Bioinformatic methods were applied to address biological questions. Two new eFP browser web tools were constructed for the intuitive visualization of data from large-scale data sets. In addition, a predicted interactome was constructed for Arabidopsis thaliana and validated using a gene coexpression analysis. The Arabidopsis Interactions Viewer was created to enable access to and visualization of predicted and confirmed interactions in the Arabidopsis interactome. In a separate analysis short sequence matches were identified between introns and coding sequences in several model systems including Arabidopsis, human, C. elegans and 12 Drosophila species. Several hundred to thousands of matches were found near each other in terms of chromosomal location, and were termed Proximal Intron N-mer (PIN) matches. Sequence matches were conserved between 11 Drosophila species and D. melanogaster, suggesting a potential functional role. Novel plasmids were designed to test whether PIN matches are functional in vivo.

Bioinformatic methods were applied to address biological questions. Two new eFP browser web tools were constructed for the intuitive visualization of data from large-scale data sets. In addition, a predicted interactome was constructed for Arabidopsis thaliana and validated using a gene coexpression analysis. The Arabidopsis Interactions Viewer was created to enable access to and visualization of predicted and confirmed interactions in the Arabidopsis interactome. In a separate analysis short sequence matches were identified between introns and coding sequences in several model systems including Arabidopsis, human, C. elegans and 12 Drosophila species. Several hundred to thousands of matches were found near each other in terms of chromosomal location, and were termed Proximal Intron N-mer (PIN) matches. Sequence matches were conserved between 11 Drosophila species and D. melanogaster, suggesting a potential functional role. Novel plasmids were designed to test whether PIN matches are functional in vivo.

Plasma membrane (PM) proteins are at the interface between the cell and the external environment and are therefore the most accessible to therapeutic drugs. I utilized cationic silica beads and mass spectrometry (MS)-based proteomics to enrich for PM proteins of human cardiomyocytes, coronary smooth muscle cells, and coronary endothelial cells. The enrichment of PM proteins was confirmed and 1006 proteins were specifically filtered and enriched into a set of known and novel cardiomyocyte PM-associated proteins of which 42% had PM-associated gene ontology annotations and/or predicted transmembrane helices. Two novel candidates, namely popeye domain-containing protein 2 (POPDC2) and protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3) were selected and found to have confirmed PM localization. In conclusion, silica bead membrane extraction combined with MS-based proteomics successfully enriched for PM proteins of the human heart of which two novel candidate proteins were shown to have confirmed PM localization.

This thesis explores chromatin modification (CM) as a biological system and uses known CM factors in four model organisms; yeast, worm, fly, and human to explore how CM factors have consistently evolved across a diverse spectrum of 111 organisms by using the InParanoid homology algorithm. Using InParanoid, phylogenetic profiles are constructed for each model organism to highlight evolutionary trajectories and which CM factors are lost, expanded, and are specific to some lineages. Phylogenetic tree construction demonstrates that peripheral subunits of CM complexes evolve independently. Accurate mapping of domains to CM factors and their homologs reveals that the architecture of domains is very well conserved, with only one potential case of a domain swap. Homology, domain architecture, and protein-protein interaction is then combined to illustrate an interolog example and potential interaction candidates. The techniques highlighted in this thesis represent a generic and powerful approach to analyzing any biological system of interest.

Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

Plasma membrane (PM) proteins are at the interface between the cell and the external environment and are therefore the most accessible to therapeutic drugs. I utilized cationic silica beads and mass spectrometry (MS)-based proteomics to enrich for PM proteins of human cardiomyocytes, coronary smooth muscle cells, and coronary endothelial cells. The enrichment of PM proteins was confirmed and 1006 proteins were specifically filtered and enriched into a set of known and novel cardiomyocyte PM-associated proteins of which 42% had PM-associated gene ontology annotations and/or predicted transmembrane helices. Two novel candidates, namely popeye domain-containing protein 2 (POPDC2) and protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3) were selected and found to have confirmed PM localization. In conclusion, silica bead membrane extraction combined with MS-based proteomics successfully enriched for PM proteins of the human heart of which two novel candidate proteins were shown to have confirmed PM localization.

This thesis explores chromatin modification (CM) as a biological system and uses known CM factors in four model organisms; yeast, worm, fly, and human to explore how CM factors have consistently evolved across a diverse spectrum of 111 organisms by using the InParanoid homology algorithm. Using InParanoid, phylogenetic profiles are constructed for each model organism to highlight evolutionary trajectories and which CM factors are lost, expanded, and are specific to some lineages. Phylogenetic tree construction demonstrates that peripheral subunits of CM complexes evolve independently. Accurate mapping of domains to CM factors and their homologs reveals that the architecture of domains is very well conserved, with only one potential case of a domain swap. Homology, domain architecture, and protein-protein interaction is then combined to illustrate an interolog example and potential interaction candidates. The techniques highlighted in this thesis represent a generic and powerful approach to analyzing any biological system of interest.

Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

Inflammatory bowel disease (IBD) is a composite classification for a range of gastrointestinal disorders. The two most common forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). The chronicity of IBD has long-term consequences on the quality of life of affected individuals. However, therapies available today are applied with limited success. It is thought that interplay between environmental triggers and commensal bacteria can cause a dysregulated inflammatory response in genetically predisposed individuals, but the exact etiology is not understood. The objective of these studies was to identify the gene expression changes which associate with differential response to therapy and with recurrence of disease following surgery. The effects of genetic variation on gene expression were also evaluated. Tissue and blood samples were collected from eligible patients. Total RNA was extracted and measured on gene expression microarrays. The raw gene expression data were analyzed in a statistical framework against variables of interest in order to assess the significance of each association. Genes were evaluated individually as well as in biological networks. A few tens of genes were identified as differentially expressed in blood between UC patients who respond to intravenous corticosteroid therapy and those who do not. Some of these genes were also shown to have high predictive value. Differentially regulated genes were also found in UC patients who experience recurrence of disease after surgery, relative to those who remain disease-free. Specifically, gene networks responsible for the regulation of cellular transport were among the major players. A comparative analysis of genotype and gene expression in the human ileum indicated that the transcription of approximately 10% of genes is influenced by variations in the genome. Results from these studies have not only contributed to our understanding of disease mechanism, but could also have medical implications. With the advance of new and less costly transcriptome technologies on the clinical stage, panels of expression markers associated with therapy response and post-operative recurrence can be used as diagnostic and prognostic tools. Data on the relationship between genotype and gene expression are already shedding new light on the function of certain genetic IBD risk variants.

Inflammatory bowel disease (IBD) is a composite classification for a range of gastrointestinal disorders. The two most common forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). The chronicity of IBD has long-term consequences on the quality of life of affected individuals. However, therapies available today are applied with limited success. It is thought that interplay between environmental triggers and commensal bacteria can cause a dysregulated inflammatory response in genetically predisposed individuals, but the exact etiology is not understood. The objective of these studies was to identify the gene expression changes which associate with differential response to therapy and with recurrence of disease following surgery. The effects of genetic variation on gene expression were also evaluated. Tissue and blood samples were collected from eligible patients. Total RNA was extracted and measured on gene expression microarrays. The raw gene expression data were analyzed in a statistical framework against variables of interest in order to assess the significance of each association. Genes were evaluated individually as well as in biological networks. A few tens of genes were identified as differentially expressed in blood between UC patients who respond to intravenous corticosteroid therapy and those who do not. Some of these genes were also shown to have high predictive value. Differentially regulated genes were also found in UC patients who experience recurrence of disease after surgery, relative to those who remain disease-free. Specifically, gene networks responsible for the regulation of cellular transport were among the major players. A comparative analysis of genotype and gene expression in the human ileum indicated that the transcription of approximately 10% of genes is influenced by variations in the genome. Results from these studies have not only contributed to our understanding of disease mechanism, but could also have medical implications. With the advance of new and less costly transcriptome technologies on the clinical stage, panels of expression markers associated with therapy response and post-operative recurrence can be used as diagnostic and prognostic tools. Data on the relationship between genotype and gene expression are already shedding new light on the function of certain genetic IBD risk variants.