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 …

Assessing Selective Plasmids For Bradyrhizobium Sp. Doa9 And Mesorhizobium Loti, Aiden J. Wilcox, Rebecca Meaney, Bogumil Karas

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No abstract provided.

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

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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 …

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 …

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 …

Hcpe, A Potential Immuno-Modulatory Protein From Helicobacter Pylori That Is Dependent On The Disulfide Bond Protein Dsbhp, Jeff Lester

Electronic Thesis and Dissertation Repository

H. pylori is a human gastric pathogen that colonizes ~ 50% of the world’s population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. H. pylori produces Helicobacter cysteine rich protein HcpE, a secreted protein which may play a role in virulence. In this study we show that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles, and may play a role in the modulation of H. pylori inflammatory responses. We identified that Dsb_HP is necessary for HcpE production and secretion in H. pylori, and …

Iron-Regulated Cyanobacterial Predominance And Siderophore Production In Oligotrophic Freshwater Lakes, Ryan J. Sorichetti

Electronic Thesis and Dissertation Repository

The frequency and intensity of cyanobacterial blooms (cyanoblooms) is increasing globally. Contrary to existing phosphorus (P) and nitrogen (N) paradigms describing cyanobloom proliferation in eutrophic (nutrient-rich) freshwater lakes, many of the recent cyanobloom reports pertain to oligotrophic (nutrient-poor) freshwater lakes with no prior history of cyanobloom occurrence. There exists a critical research need to re-visit existing conceptual models, identify regulating factors currently unaccounted for and improve our ability to effectively detect and measure cyanobacterial toxins (cyanotoxins) in lakes. Iron (Fe) is required in nearly all pathways of cyanobacterial macronutrient use, though its direct role in regulating cyanobacterial biomass is not …

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

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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 …

Protein Body Formation In Stable Transgenic Plants Of Nicotiana Tabacum Expressing Elastin-Like Polypeptide And Hydrophobin Fusion Proteins, Sonia P. Gutierrez

Electronic Thesis and Dissertation Repository

Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. However, the use of plants still faces two main limitations: the low accumulation levels of some recombinant proteins and the lack of efficient protein purification methods. Two fusion partners, elastin-like polypeptides (ELP) and hydrophobin I (HFBI) were found to increase the accumulation of recombinant proteins and induce the formation of protein bodies (PBs) when targeted to the ER in transient expression assays. In this study I examined the effect of these tags in stable transgenic plants of two Nicotiana tabacum cultivars when fused to green fluorescent …

Structure-Function Analysis Of Enzymes Of The Polyisoprenyl-Phosphate Hexose-1-Phosphate Transferase Family, Kinnari B. Patel

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Enzymes of the polyisoprenyl-phosphate hexose-1-phosphate transferase (PHPT) family are integral membrane proteins that initiate the synthesis of glycans by catalyzing the transfer of a hexose-1-phosphate sugar from UDP-hexose to the lipid carrier undecaprenyl phosphate (Und-P). These glycans such as O antigen and exopolysaccharide (EPS) provide bacteria with protection and adaptation to the environment and host immune factors. The role of PHPT proteins in initiation and the absence of any eukaryotic homologues make them an attractive target for novel antimicrobials; however study of these proteins is difficult due to the presence of multiple transmembrane helices. A requirement of the C-terminal domain …

Transactivation By Human Adenovirus Early Region 1a-Conserved Region Three, Jailal Ng Ablack

Electronic Thesis and Dissertation Repository

One of the critical functions of human adenovirus (hAd) early region 1A (E1A) protein is to activate transcription of the early viral genes. The largest isoform of E1A contains a unique region termed conserved region 3 (CR3), which includes a Cysteine-4 (C4) zinc finger domain. This region activates viral gene expression by interacting with and recruiting cellular transcription machinery to the regulatory regions of early viral genes. Although this process has been studied at length with hAd type 5 E1A, far less is known about how the E1A proteins from other hAd types activate transcription. There are dramatic differences in …