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Commercial catfish production is an inveterate industry within the southeastern United States. Bacterial disease is a significant detriment to global aquaculture, including the United States catfish industry. Among them, an atypical strain of the bacterium Aeromonas hydrophila has plagued the industry since the late 2000s. Atypical A. hydrophila (aAh) outbreaks are largely acute, resulting in catastrophic losses. The disease ecology, prevalence, and genetic distribution are poorly understood. Atypical Aeromonas hydrophila displays a rapid onset with few warning signs of the impending disease, making it difficult for early detection. At present there are two recognized haplotypes of aAh. This project aimed to investigate changes in the spatial and temporal distributions of these haplotypes. The analysis of clinical isolates from different geographic regions across multiple years revealed complete supplanting by the younger haplotype in the Mississippi Delta within 5 years of first isolation. Comparative genomics demonstrated distinct divergences in specific virulence components between the two strains, specifically the Type VI Secretion System, which may explain putative differences in outbreak dynamics and recent displacement of one strain by the other. Also, a rapid, non-lethal screening method was validated that can detect aAh within the catfish host. This method affords data collection regarding infection severity prior to onset of disease and, can predict aAh prevalence at the fish- and pond-levels. The occupancy model indicates more than half the population within a pond may be infected with aAh despite no overt signs of disease. Additionally, aAh is commonly present in approximately 10% of the population, providing the first evidence of a carrier state in this disease. Lastly, a compartmental SLIR model was used to investigate disease dynamics of aAh in catfish aquaculture ponds. Simulations suggest the introduction hypothesis does affect estimated pond profit and antibiotic intervention is an economical treatment for aAh. Routine monitoring was less economical and could dramatically reduce profit in some scenarios. Overall, this work expands our current knowledge of aAh in catfish aquaculture and lays the foundation for future studies investigating aAh management and mitigation of bacterial disease in catfish aquaculture.

The lower airways of patients with chronic airway diseases including cystic fibrosis (CF) are colonized by diverse communities of microorganisms. Over-time the airways of some 60% of CF patients become permanently colonized and dominated by Pseudomonas aeruginosa. Chronic infection of P. aeruginosa has been associated with a decline in pulmonary function, worse prognosis, and eventual patient mortality. Although P. aeruginosa evolves within the CF airways resulting in complex populations, the mechanism by which these complex populations contribute to disease progression is not well understood. Here we show diversity among isolates by observed changes in genome sequences of a strain of P. aeruginosa, known as Prairie Epidemic Strain (PES). Using whole genome sequencing and comparative genomics we identified a large core genome across 195 PES isolates from 57 CF patients of the Calgary Adult Cystic Fibrosis Clinic (CACFC) where 88% of the pangenome was categorized as core genes. Single nucleotide polymorphism (SNPs) mutations were shown to be the largest contributor of diversity at the nucleotide level compared to other polymorphism types consisting of 87% of the total polymorphisms present across the 195 PES isolates. CRISPR arrays and mobile elements such as prophage and plasmids demonstrate this strain of P. aeruginosa was stable over 30 years. In a second aim, I show variation in the populations of P. aeruginosa across an exacerbation event further highlighting the complexity of the lung bacterial community. Distinct populations of P. aeruginosa at the onset and resolution of an exacerbation within a single CF patient were identified by SNPs. These results a model where adaptive radiation as well as natural mutations contribute to the heterogeneity and diversification within populations of P. aeruginosa in CF patients. Understanding the evolution and population structure of PES through the identification of important genes and mutations through the clinical course of an exacerbation can aid in identifying new targets for patient treatment of P. aeruginosa in CF. / Thesis / Master of Science (MSc) / Cystic fibrosis is a life-threatening disease characterized by cycles of stability and respiratory illness. Bacterial species within the lungs of these patients are the main contributor to disease progression. I investigated a specific transmissible epidemic strain, Pseudomonas aeruginosa Prairie Epidemic Strain, using a unique collection of samples provided by collaborators at the adult cystic fibrosis clinic in Calgary. Using these samples, I first explored the differences between patients over a period of 34 years. I hypothesized that similar changes in genome sequences will be observed in multiple patients with a possible commonality in disease progression. Second, I explored the role this bacterial pathogen may play in cycles of respiratory illness. I hypothesize that a specific bacterial subpopulation could initiate these cycles and be identified by changes at the genome level. This research provides further knowledge of an epidemic strain of cystic fibrosis.

Chapter 1: The pluripotent state is maintained by a network of “core” transcription factors (TF). REX1 (Reduced Expression-1) is a pluripotency related TF derived from retrotransposon-mediated duplication of the zinc finger TF Yin Yang 1 (YY1). Furthermore, expression of REX1 and YY1 induces changes in genes regulated by endogenous retroviral elements (ERV), suggesting an evolutionary origin of REX1 for ERV regulation. Studies suggest that murine REX1 may act in epigenetic regulation of gene expression and ERVs, but the precise mechanism remains unelucidated, so we generated FLAG-tagged REX1 pluripotent stem cell (PSC) lines, as well as a series of truncation mutants to explore the REX1 function. Our studies indicate the presence of previously undescribed isoforms of the full-length REX1 protein, suggesting that regulation by REX1 may be more complex than initially appreciated. We hypothesize that REX1 regulates the expression of a sub-set of ERVs and REX1 isoforms regulate REX1 target genes in pluripotent stem cells. Previously, we performed REX1 ChIP-seq and found enrichment for REX1 binding at specific ERVs. Here, we show that differential expression of REX1 isoforms do not change the expression of ERVs. Furthermore, our REX1 KO lines show changes in expression of ERV family members and together with the ChIP data, suggest that REX1 may act as a negative regulator of some retroviral elements. However, further experiments reveal a potential compensation of REX1 KO, possibly by the homologous factors YY1 and YY2. Due to the limited nature and time constrain of our study, we did not find conclusive evidence to further elucidate the potential compensation mechanism and the characteristics of the REX1 isoforms. Our work provided a new avenue for exploring the functional importance of REX1 isoforms and the potential, YY1 and YY2 independent, regulatory role REX1. Chapter 2: Mitotic bookmarking describes a potential mechanism involved in the stable propagation of cellular identity through the cell cycles. Candidate based studies have identified mitotic bookmarking factors (MBFs) that are retained on the mitotic chromatin and preserve the transcriptional memory of the cell. Nevertheless, there is a poor understanding of which proteins can serve as MBFs, as well as the chromatin dynamics of bookmarked sites during mitosis and the start of G1 phase. Previously, we designed a chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) assay to develop a global unbiased approach for identifying and characterizing novel MBFs. Using ChIP-MS, we identifed putative MBFs associated with the mitotic chromatin in pluripotent stem cells (PSCs) and used ATAC-seq to identify subsets of pluripotency-associated accessible gene regions that appear to be bookmarked by a variety of transcription factors, including PARP1, PSIP1, and HDGF. Here, we characterize the interaction of a putative MBF, not found in our ChIP-MS screen, NFYa, with PARP1 and, inconclusively, another putative MBF, DNMT1. Furthermore, we found that PWWP containing putative MBF, HDGF, has a potential role in pluripotency maintenance but it is not mitosis-specific. Due to the limited nature and time constrain of our study, we did not find conclusive evidence to establish the role of PSIP1 in PSC mitotic bookmarking. Our work provided a new avenue for exploring the functional importance of mitotic bookmarks in pluripotent maintenance. / Thesis / Master of Science (MSc)

Improving the efficiency by which crops use nutrients is critical for maintaining high crop productivity while reducing fertility management costs and eutrophication related to fertilizer runoff. The native crucifer and halophyte, Yukon Eutrema salsugineum, was used in this study. Yukon E. salsugineum is closely related to important Brassica crops and thrives in its native habitat on soil that is low in available phosphate (Pi) and high in sulfur (S). To determine how Yukon E. salsugineum copes with low Pi, leaf transcriptomes were prepared from four week-old plants grown in controlled environment chambers using soil lacking or supplemented with Pi and/or S. This thesis focused on using bioinformatic approaches to assemble, analyze and compare the transcriptome profiles produced by the Yukon E. salsugineum plants undergoing four nutrient combinations of high and/or low Pi and S. The objective of the study was to identify traits associated with altered S and/or Pi with the prediction based on other species that low Pi, in particular, would pose the greatest stress and hence elicit the greatest transcriptional reprogramming. Transcriptome libraries were generated from four treatment groups with three biological replicates each. Reads in each library were mapped to 23,578 genes in the E. salsugineum transcriptome with an average unique read mapping ratio of 99.52%. Surprisingly, pairwise comparisons of the transcriptomes showed little evidence of Pi-responsive reprogramming whereas treatments differing in soil S content showed a clear S-responsive transcriptome profile. Principal Component Analysis revealed that the low variance quaternary Principal Component distinguished the transcriptomes of plants undergoing low versus high Pi treatments with differential gene expression analysis only finding 11 Pi-responsive genes. This outcome suggests that leaf transcriptomes of Yukon E. salsugineum plants under low Pi are largely undifferentiated from plants provided with Pi and is consistent with Yukon E. salsugineum maintaining Pi homeostasis through fine-tuning the expression of protein-coding and non-coding RNA rather than large-scale transcriptomic reprogramming. Previous research has shown Yukon E. salsugineum to be very efficient in its use of Pi and this work suggests that the altered expression of relatively few genes may be needed to develop Pi-efficient crops to sustain the crop demand of a growing population. / Thesis / Master of Science (MSc)

Yes / Personalized nutrition has the potential to improve health, prevent disease and reduce healthcare expenditure. Whilst research hints at positive consumer attitudes towards personalized nutrition that draws upon lifestyle, phenotypic and genotypic data, little is known about the degree to which registered dietitians (RD) are engaged in the delivery of such services. This review sought to determine possible factors associated with the integration of the emerging science of Nutritional Genomics (NGx) into the clinical practice setting by practicing registered dietitians. Scope Search of online databases (Pubmed; National Library of Medicine; Cochrane Library; Ovid Medline) was conducted on material published from January 2000 to December 2014. Studies that sampled practicing dietitians and investigated integration or application of NGx and genetics knowledge into practice were eligible. Articles were assessed according to the American Dietetic Association Quality Criteria Checklist. Key findings Application of nutritional genomics in practice has been limited. Reluctance to integrate NGx into practice is associated with low awareness of NGx, a lack of confidence in the science surrounding NGx and skepticism toward Direct to consumer (DTC) products. Successful application to practice was associated with knowledge about NGx, having confidence in the science, a positive attitude toward NGx, access to DTC products, a supportive working environment, working in the clinical setting rather than the public health domain and being in private rather than public practice. Conclusions There is a need to provide RGs with a supportive working environment that provides ongoing training in NGx and which is integrated with clinical practice.

Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented. / Master of Science

No / Concern has been expressed at successive Review Conferences of the Biological and Toxin Weapons Convention (BTWC) regarding the possible misuse of new biological knowledge; this article reviews some of these developments. Genetic manipulation, and genomics in particular, would modify existing pathogens and render previously harmless organisms pathogenic. Viruses could be modified as vectors to alter their pathogenicity in animals and man or act as carriers for genes or toxins. Plant pathogens, particularly fungi, could be modified as biological warfare agents against crops. An effective verification protocol for the BTWC is an essential part of the web of deterrence against these developments.

No / Bacterial lipoproteins are a diverse and functionally important group of proteins that are amenable to bioinformatic analyses because of their unique signal peptide features. Here we have used a dataset of sequences of experimentally verified lipoproteins of Gram-positive bacteria to refine our previously described lipoprotein recognition pattern (G+LPP). Sequenced bacterial genomes can be screened for putative lipoproteins using the G+LPP pattern. The sequences identified can then be validated using online tools for lipoprotein sequence identification. We have used our protein sequence datasets to evaluate six online tools for efficacy of lipoprotein sequence identification. Our analyses demonstrate that LipoP (http://www.cbs.dtu.dk/services/LipoP/) performs best individually but that a consensus approach, incorporating outputs from predictors of general signal peptide properties, is most informative.