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Item Examining the potential role of biogenic amines in vaginal microbial ecology and gynecological health(Montana State University - Bozeman, College of Letters & Science, 2019) Borgogna, Joanna-Lynn Claire; Chairperson, Graduate Committee: Carl Yeoman; Carl J. Yeoman was a co-author of the article, 'The application of molecular methods towards an understanding of the role of the vaginal microbiome in health and disease' in the journal 'The human microbiome' which is contained within this thesis.; Michelle D. Shardell, Elisa K. Santori, Tiffanie M. Nelson, Jessica M. Rath, Elbert D. Glover, Jacques Ravel, Patti E. Gravitt, Carl J. Yeoman and Rebecca M. Brotman were co-authors of the article, 'The vaginal metabolome and microbiota of cervical HPV-positive and HPV-negative women: a cross-sectional analysis' in the journal 'British journal of obstetrics and gynecology' which is contained within this thesis.; Michelle D. Shardell, Carl J. Yeoman, Khalil G. Ghanem, Herlin Kadriu, Alexander V. Ulanov, Charlotte A. Gaydos, Justin Hardick, Courtney K. Robinson, Patrik M. Bavoil, Jacques Ravel, Rebecca M. Brotman and Susan Tuddenham were co-authors of the article, 'The association of Chlamydia trachomatis and Mycoplasma genitalium infection with the vaginal metabolome' submitted to the journal 'Scientific reports' which is contained within this thesis.; Savannah Gray, Elisa K. Santori, Ben Americus, Zhong Li, Alexander Ulanov, Jacques Ravel, Rebecca M. Brotman and Carl J. Yeoman were co-authors of the article, 'Biogenic amines affect the growth and lactic acid productin of vaginal Lactobacillus SPP.' which is contained within this thesis.Bacterial vaginosis is the most common vaginal disorder amongst reproductive-aged women affecting nearly 1/3 of all US women. Bacterial vaginosis is associated with an increased risk of adverse sexual and reproductive health outcomes including an increased sexually transmitted infection acquisition risk. Characteristic features of bacterial vaginosis include an increase in vaginal pH (>4.5) that is associated with a depletion of vaginal Lactobacillus spp., and replacement by various strict and facultative anaerobes. The depletion of Lactobacillus spp. is an important feature as the production of lactic acid by vaginal Lactobacillus species is considered the major barrier to infection. Women with bacterial vaginosis have reduced lactic acid and higher concentrations of biogenic amines. Biogenic amines may be important biomolecules bridging important microbiological and clinical characteristics that connect shifts in the vaginal microbiome to common features of bacterial vaginosis. Herein, we utilized 16S rRNA gene amplicon sequencing combined with metabolomics to evaluate the association between biogenic amines and the most prevalent bacterial (Chlamydia trachomatis) and viral (Human papillomavirus) sexually transmitted infection. We further assessed the effect of biogenic amines upon specific growth properties of vaginal lactobacilli. We observed that women who were HPV positive or had Chlamydia trachomatis mono- or Chlamydia trachomatis /Mycoplasma genitalium co-infection had higher concentrations of biogenic amines when compared to women who were uninfected. Growth assays demonstrated that biogenic amines adversely affected the growth of the major vaginal lactobacilli. Putrescine was associated with increased doubling times and longer lag times for all tested species, while cadaverine increased the doubling times of all except L. iners, Exposure to biogenic amines was generally associated with reduced production of lactic acid, Collectively, these data provide valuable evidence that biogenic amines negatively affect the growth of vaginal Lactobacillus species, in vitro, and are associated not only with bacterial vaginosis but also with HPV and C. trachomatis mono- and co-infection. Taken together, these data provide a more refined understanding of the potential impact of biogenic amines upon the vaginal microenvironment and increased susceptibility to bacterial vaginosis, vaginal dysbiosis, and bacterial and viral STIs.Item Developing and implementing genetic tools designed to understand host takeover by Chlamydia trachomatis.(Montana State University - Bozeman, College of Letters & Science, 2019) Kessy, Enock Joel; Chairperson, Graduate Committee: Blake WiedenheftChlamydia are gram negative obligate intracellular parasites that are responsible for millions of new infections in humans and animals every year. C. trachomatis is the number one cause of bacterial sexually transmitted infections in the United States, the number one cause of infectious blindness worldwide. Since 2001, there has been a steady increase in the number of new cases of C. trachomatis infections each year. Despite the prevalence and medical importance of C. trachomatis, we still know relatively little about the lifecycle of this parasite and the host factors that are essential for the lifecycle of C. trachomatis. To address this critical gap in our knowledge, my thesis work aimed to develop and implement genetic tools to understand host takeover by C. trachomatis. In this thesis I present results suggesting that I have transformed C. trachomatis with a plasmid carrying the Cas9 gene from Campylobacter jejuni. Additional experiments are necessary to determine if the CjCas9 is expressed, nuclease active, and functional for programable editing in C. trachomatis. In addition to my work aimed at developing a CRISPR-Cas9-based genetic engineering system in C. trachomatis, I also participated in a genome wide knockout screen aimed at identifying human genes necessary for completion of the C. trachomatis lifecycle. The CRISPR-Cas9 genome wide knockout screen identified 103 genes as critical factors for C. trachomatis. To validate results for the screen I have been involved in creating clonal cell lines with deletions in three of the genes that form the Adaptor Protein (AP) Complex (i.e., AP3S2, AP1B2 and AP1G2). The genes have been deleted and future experiments are aimed at measuring the impact of these genes on the C. trachomatis lifecycle.Item The intercellular spread of alphaherpesviruses(Montana State University - Bozeman, College of Letters & Science, 2018) Herr, Alix Elise; Chairperson, Graduate Committee: Matt Taylor; Kyles S. Hain and Matthew P. Taylor were co-authors of the article, 'Limitations on the multiplicity of cellular infection during human alphaherpesviral disease' in the journal 'Current clinical microbiology reports' which is contained within this thesis.; Theresa Thornburg, Max DePartee, Ryan Waters and Matthew P. Taylor were co-authors of the article, 'The route of transmission from neurons impacts pseudorabies virus coinfection in vivo' submitted to the journal 'Journal of virology' which is contained within this thesis.There are three prominent alphaherpesviruses that infect humans: Herpes Simplex virus-1, Herpes Simplex virus-2, and Varicella Zoster virus. Each virus is harbored within 15-98% of the population. Prototypical infections involve only a handful of intercellular spread events within a host. Most spread events involve neurons. Only one virion is thought to be successfully transmitted from a neuron to another cell -- but this has yet to be verified during infectious spread within a host. In this dissertation, we used Pseudorabies virus to trace the number of virions spreading infection from infected neurons to uninfected cells. Pseudorabies virus is a prominent model alphaherpesvirus that infects mice. To quantify the number of virions transmitted between cells, we intravitreally injected different, genetically engineered Pseudorabies virus strains and quantified spread to the murine central nervous system. We calculated the average number of expressed viral genomes per infected neuron by utilizing the Poisson distributions of neurons expressing one, two, or three different viral strains. We found that when a neuronal axon transmitted infection to cells, a cell became infected with one virion on average. In contrast, when neuronal soma or dendrites transmitted the viral strains to surrounding cells, each cell expressed three viral genomes on average. Most importantly, we discovered that the absence of a specific alphaherpesviral protein, US9, diminished the capacity of the virus to infect a cell with a plurality of viral particles. This dissertation advanced the field of herpesviral research in two ways: by quantifying the number of alphaherpesviral particles transmitted between infected neurons in a host and identifying a viral protein instrumental in determining the number virions transmitted from neurons to other cells. The average number of viral particles infecting cells within a host determines the viral genetic dosage and impacts viral gene expression, viral replicative rates, and viral diversification.Item The immunobiology of calf scours(Montana State University - Bozeman, College of Agriculture, 1973) Wilson, Richard Adam