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Optimizing the production of useful macromolecules from transgenic microorganisms is crucial to biopharmaceutical companies. Improving bacterial growth and replication depends largely on the efficiency of translation, which is rate-limited by initiation. Among the most important interactions between the mRNA translation initiation region (TIR) and the translation machinery is the association between the Shine-Dalgarno (SD) sequence in the TIR and the complementary anti-SD (aSD) sequence which is located within a short unstructured segment that includes the 3’ terminus (3’ TAIL) of the mature 16S rRNA. However, the mature 3’ TAIL has been poorly characterized in the majority of bacteria, rendering optimal SD/aSD pairing unclear in these species. In light of this, we established a novel strategy to characterize the mature 3’ TAILs of bacterial species that leverages the availability of publically stored RNA sequencing (RNA-Seq) data. In chapter 2, we devised a RNA-Seq-based approach to successfully recover the experimentally verified 3’ TAIL in E. coli (5’-CCUCCUUA-3’) and resolve inconsistencies surrounding the identity of the 3’ TAIL in Bacillus subtilis. In chapter 3 we improve the method introduced in chapter 2 to clearly and more reliably define the 3’ TAIL termini for 13 bacterial species with available protein abundance data. Our results reveal considerable heterogeneity in the termini of 3’ TAILs among closely related species and that sites downstream of the canonical CCUCC aSD motif are more important to initiation than previously believed. My research contributes to advance our understanding in microbial translation efficiency in two significant ways: 1) providing an RNA-Seq-based approach to characterize rRNA transcripts, and 2) elucidating optimal recognition between protein-coding genes and the rRNA translation machinery.

In order to understand gene regulatory networks (GRNs) in mammalian cells, it is pivotal to assess the interaction between proteins and DNA. In particular, the specific DNA binding activity of transcription factors (TFs) determines the expression of target genes and in general the overall connectivity of the GRN. However, the genomic location of TF binding cannot be predicted just from the DNA sequence, and functional assays are required to detect this interaction. The investigation of the binding of TF to DNA is usually accomplished by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). While in the last 10 years this method enabled a better understanding of how transcription is regulated in living cells, it does have some drawbacks. In particular, the need for very highly specific antibodies and the large amount of starting material limit the ability of ChIP-seq to address biological questions when dealing with samples of small quantity. A technique called DNA Adenine Methyltransferase Identification (DamID) was developed in Drosophila as an alternative method for the detection of protein- DNA interactions and it is based on the fusion of a protein of interest (POI) with the DNA adenine methyltransferase (Dam). This fusion causes DNA methylation of adenines surrounding the sites where POI binds and the subsequent identification of the methylation sites allows mapping of the binding event without antibodies and using less cells as starting material. While this technology was successful in detecting the interaction between nuclear lamina and DNA in mammalian cells, to date little reports are present in the literature about TF DamID. This is mainly due to the different nature of TF binding compared to Lamin (punctuated instead of broad) and to the elevated intrinsic activity of Dam that makes the detection of real signal above the noise challenging. I here demonstrate a step-by-step optimization of the DamID technology coupled to next-generation sequencing (DamID-seq) that I used to map the binding of the mouse embryonic stem cell master regulator Oct4 in as few as 1,000 cells. This new technology paves the way for exciting new experiments where the number of cells is scarce such as in vitro cell state change or in vivo processes.

Schistosoma mansoni is a member of a genus of platyhelminths whose members cause the disease schistosomiasis. Particularly prevalent in sub-Saharan Africa, it is thought to be directly responsible for approximately 5500 deaths per year, as well as contributing significantly to morbidity, being responsible for 3.3 million lost disability-adjusted life years. Schistosomes are dioecious and male and female worms find one another and pair in the blood vessels of the host's liver. This sets in motion a unique feature of schistosome biology, the pairing-dependent sexual maturation of the female worms. Over the course of the next three weeks, the females fully develop their reproductive organs, especially ovaries and vitellarian tissue, to allow for the production of large quantities of eggs, which not only play a crucial role in the transmission of the parasites but are also responsible for much of the pathology associated with schistosomiasis. This thesis aims to explore the changes in gene expression which take place following pairing and result in the sexual maturation of females. To do so, RNA-Seq data was produced from male and female worms from mixed sex as well as single sex infections at 18, 21, 28, 35, 38 and 49 days post infection and analysed to understand when and how gene expression changes in paired worms. Then gene expression was examined in worms that had been removed from their partner to examine the process of regression, where female worms lose much of their reproductive tissue. The last experiments describe examine gene expression in the testes and ovaries of schistosomes, to reveal differences between the gonads of worms from mixed and single sex infections and understand in more detail how these worms may regulate the growth of their reproductive organs, contributing to our knowledge of schistosome biology.

The malaria pre-erythrocytic stages have been identified as an ideal therapeutic target, but complex in vitro models for Plasmodium vivax and Plasmodium falciparum lack the efficiency needed for rapid screening and evaluation of new vaccines and drugs, especially targeting the P. vivax hypnozoite. To address this challenge, we employed a multi-parameter approach using “omics’” to identify pre-erythrocytic targets and biomarkers, guide phenotypic therapeutic screening, and study parasite functionality with innovative bioassays using highcontent screening. Herein, we discuss three novel bioassays formatted in 384-well plate systems with utilization of commercially-available materials and application of high-content imaging for rapid bio-image analysis. To refine functional assessment of pre-erythrocytic targets in early infection phases, we developed a real-time, ‘live’ sporozoite motility assay and a live sporozoite hepatocyte cell traversal assay to examine chemotherapeutic and immunoprophylactic interventions in biologically relevant environments. Furthermore, our 384-well primary hepatocyte culture system and methodology maintains stable hepatocyte physiology of cryopreserved primary human hepatocytes in addition to primary non-human primate hepatocytes for greater than 30 days, thus ideal for robust liver parasite development following infection with P. vivax, P. falciparum or P. cynomolgi sporozoites. We report antimalarial drug and vaccine studies performed in all bioassays with identification of novel anti-LS inhibition mechanisms. Additionally, this research discusses the discovery of potential sporozoite and liver stage targets identified through transcriptomic profiling of freshly isolated P. vivax and P. cynomolgi sporozoites using a candid approach of recapitulating the pivotal transition period from mosquito to human through microenvironment reconstruction and exposure to biological stimuli. We further characterize sporozoite invasive phenotypes through the application of the bioassays. Together, these novel functional assays enable us to rapidly evaluate potential preerythrocytic therapeutic candidates and analyze complex Plasmodium sporozoite phenotypes.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.

Gamma-aminobutyric acid type B (GABAB) receptor is a G protein coupled receptor (GPCR) that mediates decreased neural activity. It has two subunits, GABAB1 and GABAB2. Previous complementary DNA (cDNA) microarray data showed strong GABAB1 signals from human prefrontal cortex using an intron 4 region probe, and these studies indicated that novel intron 4 containing GABAB1 splicing variants exist. We cloned GABAB1k, l, m, and n including mouse GABAB1j. Expression of these variants are much lower than other major known splicing variants, but GABAB1k, l, m, and n levels are similar across brain tissues. GABAB1l and GABAB1m impair GABAB receptor induced function. To better define GABAB1 splicing in alcoholic brains, whole transcriptome shortgun sequencing (RNA-seq) experiments were proposed. Due to the complexity of GABAB1 splicing, we used gene specific libraries as well as whole transcriptome libraries to maximize GABAB1 specific splicing junction search. The splicing junction search data found that GABAB1 gene is 2 to 3 times longer than the previous known gene length. Extremely low expression at 5’ end exons was found, and GABAB1 exons were grouped based on expression levels. Chronic alcohol altered exon/intron expression and splicing junctions more than overall gene expression. Decreased exon expression at a GABA binding site, a transmembrane domain (TM), and a microRNA (miRNA) binding site may diminish the normal GABAB1 transcript population and compromise signal transduction following chronic alcohol exposure. This may explain why GABAB receptor agonists have therapeutic benefit in treating alcoholism. During the sequence mapping, read pile-ups and gaps were found from whole transcriptome libraries in known exons. These may prevent single nucleotide polymorphism (SNP) and splicing junction identification and gene expression calculations. Sequence analysis found sequence biases from their mapped reads. The major sequence biases were from RNaseIII RNA fragmentation and T4 polynucleotide kinase (T4PNK) reaction. Heat fragmentation and OptiKinase treatment removed the read pile-ups and gaps including the sequence biases. The identification of RNaseIII target sequences can be incorporated into methods of miRNA gene prediction. These data showed the complexity of GABAB1 receptor splicing and the perturbation of splicing by chronic alcohol abuse demonstrate the power of RNA-seq to provide new insight into gene expression and the role of GABAB receptors in alcoholism. In addition, many other important brain genes may have unexplored splicing variants which will be important for alcoholism and other psychiatric diseases. Also, new RNA-seq library constructions improved the quality of gene expression studies. / text

<p>Abstract</p><p>Transcription factors control numerous vital processes in the cell through their ability to control gene expression. Dysfunctional regulation by transcription factors lead to disorders and disease. Transcription factors regulate gene expression by binding to DNA sequences (motifs) on the genome and altering chromatin. DNase-seq footprinting is a well-established assay for identification of DNA sequences that bind to transcription factors. We developed computational techniques to analyze footprints and predict transcription factor binding. These transcription factor specific predictive models are able to correct for DNase sequence bias and characterize variation in DNA binding sequence. We found that DNase-seq footprints are able to identify cell-type or condition specific transcription factor activity and may offer information about the type of the interaction between DNA and transcription factor. Our DNase-seq footprint model is able to accurately discover high confidence transcription factor binding sites and discover alternative interactions between transcription factors and DNA. DNase-seq footprints can be used with ChIP-seq data to discover true binding sites and better understand transcription regulation.</p> / Dissertation

Campylobacter jejuni is an avian commensal bacterium and causes gastrointestinal diarrhea in humans called campylobacteriosis. Campylobacteriosis is acquired by consumption of undercooked poultry contamined with C. jejuni. Poultry can become colonized from contaminated drinking water. The chicken flock and drinking water of 4 poultry farms in Ontario were sampled and the prevalence of C. jejuni in these flocks was determined to be 16.7% over a 1 year sampling period. We determined that contamined- water was a significant risk factor for Campylobacter-positive flocks from flaA typing, PFGE analysis, and genomotyping several isolated strains. Free living amoebae, such as Acanthamoeba species, live in the drinking water of poultry farms. It is hypothesized that Acanthamoeba in the drinking water of poultry farms can take up and act as environmental reservoirs of C. jejuni. Acanthamoeba species were isolated from the drinking water. Acanthamoeba strains were found to act as a vehicle for protection, persistence and growth of C. jejuni isolated from the farm water. The transcriptome of both C. jejuni and A. castellanii during the initial stages of C. jejuni internalization were described by RNA-seq. C. jejuni oxidative defence genes (such as katA, sodB, fdxA) and some other unknown genes (Cj0170, Cj1325, Cj1725) were found to be essential in the interaction with A. castellanii. Our findings suggest that Acanthamoebae act as a C. jejuni reservoir and could be a contributing source of C. jejuni in the environment. Through transcriptomics studies, we have begun to uncover some genetic clues involved in this interaction.