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

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.

Ageing is difficult to study because of the complexity and multi-factorial nature of traits that result from a combination of environmental, genetic, epigenetic and stochastic factors, each contributing to the overall phenotype. In light of this challenge, transcriptomic studies of aging organisms are of particular interest, since transcription is an intermediate step that links genotype and phenotype. In recent years microarrays have been widely used for elucidation of changes that occur with age in the transcriptome in Caenorhabditis elegans. However, different microarray studies of C. elegans report sets of differentially expressed genes of varying consistence, with different functional annotations. Failures to find a consistent set of transcriptomic alterations may reflect the absence of a specific genetic program that would guide age-related changes but may also, to some extent, be a consequence of a small sample sizes and a lack of study power in transcriptomic researches. To tackle this issue we analyzed RNA sequences of samples from a time-series experiment of normal aging of C. elegans, performing the first, to our knowledge, NGS-based study of such kind. As a result, evidences were collected that promote a union of two competing theories: the theory of DNA damage accumulation and the theory of programmed aging. Next, we applied two alternative methods, namely the Short Time-series Expression Mining and the Network Smoothing algorithm, in order to obtain and analyze sets of genes that represent distinct modules of age-related changes in the transcriptome. Besides characterization of age-related changes, we were also interested in assessment and validation of the Network Smoothing algorithm. Generally, results of clustering of smoothed scores are consistent with results of short time-series clustering, allowing robust elucidation of functions that are perturbed during aging. At the last phase of the project we questioned if observed changes in the transcriptome can be controlled by specific transcription factors. Thus we used Chip-seq data to predict plausible transcription factor regulators of gene sets obtained using time series clustering and Network smoothing. On the one hand, all predicted transcription factors had documented relevance to aging. On the other hand, we did not achieve gene set specific prediction of transcription factors. In fact, genes with the opposite dynamics were predicted to respond to the same transcription factors.  To summarize, we characterized in details age-related changes in the transcriptome of C. elegans, validated the performance of the Network Smoothing algorithm and showed that integration of gene expression with Chip-seq data allows to predict transcription factors that are capable to modulate the lifespan of C. elegans.

No description available.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy Durrett / In plants including the members of Brassicaceae family, such as Arabidopsis thaliana and Brassica juncea, seed storage reserves, which include lipids and proteins, accumulate in seeds during development. Triacylglycerols (TAG) are the major storage lipids found in the developing seeds, petals, pollen grains, and fruits of plants. In Arabidopsis seeds, acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) is the major enzyme contributing to TAG biosynthesis. In Arabidopsis, dgat1-1 mutants retain 60-80% seed TAG content due to the involvement of phospholipid: diacylglycerol acyltransferase (PDAT1) in acyl-CoA independent TAG biosynthesis. My study focuses on the elucidation and functional characterization of novel genes involved in the regulation of the TAG biosynthesis pathway. In developing seeds of the dgat1-1 mutant, altered fatty acid composition was observed with reduced TAG content and increased polar lipid content as compared to wild type. RNA-Seq of developing Arabidopsis seeds was employed to detect differentially expressed genes in dgat1-1. An empirical analysis for differential gene expression revealed a significant number of differentially expressed genes among all developmental stages in dgat1-1. Significant changes in gene expression profile were detected in lipid-related genes such as lipases and desaturases. RT-PCR was used to confirm the differential expression of major lipid-related genes including DGAT1, PDAT, and FAD2. Lipid profiling of T-DNA insertion mutants for differentially expressed genes revealed significant changes in lipid content and composition. Mutations in a member of the α, β-hydrolase family, encoded by gene named PLIP1, resulted in smaller seed and an altered seed oil phenotype. Also, combining the dgat1-1 and plip1-2 mutations resulted in a lethal phenotype, demonstrating the important role of this enzyme in embryo development and TAG biosynthesis. To identify key components in the regulation of storage lipid biosynthesis, correlation analysis using differential transcript abundance and lipid profile during different stages of seed development from dgat1-1 and wild type lines of Arabidopsis was performed. Using clustering analysis with Pearson correlation coefficient and single linkage identified one cluster of genes which included PLIP1, FAD2, FAD3, and PDCT . Similar analysis using combined data from the neutral and polar fractions resulted in clustering of lipids containing polyunsaturated fatty acids. To investigate the reduced seed germination phenotype for mature seeds of dgat1-1 and non-germinating green seed phenotype of dgat1-1 plip1-2 lines, differential expression (DE) analysis for genes involved in hormone metabolism was performed. Upregulation of expression was observed for genes involved in promoting abscisic acid (ABA) response, which led us to specuate the role of altered hormone metabolism in delayed germination of dgat1-1 seeds. Development of allopolyploid Brassica species from its diploid progenitors involves duplication, loss, and reshuffling of genes leading to massive genetic redundancy. It leads to selective expression or newly acquired role for duplicated homeologs. Differential expression (DE) analysis for homoeologous genes from A and B subgenomes of allopolyploid B. juncea implicated in FA synthesis, acyl editing, and TAG biosynthesis and metabolism was performed. Differential expression (DE) analysis identified the transcriptional dominance of A subgenome homoeologs. Identification of these homoeologs will enable their use in breeding programs directed towards improvement of lipid content and composition in seeds.

Pt(II) chemotherapies, including cisplatin and oxaliplatin, have been used in cancer treatment since the 1970s, however, a full understanding of the mechanism by which these drugs function is still lacking. While the interaction between Pt(II) drugs and DNA has been extensively studied and subsequently indicted in the cellular response to Pt(II) drugs, recent data indicates non-DNA targets play important roles as well. To gain insight into the non-DNA damage-based effects induced by these drugs, MDA-MB-468 cells were treated at therapeutic concentrations of cisplatin between 30 minutes and 24 hours. Not only does this data provide insight into the complex time-dependent nature of the cellular response to cisplatin, but novel responses were also observed. First, I describe how the expression of numerous snoRNAs decreases as early as 30 minutes post-treatment with either cisplatin or oxaliplatin, and differential expression analysis indicates this occurs before activation of the DNA damage response. Since snoRNAs are necessary components in ribosome processing, we sought to determine the role snoRNAs play in the cellular response to Pt(II) drugs. A subgroup of our identified snoRNAs direct modification of helix 69 on the 28S ribosome. Quantification of methylation of helix 69 and other locations suggests cisplatin induced changes in snoRNA expression leads to dysregulation of rRNA modification, likely altering ribosome activity. I also observe varied activation of different types of DNA damage and cell cycle arrest between 3 and 12 hours of cisplatin treatment while early expression changes show downregulation of mitochondrial genes. We also identify a number of lncRNAs previously associated with TNBC that are downregulated after cisplatin treatment. This study establishes a gene expression profile induced by cisplatin treatment of triple-negative breast cancer that demonstrates the complex interplay of multiple means of stress induction. Lastly, we establish a method for analyzing direct DNA binding targets of platinum(II) chemotherapeutics. This pilot study confirms high accumulation of platinum(II) compounds on guanine-rich DNA and suggests DNA binding of significant genes leads to changes in their RNA expression. / 10000-01-01