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.

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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

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding.

Philosophiae Doctor - PhD / Apple (Malus x domestica) production in the Western Cape, South Africa, is one of the major contributors to the gross domestic product (GDP) of the region. The production of apples is affected by a number of diseases. One of the economically important diseases is apple scab that is caused by the pathogenic fungus, Venturia inaequalis. Research to introduce disease resistance ranges from traditional plant breeding through to genetic manipulation. Parallel disease management regimes are also implemented to combat the disease, however, such strategies are increasingly becoming more ineffective since some fungal strains have become resistant to fungicides. The recently sequenced apple genome has opened the door to study the plant pathogen interaction at a molecular level. This study reports on proteomic and transcriptomic analyses of apple seedlings infected with Venturia inaequalis. In the proteomic analysis, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify apple leaf proteins extracted from infected and uninfected apple seedlings. Using MelanieTM 2-DE Gel Analysis Software version 7.0 (Genebio, Geneva, Switzerland), a comparative analysis of leaf proteome expression patterns between the uninfected and infected apple leaves were conducted. The results indicated proteins with similar expression profiles as well as qualitative and quantitative differences between the two leaf proteomes. Thirty proteins from the apple leaf proteome were identified as differentially expressed. These were selected for analysis using a combination of MALDI-TOF and MALDI-TOF-TOF MS, followed by database searching. Of these spots, 28 were positively identified with known functions in photosynthesis and carbon metabolism (61%), protein destination and storage (11%), as well as those involved in redox/response to stress, followed by proteins involved in protein synthesis and disease/defence (7%), nucleotide and transport (3%). RNA-Seq was used to identify differentially expressed genes in response to the fungal infection over five time points namely Day 0, 2, 4, 8 and 12. cDNA libraries were constructed, sequenced using Illumina HiScan SQTM and MiSeqTM instruments. Nucleotide reads were analysed by aligning it to the apple genome using TopHat spliceaware aligner software, followed by analysis with limma/voom and edgeR, R statistical packages for finding differentially expressed genes. These results showed that 398 genes were differentially expressed in response to fungal infection over the five time points. These mapped to 1164 transcripts in the apple transcripts database, which were submitted to BLAST2GO. Eighty-six percent of the genes obtained a BLAST hit to which 77% of the BLAST hits were assigned GO terms. These were classed into three ontology categories i.e. biological processes, molecular function and cellular components. By focussing on the host responsive genes, modulation of genes involved in signal perception, transcription, stress/detoxification, defence related proteins, transport and secondary metabolites have been observed. A comparative analysis was performed between the Day 4 proteomic and Day 4 transcriptomic data. In the infected and uninfected apple leaf proteome of Day 4, we found 9 proteins responsive to fungal infection were up-regulated. From the transcriptome data of Day 4, 162 genes were extracted, which mapped to 395 transcripts in the apple transcripts. These were submitted to BLAST2GO for functional annotation. Proteins encoded by the up-regulated transcripts were functionally categorised. Pathways affected by the up-regulated genes are carbon metabolism, protein synthesis, defence, redox/response to stress. Up-regulated genes were involved in signal perception, transcription factors, stress/detoxification, defence related proteins, disease resistance proteins, transport and secondary metabolites. We found that the same pathways including energy, disease/defence and redox/response to stress were affected for the comparative analysis. The results of this study can be used as a starting point for targeting host responsive genes in genetic manipulation of apple cultivars.

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.

Neuroblastoma (NBL), the most common non-Central Nervous System (CNS) solid tumor of childhood, characteristically displays heterogeneous clinical presentation and biological behavior. Previous work has studied the genetic basis of the disease and revealed a low somatic mutation burden. In order to identify novel therapeutic targets and better understand the biology of high-risk NBLs, I investigated whole transcriptome profiles of two cohorts of metastatic NBLs using RNA sequencing. First, I studied changes in splicing pattern in a cohort of 29 patients. V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplified NBLs showed a distinct splicing pattern affecting multiple cancer hallmarks. Six splicing factors have altered expression patterns in MYCN-amplified tumors and cell lines, and binding motifs for these factors were significantly enriched in differentially-spliced genes. ChIP-seq analysis showed direct binding of MYCN to promoter regions of splicing factors PTBP1 and HNRNPA1, demonstrating that MYCN regulates splicing by directly regulating expression of key splicing factors. Furthermore, high expression of PTBP1 and HNRNPA1 was significantly associated with poor overall survival of stage 4 NBL patients (p≤0.05). Knocking down PTBP1, HNRNPA1 and their downstream target PKM2, a pro-tumor-growth isoform, resulted in repression of NBL cell growth. Second, I used whole transcriptome sequencing in a cohort of 150 patients to assess expressed mutations, fusion genes, and gene expression including long non-coding genes to provide clinically-relevant classification and to offer insights into NBL tumor biology. Twenty-four genes including ALK, ATRX and MYCN were recurrently mutated in NBL transcriptomes. In-frame FOXR1 fusions were detected in 4 samples, including 3 cases or 14% of stage 4S NBLs. Unsupervised gene expression analysis revealed four molecular subgroups. MYCN and tumor microenvironment were the primary discriminating signatures in these molecular subgroups. Fifty-eight percent of MYCN-not-amplified samples showed high MYCN signatures, which were potentially contributed by various genomic events such as MYCN activating mutations and FOXR1 fusions. High MYCN signature was significantly associated with poor overall survival in MYCN-not-amplified tumors (p=0.0017). In addition, the tumor microenvironment including stromal and immune cell infiltration significantly contributed to the NBL transcriptional landscape and tumor progression.

Obesity is characterized by the accumulation of excess adipose tissue and has become a global health burden as the rates of obesity in both adults and children continue an upward trend year after year. Obesity is an important public health concern as it has been linked to increased prevalence of comorbidities such as type 2 diabetes, fatty liver, cardiovascular complications and cancer. Adipocytes are exquisitely sensitive to energy demands, quickly responding by releasing fatty acids, storing excess calories as triglycerides and/or secreting adipokines. The ability of the adipocyte to carry out its important functions requires perpetual remodeling of the extracellular matrix (ECM) surrounding the adipocyte. Metabolic dysfunction and fibrosis arise when the healthy balance of remodeling becomes dysregulated. The long-term goal of this project was to gain a deeper understanding of the events occurring within an adipocyte in the context of weight gain. I hypothesized that the adipocyte would play an active role in remodeling of the ECM. To test this hypothesis, I fed C57/Bl6 mice either high fat diet (HFD) or chow diet for 8, 20 and 34 weeks, at which time, the perigonadal adipose tissue was digested to isolate the adipocyte fraction and RNA-seq analysis was performed. My data demonstrate that adipocytes responded to their obesogenic, pro-inflammatory environment through upregulation of ECM-related genes after only 8 weeks of HFD coupled with declining expression of mitochondrial genes and increasing genes associated with endoplasmic reticulum (ER) stress after 20 and 34 weeks of HFD. Overall, these data give a novel view into the dysfunctional state of isolated adipocytes over a time course of HFD and response to the changing microenvironment. MICAL2 is an atypical actin-modulating protein that has been shown to be involved in the regulation of MRTFA/SRF signaling and in cancer progression. I demonstrated for the first time that MICAL2 expression is enriched in the stromal vascular fraction of adipose tissue and is upregulated with HFD and pro-fibrotic stimulus, TGF-β1. I also demonstrated that MICAL2 is an anti-adipogenic and pro-fibrogenic protein. Altogether, the novel biology uncovered suggests a role for MICAL2 in adipose tissue remodeling which warrants further investigation. / 2021-06-14T00:00:00Z

Epilepsy, a disease characterized by recurring spontaneous seizures, affects over 65 million people, 2% of the world’s population. Over 30% of patients are refractory to all current medical therapy, and for those that can be treated, many suffer from severe drug side-effects. Understanding the molecular basis of epilepsy is vital to the advancement of better therapeutic options and an eventual cure. Upregulation of brain-derived neurotrophic factor (BDNF) is highly associated with epileptogenesis in human patients, as well as animal models. Our laboratory discovered that BDNF induces the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway in neurons and that inhibition attenuates spontaneous seizures in a temporal lobe epilepsy model. The mechanism behind JAK/STAT signaling in neurons and its relationship to epilepsy still remains to be elucidated and is the subject of my thesis. Surprisingly, even though BDNF is such a major signaling molecule, its full genomic impact has never been assessed. We conducted a high-density RNA-sequencing analysis of the BDNF transcriptome in cortical neurons and probed such regulation with selective JAK inhibitors. Results suggest that 68% of BDNF-induced changes in gene expression implicated in epilepsy are regulated by JAK/STAT signaling. Eighty percent of BDNF-induced changes coding for proteins involved in synaptic neurotransmission (receptor subunits and ion channels) involve JAK/STATs. Additionally, these datasets include genes that have never been associated with BDNF regulation (such as Dopamine Receptor D5 and Galanin Receptor 1). Most interestingly, the datasets reveal that BDNF-induced JAK/STAT signaling in neurons is non-canonical, as STAT3 phosphorylation at tyrosine 705 is not required for action. To directly examine STAT3’s role in epileptogenesis, we studied the transcriptome of transgenic mice that express lower levels of STAT3 specifically in neurons. Using the intrahippocampal kainic-acid (KA) model of epilepsy, our datasets suggest that STAT3 knockdown in vivo, and selectively in neurons, protects mice from KA-induced dysregulation of the sphingolipid metabolism pathway that is associated with the trafficking, sorting, and stability of membrane-bound proteins, including neurotransmitter receptors and ion channels. Finally, we discuss a model for JAK/STAT signaling in neurons that includes structural aspects of an intracellular BDNF receptor (p75NTR) associated with JAK2. / 2021-06-14T00:00:00Z