and Health Sciences

Sickle cell anemia (SCA) is a genetic disease that affects mostly individuals of African and/or Hispanic descent, with the majority of cases in sub-Saharan Africa. Individuals with this disease show slowed growth, delayed sexual maturity, and poor immunologic function. These complications could partly be explained by the state of undernutrition associated with the disease. Proposed mechanism of undernutrition include protein hypermetabolism, decreased dietary intake possibly from interleukin-6-related appetite suppression, increased cardiac energy demand/expenditure, and increased red cell turnover. All the above mechanisms manifest as increased resting energy expenditure. Nutritional intervention utilizing single or multiple nutrient supplementation has led to improved …

Dna-Binding Site Recognition By Bhlh And Mads-Domain Transcription Factors, Joshua R. Werkman

Theses and Dissertations--Plant and Soil Sciences

Herewithin, two transcription factor (TF) regulatory complexes were investigated. A bHLH–MYB–WDR (BMW) DNA-binding complex from maize was the first complex to be studied. R, a maize bHLH involved in the activation of genes in the anthocyanin pathway, had been characterized to indirectly bind DNA despite the presence of a functional DNA-binding domain. Findings presented here reveal that this is only partially correct. Direct DNA-binding by R was found to be dependent upon two distinct dimerization domains that function as a switch. This switch-like mechanism allows R to be repurposed for the activation of promoters of differing cis-element structure.

The …

Characterization Of G-Patch Motif Contribution To Prp43 Function In The Pre-Messenger Rna Splicing And Ribosomal Rna Biogenesis Pathways, Daipayan Banerjee

Theses and Dissertations--Biology

The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 …

The Role Of Cardiolipin In Iron Homeostasis And Glutathione Metabolism, Vinay A. Patil

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. Inside the mitochondria, CL is a critical target of mitochondrial generated reactive oxygen species (ROS) and regulates signaling events related to apoptosis and aging. CL deficiency causes perturbation of signaling pathways outside the mitochondria, including the PKC-Slt2 cell integrity pathway and the high osmolarity glycerol (HOG) pathway, and is a key player in the cross-talk between the mitochondria and the vacuole. The importance of CL in human health is underscored by the observation that perturbation of CL …

The Role Of Polyadenylation In Seed Germination, Liuyin Ma

Theses and Dissertations--Plant and Soil Sciences

Seed germination has many impacts on the uses of seeds, and is an important subject for study. Seed germination is regulated at both transcriptional and post-transcriptional levels. Therefore, it is important to study how polyadenylation regulates gene expression during seed germination. To this end, a modified Illumina GAIIx sequencing protocol (described in Chapter Two) was developed that allows deep coverage of poly(A) site position and distribution.

Alternative polyadenylation (APA) regulates gene expression by choosing one potential poly(A) site on a precursor RNA consequentially shortening/lengthening the mRNA relative to other possible sites. To further explore this phenomenon, genes affected by APA …