Browsing by Author "Hynes, Michael Francis"

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    An Exploration of Cell Death and Lysis in an Alkaliphilic Cyanobacterial Consortium using Multi-'Omics Approaches
    (2023-12-01) Khot, Varada Milind; Strous, Marc; Hynes, Michael Francis; de Koning, Jason; Ryan, Cathryn; Petersen, Jillian
    Cyanobacteria are important primary producers in many ecosystems, converting light into chemical energy. Their death plays a vital role in releasing organic carbon and nitrogen into the environment, supporting the life of other, heterotrophic organisms, and facilitating carbon sequestration in sediments. With warming aquatic systems, algal blooms are becoming more frequent, persistent, and larger in size, highlighting the need to understand the mechanisms of their death. Cyanobacterial death, whether via viral predation or other mechanisms such as programmed cell death, remains an underexplored topic in non-marine aquatic ecosystems. In this thesis, cell lysis and death of an alkaliphilic Cyanobacterium, Candidatus Sodalinema alkaliphilum, is explored primarily from the perspective of viral infections. Ca. Sodalinema alkaliphilum was enriched, along with its associated heterotrophic microbial community, from alkaline soda lakes on Cariboo Plateau in British Columbia, Canada. This robust cyanobacterial consortium has also been the focus of biotechnological research aimed at producing sustainable bioproducts. Robustness of this consortium can be defined as its capacity to maintain phototrophic function in spite of (a)biotic disturbances. First, bioinformatic techniques to investigate the viral ecology and dynamics of microbial communities in silico were reviewed in Chapter 2. These included identifying viral sequences, classifying their taxonomy, performing phylogenomic analyses, and predicting their hosts. Methods from Chapter 2 were subsequently applied to characterize viral populations in the alkaliphilic cyanobacterial consortium and to investigate viral dynamics by comparing bacterial defence mechanisms across multiple years. This study was the first metagenomic survey of viruses in an alkaliphilic phototrophic consortium and provided valuable insight into the viral ecology of a phototrophic enrichment culture. Finally, based on findings from Chapter 3, the same cyanobacterial consortium was challenged with threats from its natural environment, the alkaline soda lakes. In Chapter 4, the trigger and mechanism of a sudden and rapid cell lysis of the Ca. Sodalinema alkaliphilum was explored using metagenomic, metatranscriptomics analysis, and transmission electron microscopy. This work enhanced our understanding of viral dynamics within an alkaliphilic phototrophic microbial community and highlighted the complexity of cell death in the natural environment.
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    Analysis of Viral Variants and Biomarkers in Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) Coinfected Individuals
    (2024-07-02) Presbitero, Alexandra; Coffin, Carla; Hynes, Michael Francis; van Marle, Guido; Jenne, Craig
    Hepatitis B virus (HBV) and hepatitis Delta virus (HDV) coinfection can cause the most severe form of viral hepatitis in humans. The HDV needs HBV to disseminate, co-opting HBV surface antigen (HBsAg) during assembly. Both viruses have error-prone replication leading to increased variability. We aim to compare clinical and viral outcomes in a contemporary cohort of HBV/HDV coinfected (HBsAg positive and HDV antibody (anti-HDV) positive) vs. HBV monoinfected (HBsAg positive, anti-HDV negative) individuals. Viral variants, HBV biomarkers, HBV DNA, and quantitative HBV surface antigen (qHBsAg) were assessed. Nucleic acid related antigen (NRAg) and HBV core antibody (qAHBc) were quantified by ELISA. HDV RNA/HBV DNA was tested by qPCR. HBV S, X, and HDV delta gene were analyzed by Sanger and Next Generation Sequencing (NGS) and MEGAX. In this prospective study, 25 anti-HDV+/HBsAg+ patients (median age 41y (SD10), 10 females, mostly Asian (n=10) and HBeAg negative (n=19), with median 5 years follow-up (range 1-10). The known HBV genotypes (GT) (n=16) were 3A, 10D, 3E, and known HDV GT (n=19) were 1 (n=16), 2 (n=1), 5 (n=2). Data was compared to a contemporary cohort of 20 HBV monoinfected patients (median age 45y (SD10), 7F, mostly Asian (n=15), with HBV GT 2A, 3B, 12C, 2D, 1E, all HBeAg (-). Single nucleotide polymorphism (SNP) analysis of the HBV S to date in 11/20 by Sanger sequencing showed no mutations, but 3/20 showed X gene variants. Similarly, HBV X NGS analysis in 2 patients showed 16 mutations and 18 S variants in 9 patients. NGS analysis of S gene showed 6 unique mutations not found in the HBV/HDV coinfected cohort. In 25 HBsAg+/anti-HDV+ patients, 19 are HBeAg(-), with low-level HBV viremia, high qHBsAg, and 23/25 HDV RNA+. NGS and SNP analysis showed unique HBV S/X variants. The X and/or S mutations detected correlate with severe liver disease, impaired replication, HCC development, decreased antigenicity and vaccine escape. We found unique SNPs/variants and increased diversity over time in the HDV gene. In summary, this study provides novel data on HBV and/or HDV viral sequence changes in a cohort with high risk of liver disease progression over long-term follow-up.
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    The Virus Resistance Mechanism of Abortive Infection K in Lactococcus lactis
    (2023-07) Du, Amy; Hynes, Michael Francis; Fraser, Marie Elizabeth; Harrison, Joe Jonathan; Niu, Dongyan; Fortier, Louis-Charles
    In nature, there is a constant battle between viruses and their hosts known as the evolutionary arms race, where they both continuously evolve to attempt to gain an advantage over the other. This evolutionary arms race has resulted in many mechanistic inventions, such as abortive infection (Abi) mechanisms in bacteria. In Abi mechanisms, viruses inject their genetic material into cells, but bacteria block phage replication by preventing one of the steps of phage maturation, typically resulting in cell death. This study looks at the Abi mechanism called AbiK, which is controlled by a protein of the same name that was discovered on a plasmid in Lactococcus lactis. A large portion of the study focuses on what happens in vivo during the AbiK mechanism. The normal wild-type infection, the abortive infection mediated by the AbiK protein, and the escape of the AbiK mechanism by a mutant phage are all studied for comparative analyses. Observations have shown that phage DNA replication is inhibited, and that transcription of the phage genes is delayed during the AbiK mechanism. The mutant phage can bypass these inhibitions, and successfully replicate its DNA and complete the phage lytic cycle. RNA-Sequencing experiments were conducted to narrow down the mechanism behind AbiK, and preliminary results indicate that AbiK depletes the nucleotide resources available in the cell, eventually resulting in cell death and inhibition of the phage replication cycle. A second part of this study focuses on the biochemistry of the AbiK protein. The AbiK protein has a polymerase activity that polymerizes an untemplated long single-stranded DNA that is covalently attached to the AbiK protein. This study identified which amino acid is used as a primer, which is tyrosine 44 on the AbiK protein. The release of protein structure prediction databases allowed for the analysis of a generated model of the AbiK protein, allowing for the elucidation of other biochemical functions the AbiK protein has. In addition to the polymerization activity, AbiK is hypothesized to bind to nucleotides or other proteins. The culmination of these studies allowed for insight on the AbiK mechanism, generating a hypothesis for future studies.