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Familial Pancreatic Cancer (FPC) is an autosomal dominant condition with greater then 80% of genetic causes unknown. We hypothesize that an integrative approach employing germline exome sequencing and somatic microarray analysis of FFPE DNA will identify novel tumour suppressor genes (TSGs). 55 FPC and 21 sporadic PDAC tumours were analyzed on the Affymetrix Oncoscan FFPE Express 2.0 SNP microarray and compared to data from 33 germline FPC cases analyzed on the Illumina GAIIx Analyzer. We have demonstrated that FPC is genetically heterogeneous with recurrent loss at CDKN2A/p16, TP53 and SMAD4. Analysis of 2 sisters has shown a shared loss region involving DCLK3 and SERPINF1. By an integrative approach, we have identified ATPAF1-AS1 and MAP3K6 as potential TSGs. Germline variants of these genes have been confirmed by Sanger sequencing and somatic fluorescence in-situ hybridization. Future functional studies will better characterize the importance of these regions and novel putative TSGs in FPC.

Familial Pancreatic Cancer (FPC) is an autosomal dominant condition with greater then 80% of genetic causes unknown. We hypothesize that an integrative approach employing germline exome sequencing and somatic microarray analysis of FFPE DNA will identify novel tumour suppressor genes (TSGs). 55 FPC and 21 sporadic PDAC tumours were analyzed on the Affymetrix Oncoscan FFPE Express 2.0 SNP microarray and compared to data from 33 germline FPC cases analyzed on the Illumina GAIIx Analyzer. We have demonstrated that FPC is genetically heterogeneous with recurrent loss at CDKN2A/p16, TP53 and SMAD4. Analysis of 2 sisters has shown a shared loss region involving DCLK3 and SERPINF1. By an integrative approach, we have identified ATPAF1-AS1 and MAP3K6 as potential TSGs. Germline variants of these genes have been confirmed by Sanger sequencing and somatic fluorescence in-situ hybridization. Future functional studies will better characterize the importance of these regions and novel putative TSGs in FPC.

Technological advancements have sparked discovery in biology, enabling important questions to be addressed experimentally at unprecedented depth and scale. One such advance, the development of large-scale approaches to study gene expression, has transformed the way we view the transcriptome. In recent years, these approaches have been applied to studies of alternative RNA splicing, a process where multiple distinct messenger RNAs can be generated from precursor transcripts to produce extensive transcriptomic diversity from a limited repertoire of genes. Global analyses have not only reinforced models initially based on single gene studies, they have also led to numerous insights into general principles governing the regulation and evolution of alternative splicing. In this thesis, I describe how I have combined both large-scale and focused approaches to study alternative splicing regulation during development and in an evolutionary context. Using microarray profiling and comparative genomics approaches, I describe the first large-scale comparative analysis of alternative splicing patterns between humans and chimpanzees. Next, I describe the discovery of a novel neural-specific RS domain splicing factor and the network of alternative exons it regulates to promote nervous system development in vertebrates. Finally, I describe the profiling of alternative splicing patterns during C. elegans development using splicing microarrays and high-throughput sequencing. In this latter study, I also describe two resources that facilitate the analysis of tissue- or cell type-specific splicing events, and enable the function of isoforms to be assessed in vivo. Collectively, these studies have shed light on how differential regulation of alternative splicing has contributed to the evolution of complexity and diversity in biological systems.

Technological advancements have sparked discovery in biology, enabling important questions to be addressed experimentally at unprecedented depth and scale. One such advance, the development of large-scale approaches to study gene expression, has transformed the way we view the transcriptome. In recent years, these approaches have been applied to studies of alternative RNA splicing, a process where multiple distinct messenger RNAs can be generated from precursor transcripts to produce extensive transcriptomic diversity from a limited repertoire of genes. Global analyses have not only reinforced models initially based on single gene studies, they have also led to numerous insights into general principles governing the regulation and evolution of alternative splicing. In this thesis, I describe how I have combined both large-scale and focused approaches to study alternative splicing regulation during development and in an evolutionary context. Using microarray profiling and comparative genomics approaches, I describe the first large-scale comparative analysis of alternative splicing patterns between humans and chimpanzees. Next, I describe the discovery of a novel neural-specific RS domain splicing factor and the network of alternative exons it regulates to promote nervous system development in vertebrates. Finally, I describe the profiling of alternative splicing patterns during C. elegans development using splicing microarrays and high-throughput sequencing. In this latter study, I also describe two resources that facilitate the analysis of tissue- or cell type-specific splicing events, and enable the function of isoforms to be assessed in vivo. Collectively, these studies have shed light on how differential regulation of alternative splicing has contributed to the evolution of complexity and diversity in biological systems.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

This dissertation consists of four different topics in the areas of proteomics, genomics, and cardiology. First, a data-based method was developed to assign the subcellular localization of proteins. We applied the method to data on the bacteria Rhodobacter sphaeroides 2.4.1 and compared the results to PSORTb v.3.0. We found that the method compares well to PSORTb and a simulation study revealed that the method is sound and produces accurate results. Next, we investigated genomic features involved in the lethality of the knockout mouse using the random forest technique. We achieved an accuracy rate of 0.725 and found that among other features, the evolutionary age of the gene was a good predictor of lethality. Third, we analyzed DNA breakpoints across eight different cancer types to determine if common hotspots or cancer-type specific hotspots can be well-predicted by various genomic features and investigated which of the genomic features best predict the number of breakpoints. Using the random forest technique, we found that cancer- type specific hotspots are poorly predicted by genomic features but common hotspots can be predicted using the relevant genomic features. Additionally, we found that among the genomic features analyzed, indel rate and substitution rate were consistently chosen as the top predictors of breakpoint frequency. Lastly, we developed a method to predict the hypothetical heart age of a subject based on the subject’s electrocardiogram (ECG). The heart age predictions are consistent with current ECG science and knowledge of cardiac health.

Previous studies of the grey wolf (Canis lupus) using microsatellites have showed strong population structure despite the high mobility of individuals. I re-assessed the structure of North American grey wolves by genotyping 132 wolves at a genome-wide set of >26 000 single nucleotide polymorphisms (SNPs), and found less population structure, a strong pattern of isolation by distance, and determined that gene flow between subpopulations relates to prey specialization. To assess how accurately smaller data sets assign individuals, I analyzed sub-sets of SNPs and found that small marker sets varied greatly in estimates of subpopulation assignment, and showed high discordance with assignments determined when using all 26k markers. Finally, using a genome scan to detect natural selection I identified SNPs in three genes that may have undergone directional selection, contain variation with observed phenotypic consequences in other mammal species and may be related to adaptation in grey wolves. / Systematics and Evolution

Small Cell Lung Cancer (SCLC) is a very aggressive neuroendocrine tumour of the lung, which demonstrates a 5 year survival of only 10% for extensive stage disease (20-30% for limited stage), with only modest improvement over the last few decades. Identification of new molecular diagnostic and therapeutic targets is thus imperative. Previous efforts in identifying molecular changes in SCLC by gene expression profiling using microarrays have facilitated disease classification but yielded very limited information on SCLC biology. Previous DNA studies have been successful in identifying several loci important to SCLC. However the low resolution of conventional chromosomal Comparative Genomic Hybridization (CGH) has limited the findings to large chromosomal regions with only a few specific candidate genes discovered to date. Thus, to further understand the biological behaviour of SCLC, better methods for studying the genomic alterations in SCLC are necessary. This thesis highlights the development of array CGH technology for the high resolution dissection of aneuploidy in cancer genomes and the application of this new technology to the study of SCLC. I present the development of the first whole genome CGH array which offered unprecedented resolution in the profiling of cancer genomes allowing fine mapping of genes in a single experiment. Through application of DNA based analysis in conjunction with integrated expression analysis and comparison of SCLC to less aggressive non-small cell lung tumours I have identified novel patterns of pathway disruption specific to SCLC. This included alteration to Wnt pathway members and striking patterns of cell cycle activation through predominantly downstream disruption of signalling pathways including direct activation of the E2F transcription factors, which are normally repressed by the Rb gene. Analysis of targets of the E2F/Rb pathway identified EZH2 as being specifically hyper-activated in SCLC, compared to NSCLC. EZH2 is a polycomb group gene involved in the control of many cellular functions including targeted DNA methylation and escape from senescence in hematopoietic stem cells. Taken together these results suggest that in SCLC, downstream disruption may replace multiple upstream alterations leading to activation independent of a specific mitogenic pathway, and that EZH2 represents a potentially important therapeutic target.