Microbiology Commons^™

Investigating Soil Microbiome Changes During Panax Quinquefolius Cultivation In New Gardens, Pok Man Paul Wan

Electronic Thesis and Dissertation Repository

Ginseng (Panax quinquefolius) is susceptible to ginseng replant disease (GRD), resulting in root rot and decreased yield when ginseng is grown in soils previously used for ginseng crops. Although fungal and oomycete pathogens are implicated, GRD is a complex disease and the changes in the soil when initially cultivating ginseng that lead to GRD remain unclear. This thesis reported changes in the soil microbiome from three new gardens in Norfolk County, Ontario over three years starting from seeding with ginseng. Over this period of cultivation, metabarcoding of the V4 region of the 16S ribosomal RNA gene clearly showed …

Evolution Of Overlapping Reading Frames In Virus Genomes, Laura Muñoz Baena

Electronic Thesis and Dissertation Repository

Viruses are formidable pathogens that represent the majority of biological entities in our planet, and their genomes are a source of interesting enigmas. One feature in which virus genomes are usually rich, is the presence of overlapping reading frames (OvRFs) — portions of the genome where the same nucleotide sequence encodes more than one protein. OvRFs are hypothesized to be used by viruses to encode proteins more compactly and to regulate transcription. In addition, OvRFs might be a source of gene novelty, facilitating the creation of new open reading frames (ORF) within the transcriptional context of existing ones.

To characterize …

Exploration Of The Immune Landscape Of Ebv-Associated Gastric Cancers, Mikhail Salnikov

Electronic Thesis and Dissertation Repository

Epstein–Barr virus (EBV) is a gammaherpesvirus associated with 9% of all gastric cancers (GCs). EBV-associated GCs (EBVaGCs) are pathologically and clinically distinct entities from EBV-negative GCs (EBVnGCs), with EBVaGCs exhibiting differential molecular pathology and patient prognosis. The purpose of this thesis is to investigate the tumor microenvironment (TME) of EBVaGCs, which has not been explored in-depth. We hypothesize that EBVaGCs and EBVnGCs are also distinct in terms of the molecular immune landscape. We employed over 400 stomach adenocarcinoma (STAD) samples from The Cancer Genome Atlas (TCGA), as well as a single cell dataset, for the construction of a web suite …

Development Of A High-Throughput Approach For Testing Optimal Fecal Microbiota Transplant Material, Shaeley Jae Gibbons

Electronic Thesis and Dissertation Repository

Historically used to treat Clostridioides difficile infection, fecal microbiota transplants (FMT) are now being tested to treat gut and systemic diseases, including (but not limited to) autoimmune, liver, and cardiovascular. Successful FMT relies on components within the donor sample, which may have specific conserved properties that vary greatly between individuals. Screening donors for potentially transmittable conditions is a well-established practice, however, screening material for the likeliness of a successful clinical outcome or finding the best match of material for patients has not been undertaken. We hypothesized an optimal donor may be selected by high-throughput in vitro screening of fecal microbiota. …

From Micro To Macro: Examining Potential Microbiome Mediated Influences On Human Growth And Health Outcomes Through Breastfeeding And Antibiotic Exposures, Nicole K. Phillips

Electronic Thesis and Dissertation Repository

Human microbiome research has rapidly developed over the past two decades yet absent from most research is the composition and dynamics of microbiomes within human populations. Given the limitations in longitudinal studies which requires decades of repeated microbe taxonomic testing of a population sample, an alternative option is to examine microbiomes and their influences via proxies using pre-existing health datasets. This research demonstrates preliminary associations between presumed disrupted and supportive microbiomes dynamics proxied by antibiotic and breastmilk exposure respectively. Using health record data across the life span from approximately 500,000 U.K. participants, this research demonstrates variable altered growth and health …

Using A One Health Approach For The Assessment Of Rabies Control In Rural Victoria Falls, Zimbabwe, Ryan Lapenna

Electronic Thesis and Dissertation Repository

Rabies, a viral zoonotic disease, is widespread in Zimbabwe, with human fatalities reported annually in the country. To supplement the Government’s effort on rabies control, the Victoria Falls Wildlife Trust and Veterinarians for Animal Welfare Zimbabwe have joined forces to carry out dog rabies vaccinations in rural communities adjacent to the major tourist attraction of The Victoria Falls. A One Health approach was used to 1) determine the level of protective rabies antibodies among a sample of rural dogs and 2) collect information and describe the knowledge, attitudes, and practices related to rabies among dog owners. In 2020, blood was …

Towards More Complete Metagenomic Analyses Through Circularized Genomes And Conjugative Elements, Benjamin R. Joris

Electronic Thesis and Dissertation Repository

Advancements in sequencing technologies have revolutionized biological sciences and led to the emergence of a number of fields of research. One such field of research is metagenomics, which is the study of the genomic content of complex communities of bacteria. The goal of this thesis was to contribute computational methodology that can maximize the data generated in these studies and to apply these protocols human and environmental metagenomic samples.

Standard metagenomic analyses include a step for binning of assembled contigs, which has previously been shown to exclude mobile genetic elements, and I demonstrated that this phenomenon extends to all conjugative …

Regulation Of Mhc Ii Trafficking And Expression By Host And Viral Factors, Alex Lac

Electronic Thesis and Dissertation Repository

Antigen presentation by major histocompatibility complex class II (MHC II) to the adaptive immune system is crucial for mounting sterilizing immune responses. This central role has made antigen presentation a target for antagonism by many pathogens. Notably, infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) decrease MHC II expression in several immune cells. The mechanisms responsible for this suppression are unknown but involves either redirecting MHC II molecules away from the cell surface or inhibiting MHC II expression. To understand how pathogens manipulate intracellular MHC II trafficking, we first investigated the role of the Golgi trafficking regulator, ERC1, in …

Developing A Toolbox To Engineer Quantitative Trait Variation In Legume Species Using Crispr/Cas Technologies., Petar Miletic

Electronic Thesis and Dissertation Repository

The impact of intensive agriculture on the environment is immense. This is especially dire with regard to the natural nitrogen (N) cycle, where the human driven interference, primarily associated with industrial fertilization, has reached unsustainable levels. Unlike cereals, legumes, such as soybean, alfalfa and common bean, have the ability to use atmospheric nitrogen, which limits the need for industrial fertilization. A more wide-spread use of legumes could alleviate some of the negative impacts on the biogeochemical cycle while also providing a useful alternative to meat consumption, an important factor in sustainability. To reach this goal, further improvements of legume crops …

Response Of The Soil Bacterial Community, Resistome, And Mobilome To A Decade Of Macrolide Antibiotic Contamination, Liam Paul Brown

Electronic Thesis and Dissertation Repository

Biosolids (treated sewage sludge) are used as agricultural fertilizer but are frequently contaminated with macrolide antibiotics, to which resistance is rising among historically susceptible bacteria. To determine if the land-application of macrolides carried in biosolids could promote antibiotic resistance in soil bacteria, soil plots were exposed annually to environmentally realistic or high doses of macrolides for ten years. I sequenced the bacterial 16S ribosomal DNA, metagenomic DNA, and integron gene cassettes within the treated and antibiotic-free soil to compare the compositions and diversities of the bacterial communities, antibiotic resistance genes, and mobile genetic elements. I determined that the high dose …

Fishing For The Right Probiotic: Investigating The Mechanism Of Mucosal-Bacterial Interactions At The Interface Of Health And Productivity In Salmonid Aquaculture, Luana Langlois

Electronic Thesis and Dissertation Repository

Aquaculture is vital for the global food supply, but the high incidence of infectious diseases threatens the industry’s productivity. The intestinal mucosa is a key port of entry for pathogens and provides an extensive interface for host-microbe interactions. Tight junctions are at the core of gut barrier function and the mucosal health of finfish. Disruption of these complexes gives rise to sepsis, which leads to systemic inflammation and death. The present study employs a combinatorial approach that integrates in vitro and in vivo analyses to gain actionable insights into the mechanism of microbial-mediated modulation of host health. The experiments outlined …

Microbial Innovations For Disease Management In Honey Bees, Brendan A. Daisley

Electronic Thesis and Dissertation Repository

Arguably the most important insect pollinator, honey bees (Apis mellifera) are threatened by infectious disease, pesticide exposure, and nutritional deficits resulting from habitat loss.

The major goals of this thesis were to advance our understanding of the immune and microbiota factors underpinning synergistic interactions between the threats, and to develop a honey bee-specific probiotic with strain-level functions to alleviate cumulative stress burden.

Drosophila melanogaster was used as a high-throughput discovery platform in molecularly characterizing the effects of candidate probiotic lactobacilli and neonicotinoid-neuroimmune-microbiota mechanisms prior to experimentation in honey bees. Imd pathway activation by lactobacilli was identified to be …

The Ability Of Lactobacillus Spp. To Limit Extracellular Atp Release By Urogenital Bacteria, Hannah Wilcox

Electronic Thesis and Dissertation Repository

Urgency urinary incontinence is a common symptom of overactive bladder (OAB) and urinary tract infection (UTI), which can be triggered by extracellular adenosine triphosphate (eATP) through purinergic signaling. Current treatments for OAB and UTI fail to consider the potential impact of eATP. It is hypothesizes that certain commensal Lactobacillus spp. can modulate levels of eATP released by pathogenic bacteria. This project examined bacterial modulation of eATP during growth, the impact of prebiotics on lactobacilli growth, and studied its reduction in an in vitro bladder infection model. Release and uptake of ATP by a range of commensal and pathogenic bacteria was …

Investigation Of Shocked Basalts From Vargeão Dome And Vista Alegre: Implications For The Search For Life On Mars, Nikol Posnov

Electronic Thesis and Dissertation Repository

Impact craters represent excellent astrobiological targets for planetary exploration missions to Mars. The impact of an asteroid or comet into a crystalline, H₂O-bearing target may result in development of a hydrothermal system and increase the habitability of the substrate for the colonization of endolithic microorganisms. Given that Mars’ surface is covered by cratered basaltic rock, this study investigated target rocks and impact breccias from Vargeão Dome and Vista Alegre impact structures that formed in basalt in the Paraná Basin of Brazil.

Utilizing petrography and micro-X-ray diffraction (μXRD), the degree of shock metamorphism in plagioclase was quantitively determined. Measuring …

Generation And Characterization Of An In Vitro Organotypic Foreskin Model For Future Study Of The Penile Microbiome, Geoffrey J. Rempel

Electronic Thesis and Dissertation Repository

The foreskin is a site of HIV-1 acquisition in heterosexual males. The lack of relevant in vitro models that mimic the foreskin microenvironment, including innate immune and barrier functions, has limited our understanding of susceptibility at this site. We hypothesize that we can establish organotypic in vitro foreskin that mimics in vivo tissue. Organotypic foreskin models were generated and are composed of stratified cell layers that express E-cadherin suprabasally, filaggrin apically, and expressed TLR1, TLR2, and β-defensin-1 mRNA similar to in vivo foreskin. Preliminary results suggest organotypic foreskin tissues retain the ability to respond to LPS. Lastly, organotypic foreskin permeability …

Hiv-1 Drug Resistance To Integrase Strand Transfer Inhibitors In Hiv-1 Non-B Subtypes, Emmanuel Ndashimye

Electronic Thesis and Dissertation Repository

Human immunodeficiency syndrome (HIV-1) has infected over 75 million people and over 35 million have succumbed to virus related illnesses. Despite access to a variety of antiretroviral therapy (ART) options, ART programs have been disproportionally spread in the world with low-and middle-income countries (LMICs) facing challenges to access the most potent ART options. With less potent ART remaining in use in LMICs, HIV-1 drug resistance (HIVDR) presents a growing challenge in LMICs. Since approval of the first-generation integrase strand transfer inhibitor (INSTIs), Raltegravir (RAL) in 2007, INSTIs remain the best choice as a backbone of ART. Access to second generation …

A Workflow To Analyze Ethcd Mass Spectrometry Data For Studying Hiv Gp120 Glycosylation, Yingxue Sun

Electronic Thesis and Dissertation Repository

The great heterogeneity of HIV populations and richness of surface glycan clouds makes it difficult to locate a conserved and exposed protein epitope as an effective vaccine target. However, more than 80% new infections result from single transmitted founder (T/F) viruses. We set out to design a workflow to study the traits of T/Fs that allow for their superior infectivity, specifically, the glycosylation patterns of gp120, a subunit of HIV envelope protein responsible for binding to host cell receptors. Our main research methods include Western blot and mass spectrometry. Our current understanding of the mass spectrometry data indicates that our …

Characterization And Discovery Of Short Linear Motifs Mediating Protein Nuclear Import, Tanner M. Tessier

Electronic Thesis and Dissertation Repository

Protein-protein interactions (PPI) mediated through short linear motifs (SLiMs) are ubiquitous throughout the human proteome and are involved in many essential cellular processes. One such type of SLiM is the classical nuclear localization sequence (cNLS), which facilitates nuclear import by binding importin-α (Imp-α). This pathway is indispensable to many cellular processes and is extensively used by viral proteins that function within the nucleus of infected cells. Based on this, I demonstrated that the classical nuclear import pathway inhibitor, ivermectin, can inhibit replication of human adenovirus. Treatment with ivermectin blocks nuclear localization of the E1A protein, an essential viral nuclear protein …

Investigating The Impact Of Environmentally Relevant Imidazole Concentrations On The Antifungal Susceptibility And Community Composition Of Soil Fungi, Farhaan Kanji

Electronic Thesis and Dissertation Repository

Miconazole and clotrimazole are environmentally-persistent drugs that are entrained into crop soils through the application of biosolids. There is concern that environmental exposure to such azole antifungals, which inhibit fungal growth by disrupting the production of the fungal cell membrane component ergosterol, promotes resistance in clinically or agriculturally relevant fungi. Thus, either environmentally-relevant or excessive levels of these drugs were applied to microplots over ten years and compared with drug-free plots. Overall, ergosterol quantification, plates counts, and identification of >250 fungal isolates showed lower fungal counts and species richness in plots receiving excessive drug amounts. In addition, fungi from treated …

Development Of A Thermosensitive Endonuclease To Act As A Plasmid Kill-Switch, Christopher D. Leichthammer

Electronic Thesis and Dissertation Repository

Biocontainment is an integral part of biomedical research that aims to protect the environment and human health by containing hazardous or invasive organisms in the laboratory. Containment systems often rely on elaborate genetic circuits; however, cells may escape containment by developing mutations that render the genetic circuits inviable or resistant to killing mechanisms. The aim of this thesis was to create a site-specific nuclease for biocontainment of plasmids in the mammalian gastrointestinal tract. LAGLIDADG homing endonucleases would be good candidate nucleases for a biocontainment system as they are resistant to mutations in their coding sequence and their target sequence in …

Characterizing The Diversity Of The Eukaryotic Microbiome In Marine Crustacean Zooplankton., Rose-Lynne Savage

Electronic Thesis and Dissertation Repository

Understanding zooplankton productivity is critical for modeling marine food web function, of which one poorly known factor is the influence of zooplankton symbionts. Zooplankton protist symbiont diversity is underestimated due to the limited surveys and techniques previously used. Using 18S V4 metabarcoding, I characterized the eukaryotic microbiomes associated with crustacean zooplankton from the northern Strait of Georgia, BC. Apostome ciliates were most abundant in all hosts except for cyclopoid copepods, which were dominated by Syndiniales. Most symbiont lineages were more abundant in one or two hosts, suggesting some degree of host preference. Microbiome data also provided information on diet, confirming …

Molecular Identification And Characterization Of Viral Pathogens Infecting Sweet Cherry, Aaron J. Simkovich

Electronic Thesis and Dissertation Repository

Stone fruits are a valuable crop grown worldwide, however pathogens such as viruses threaten fruit production by reducing tree health and fruit yield. In an orchard within the Niagara region of Ontario, symptoms typical of viral infection such as chlorosis and leaf deformation were seen on sweet cherry (Prunus avium L.) trees. Next generation sequencing was performed on symptomatic and asymptomatic leaves and four viruses were identified. On the tree displaying the most severe symptoms, Prune dwarf virus (PDV), was the only virus detected. A survey conducted during this work showed 42% of cherry trees on a single …

Staphylococcus Aureus Small Colony Variants Use Heme And Staphyloferrin B For Iron Acquisition, Izabela Z. Batko

Electronic Thesis and Dissertation Repository

Iron is an essential nutrient for the bacterium Staphylococcus aureus. Wild-type S. aureus utilizes various iron acquisition systems to support growth in iron deplete conditions. S. aureus small colony variants (SCVs) are associated with chronic infections, yet the mechanisms by which these variants acquire iron are unknown. Mutation of hemB, involved in heme biosynthesis, generated a stable SCV that was auxotrophic for hemin and formed small colonies on solid media. To support growth under iron deplete conditions, my data revealed that S. aureus hemB synthesizes the siderophore staphyloferrin B, but not staphyloferrin A, although both siderophores could be …

Investigation Of The Growth And Survival Of Staphylococcus Aureus In Cftr-Deficient Macrophages, Bita Azad

Electronic Thesis and Dissertation Repository

Staphylococcus aureus and its small colony variants (SCVs) are commonly isolated from the lungs of cystic fibrosis (CF) patients. Although studies have suggested that cystic fibrosis transmembrane regulator (CFTR)-deficient macrophages are diminished in their ability to kill intracellular pathogens, whether this is true for CFTR-deficient macrophages infected with S. aureus or its SCVs is unknown. I employed gentamicin protection and eFluorÔ-670-based proliferation assays to assess the intracellular replication of S. aureus in CFTR inhibitor treated THP-1 and primary human macrophages, and in primary macrophages derived from CF patient blood. My work shows that the susceptibility of CFTR-deficient macrophages to the …

Investigating The Regulation Of Fatty Acid Efflux Pump Fare By Tetr Family Regulator Farr In Staphylococcus Aureus, Katherine A. Ferguson

Electronic Thesis and Dissertation Repository

The success of the USA300 strain of methicillin resistant Staphylococcus aureus can be attributed in part to its enhanced ability to overcome innate defenses of the skin including sebum, which provides a source of antimicrobial unsaturated free fatty acids (uFFA). We have previously identified farE and farR genes that confer S. aureus resistance to uFFA, respectively encoding a uFFA efflux pump and a TetR family regulator required for farE expression. However, the exact regulatory mechanism of FarR remains to be elucidated. Here, we show the importance of a conserved TAGWTTA motif in FarR operator sites, such that the loss of …

Contraction Analysis Of Functional Competitive Lotka-Volterra Systems: Understanding Competition Between Modified Bacteria And Plasmodium Within Mosquitoes., Nickolas Goncharenko

Electronic Thesis and Dissertation Repository

We propose and analyze an extension to the classic Competitive Lotka-Volterra (CLV) model. The goal is to model competition between species, with a response from the environment. This response is a function of the population of all species and can represent numerous physical phenomena including resource limitation and immune response of a host due to infection. We name this new system a Functional Competitive Lotka-Volterra (FCLV) model. We mainly use the construction of contraction metrics, to determine global properties of the model. We use this result to analyze the competition between Plasmodium sp. and genetically engineered bacteria within the midgut …

Designing A Novel Hiv-1 Candidate Vaccine, Rahul Pawa

Electronic Thesis and Dissertation Repository

Currently no vaccine has been developed that can prevent the spread of HIV-1. During sexual transmission, a single viral variant called the Transmitted/Founder (T/F) purportedly with unique physical properties, establishes infection in 70-80% of individuals. Unlike previous studies that have tried to identify T/F viruses based on their structure glycan composition and amino acid sequence, we have analyzed the RNA sequences of HIV-1 to help identify T/F variants. Using a combination of both in silico data analysis and in vitro assays, we have identified that T/F viruses have higher numbers of immunostimulatory motifs than HIV virions that fail to infect. …

Utilizing Fiv (Feline Immunodeficiency Virus) To Develop A Novel Animal Model To Study Hiv (Human Immunodeficiency Virus), Ankita Suryakant Kambli

Electronic Thesis and Dissertation Repository

This project sought to perform the in vitro work needed to accomplish the long-term vision of harnessing the similarities between HIV (Human Immunodeficiency Virus) and FIV (Feline Immunodeficiency Virus) to develop an animal model whereby cats can be used to study HIV pathogenesis and therapeutics. We transfected CRFK (Crandell Rees Feline Kidney) fibroblasts with plasmids that could express human or feline CD4, CCR5, or both, and determined receptor surface expression through flow cytometry. We discovered that HIV envelope expressed on 293T can fuse with huCD4/huCCR5 on CRFK. These cat cell lines were also capable of supporting HIV infection. Additionally, we …

Encountering Lectins In The Recipient Mucosa: Implications Of N-Linked Glycosylation On Hiv-1 Transmission, Adam Meadows

Electronic Thesis and Dissertation Repository

Although several studies have determined key differences in envelope motifs between TF and chronic HIV-1, it is still not known what the overall glycosylation profile is that is selected for in a transmission event, as well as what contributes to this selection. Using a bottom-up approach of modifying specific viruses, determining their transmission fitness in an ex vivo tissue explant assay, and determining their glycan content, we have laid the basis for determining the overall glycan structure which is selected for in TF HIV-1. Preliminarily, we have shown that C-type lectins represent a stringent barrier to transmission and have several …

Determining The Relative Transmission Fitness Of Hiv-1 Subtypes A, B, C, And D, Spencer Yeung

Electronic Thesis and Dissertation Repository

There is in vivo evidence that suggests the genetic diversity of HIV-1 subtypes influence heterosexual transmission efficiency. To recapitulate sexual transmission in vitro, blocks of genital tissue were exposed to mixtures of genetically different subtype viruses. Migrating immune cells were collected and co-cultured with a CD4+ T-cell line permissive to HIV infection (PM1) to measure dendritic cell virus transfer; HIV-exposed tissues were cultured separately. Next generation sequencing (NGS) of HIV-1 DNA was used to quantify relative infection rates of the various challenge viruses, and to assess fitness differences in infection of the tissue vs. migratory/T cell co-cultures. Our results …