We previously reported that in inherited photoreceptor degenerations (IPDs), the mutant photoreceptors (PRs) are at a constant risk of death (Pacione, Szego et al, 2003). Using microarrays to identify genes that may mediate the constant risk, I identified 145 differentially expressed transcripts in the Rds+/- mouse model of IPD at a time when 90% of the PRs were alive. A major finding was the up-regulation, quantified by qPCR, of four components of a putative IL-6 cytokine signaling pathway: Oncostatin M (Osm) (2-fold increased), Oncostatin M receptor (Osmr)(2.6-fold increased), Stat-3 (2.3-fold increased), C/EBP delta(3.2-fold increased). Similarly, I found increases in the cognate proteins Osmr (3-fold), Stat-3 (2.6-fold), and the phosphorylated, transcriptionally active form of Stat-3, pStat-3 (5.8-fold)(all p<0.01). Other Il-6 cytokine signaling molecules were largely unchanged, but the mRNA of leukemia inhibitory factor (Lif), was increased (3.0-fold). Comparable increases of most transcripts were also present in the Rd1-/- and mutant rhodopsin P347S transgenic (P347S) IPD models. The increases in cytokine signaling molecules occurred predominantly in Müller glia, although C/EBP delta transcript was increased in PRs. Because exogenous IL-6 cytokine treatment slows PR death in IPDs, I asked whether the endogenous increases in IL-6 pathway proteins in IPD retinas were a survival response, and generated IPD models with Osmr, Lif or C/EBP delta loss-of-function (LOF) mutations. Osmr LOF decreased PR survival in the retinas of Rds+/-;Osmr-/- mice, which had 12.5% fewer PRs than those of Rds+/-;Osmr+/+ mice (n=9, p<0.05) at 4 month of age, and Tg-RHO(P347S);Osmr-/- mice had 13.5% fewer PRs (n=6, p<0.01) at 31 days of age. Unexpectedly, Osmr LOF had no effect on pStat3 levels in Rds+/-;Osmr-/- retinas, indicating that retinal Stat3 activation may be predominantly regulated by other molecules. In contrast to the Osmr LOF, Lif or C/EBP delta LOF unexpectedly increased mutant PR survival. Rd1-/-;Lif -/- mice at 13 days had 14% more PRs than Rd1-/-;Lif+/+ mice (n=6, p<0.003) and a 1.7 fold decrease in pStat-3 (n=4, p<.05). Similarly, 8 month-old Rds+/-; C/EBP delta-/- mice had 18% more PRs than Rds+/-; C/EBP delta+/+ mice (n=5, p<0.005). These findings suggest that in mutant PRs: 1) up-regulation of the Osmr receptor is protective; 2) the presence of Lif or C/EBP delta is pathogenic, and therefore 3) Osmr, Lif and C/EBP delta act either in different pathways or different cells, to account for the differing effects of their LOF on PR cell death; and 4) the partial effects of Osmr, Lif and C/EBP delta LOF indicate that other genes also mediate the constant risk of death of mutant PRs in IPDs.

In Arabidopsis thaliana (Arabidopsis), by screening for the inability to germinate on low concentrations of exogenous abscisic acid (ABA), loss-of-function mutations in the β-subunit of a protein farnesyltransferase (FTase) were identified (Cutler et al., 1996). Designated era1-2, these mutants are pleiotropic and show a hypersensitive ABA response at the level of germination and stomatal closure, thereby conferring drought resistance, besides having particular developmental phenotypes (Pei et al., 1998; Bonetta et al., 2000). Although a number of proteins have been shown to be farnesylated in plants, which has provided some insight into how farnesylation regulates various processes, there is still no clear understanding of how loss of farnesylation can confer ABA hypersensitivity, for example. The simplest interpretation is that farnesylation acts as a negative regulator of ABA signal transduction. The primary goal of this thesis is to carry out several reverse genetic screens using a Arabidopsis homozygous T-DNA knockout collection to discover potential targets of farnesylation as well as to determine the overall function of these farnesylated targets in plant growth and development. This included screening for morphological changes related to era1-2, altered responses to ABA at the level of germination, and altered drought responses. In total, 15 unique mutants were identified from the aforementioned reverse genetic screens. A knockout in the gene At3g30180 became particularly interesting for further study since it exhibited several phenotypes that resemble era1-2, including ABA hypersensitivity in germination, drought resistance, protruding carpels, reduced fertility, and round and broadened leaves. At3g30180, otherwise known as CYP85A2, is a cytochrome P450 that mediates the final step in the biosynthesis of brassinolide (BL), a brassinosteroid (Kim et al., 2005). At3g30180 was also identified through a bioinformatic screen (Brady and Provart, 2009; Usadel et al., 2009). Overall, ERA1 positively regulates CYP85A2 function through farnesylation, and therefore BL production, which negatively regulates ABA signaling.

Vascular smooth muscle cells (VSMC) serve as key constituents of the developing vasculature, ensuring stability of the nascent vessels, and are essential for the proper performance of the mature cardiovascular system. Pathological alterations in SMC biology, manifesting as perturbations in the differentiation state of SMC, play a pivotal role in the development and progression of various vascular diseases. Thus an understanding of the mechanisms and factors that control SMC differentiation underlies the potential for diagnostic and therapeutic advances. As such, this thesis aimed to implicate the c-Myb transcription factor in the process of SMC differentiation. For this purpose, in vitro assays were developed using the embryoid body (EB) model system, suitable for clarifying the molecular pathways and extrinsic factors that promote or obstruct SMC lineage specification and maturation to the contractile phenotype. Contractile activity was observed in the developing EBs, and further characterized as smooth muscle and cardiac contractions. Temporal induction of SM-specific markers preceded and paralleled contractile SMC appearance and their mRNA levels predicted the relative ability of distinct mouse ES cell (mESC) lines to generate EBs with contracting SMC. Using fluorescent SM-specific promoter-reporter gene strategy it was shown that spontaneous SM-contractions coincide with SM-myosin-heavy-chain (SM-MHC) promoter activity. Moreover, this technology was employed to generate SMC (SM-MHC+) in a scalable stirred-suspension culture system. This thesis addressed the effects of c-myb ablation on SMC differentiation both in vitro and in vivo. c-myb-/- mESC were unable to produce EBs with spontaneously contracting SMC, but gave rise to contracting cardiomyocytes unimpaired. Temporal patterns of myogenic and SM-specific gene expression levels during the course of differentiation revealed that, unlike wild-type EBs, c-myb-/- EBs lacked robust induction of these critical markers. Accordingly, c-myb-/- EBs exhibited significantly reduced SMC numbers. Additionally, in chimaeric EB model substantial contribution by c-myb-/- mESC was detrimental to the development of SM-contractions. To corroborate the in vitro data, chimaeric embryos and adult mice were used to demonstrate significantly reduced c-myb-/- cell contribution to vascular and visceral SMC lineages in vivo. Collectively, this thesis assigns a novel role for the c-Myb transcription factor and advances knowledge of in vitro SMC differentiation.

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.

Deletion mutant collections allow for the systematic study of gene function by linking a genotype to a phenotype. Furthermore, these collections permit the parallel and quantitative study of phenotypes, which is the foundation of functional genomics. I begin by summarizing the methods used and data derived from the field of functional genomics using the Baker’s yeast Saccharomyces cerevisiae, and provide important background information on the origins of the filamentous growth-competent S.cerevisiae strain Σ1278b, and the developmental process of fungal dimorphism. I describe my efforts in creating a complete deletion mutant collection in the filamentous growth-competent S.cerevisiae strain Σ1278b, and the subsequent phenotypic analysis of that deletion mutant collection. By quantitatively measuring mutant phenotypes of cells undergoing haploid invasive growth, biofilm mat formation and diploid pseudohyphal growth, I studied the clinically relevant developmental process of fungal dimorphism. I present the first genome-wide and quantitative phenotypic analysis of fungal dimorphism and identify a novel transcription factor encoded by the open reading frame YDL233W, which I named FMR1for Filamentation Master Regulator 1. By performing genetic, cell biological, biochemical, and expression analysis, I demonstrate that Ydl233w acts by forming a protein complex with the DNA-binding transcription factors Flo8 and Mss11 and this complex binds to a specific element within the promoter of the surface adherence mediating gene FLO11. I directly compare the essential gene sets between the Σ1278b deletion collection and the reference deletion collection made in the S288c genetic background completed by the Yeast Deletion Consortium in 2002. I find that most essential genes are shared between these two strains but a number of genes are essential for viability in only one genetic background, a phenomenon termed conditional essentiality. I describe the genetic basis of conditional essentiality as a consequence of the complex inheritance of background-specific alleles. Lastly, I summarize the scientific advancements of my research using the Σ1278b deletion collection, and highlight some potential applications for both the data derived from my research and the deletion mutant collection itself. The Σ1278b deletion collection provides a valuable resource for yeast geneticists, evolutionary biologists, researchers of fungal disease, and researchers interested in modeling the genetics that underlie complex traits and diseases.

Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Known mutations account for only a subset of MB cases. We hypothesized that CpG island methylation-mediated tumour suppressor gene (TSG) silencing contributes to MB pathogenesis, either alone, or in combination with genetic events such as loss of heterozygosity (LOH). We performed a microarray-based genome-wide screen of MB cell lines treated with 5-aza-2’deoxycytidine, identifying genes exhibiting increased expression following treatment. Using this strategy, we identified inhibitors of WNT signalling (SFRP1, SFRP2, and SFRP3) and an inhibitor of the HGF/MET signalling pathway (SPINT2) as putative TSGs silenced by promoter region methylation in MB. Methylation of the WNT signalling inhibitors SFRP1, SFRP2, and SFRP3 was identified using bisulfite sequencing and methylation-specific PCR (MSP). Stable re-expression of SFRP1, SFRP2, and SFRP3 reduced proliferation, impaired anchorage-independent growth, and limited WNT signalling activity. SFRP1 re-expression reduced tumour growth in vivo in xenograft models. Aberrant WNT signalling plays a role in the pathogenesis of a subset of sporadic human MB, as well as MB in cases of Turcot syndrome with germline mutations of APC. Activating mutations of β-catenin are also implicated in a subset of MB. We have identified for the first time an additional mechanism – loss of normal pathway inhibition by SFRP gene silencing – that contributes to MB pathogenesis. SPINT2 methylation was confirmed with bisulfite sequencing and MSP. Stable re-expression of SPINT2 reduced proliferation, impaired cell migratory ability, and decreased the capacity for anchorage-independent growth. In vivo, re-expression of SPINT2 reduced tumour formation in xenograft models. This study identified for the first time SPINT2 as a putative TSG in human MB, and further implicated aberrant HGF/MET oncogenic signalling in the pathogenesis of this disease. The efficacy of targeting the HGF/MET pathway as a novel therapeutic strategy was tested in vitro using the small molecule MET kinase inhibitor PHA665752. Treatment of MB cell lines with PHA665752 reduced cell proliferation, anchorage-independent growth, migration, and limited downstream signalling via the MAPK and PI3K/AKT pathways.

This thesis examines how natural variation in the foraging gene (for) of Drosophila melanogaster influences several learning and memory phenotypes in adult flies. These studies are undertaken using aversive olfactory associative conditioning paradigms. Novel approaches to standard Pavlovian conditioning paradigms are used in order to test hypotheses that were formulated based on what is known concerning movement- and feeding-related behaviour in the rover (forR) and sitter (fors) variants of for. The results show that natural variation in for, which is thought to have been under balancing selection in the wild, influences adult learning and memory traits appreciably. More specifically, forR flies, who are exposed to greater environmental heterogeneity than sitters, display retroactive interference. As might be expected from their foraging behaviour, rover responses are biased towards more recent learning events. Additionally, results indicate that individual performance in a learning task was affected by allelic variation in for and through pharmacological manipulations of PKG activity levels. Interestingly, in fors, but not forR, the acquisition of information was facilitated by social interaction (i.e. being in a group). In forR, but not in fors, the type of social interaction (being with other forR or with other fors) affected learning and memory. Also, naive individual forR flies tended to follow groups of conditioned fors but not groups of conditioned forR. In several other chapters, the thesis explores recent issues in behaviour genetics. In particular, the following concepts are explored: 1) the fruit fly as model for ecological and evolutionary studies; 2) cGMP-dependent protein kinase as a modifier of behaviour in disparate species; and 3) conservation of gene function in behaviour.

This thesis examines how natural variation in the foraging gene (for) of Drosophila melanogaster influences several learning and memory phenotypes in adult flies. These studies are undertaken using aversive olfactory associative conditioning paradigms. Novel approaches to standard Pavlovian conditioning paradigms are used in order to test hypotheses that were formulated based on what is known concerning movement- and feeding-related behaviour in the rover (forR) and sitter (fors) variants of for. The results show that natural variation in for, which is thought to have been under balancing selection in the wild, influences adult learning and memory traits appreciably. More specifically, forR flies, who are exposed to greater environmental heterogeneity than sitters, display retroactive interference. As might be expected from their foraging behaviour, rover responses are biased towards more recent learning events. Additionally, results indicate that individual performance in a learning task was affected by allelic variation in for and through pharmacological manipulations of PKG activity levels. Interestingly, in fors, but not forR, the acquisition of information was facilitated by social interaction (i.e. being in a group). In forR, but not in fors, the type of social interaction (being with other forR or with other fors) affected learning and memory. Also, naive individual forR flies tended to follow groups of conditioned fors but not groups of conditioned forR. In several other chapters, the thesis explores recent issues in behaviour genetics. In particular, the following concepts are explored: 1) the fruit fly as model for ecological and evolutionary studies; 2) cGMP-dependent protein kinase as a modifier of behaviour in disparate species; and 3) conservation of gene function in behaviour.

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.

Medulloblastoma is the most common pediatric brain tumour, yet many of the underlying genetic and epigenetic factors have yet to be discovered. After a genome wide screen of a large cohort of primary medulloblastomas, we discovered that many of the genes within the cell adhesion family are affected by either copy number loss and/or decreased expression unexplained by copy number change. This led us to believe that both genetic and epigenetic factors were affecting this gene family. Through methylation-specific PCR, RT-PCR and high-throughput methylation status analysis, we have concluded that promoter CpG methylation plays a role in the expression of the PCDH10 protein in both medulloblastoma cell lines and primary tumours. Through functional validation with a stable cell line re-expressing PCDH10, I show that cell cycle and proliferation remain unchanged but migration is decreased in cells with PCDH10 re-expression. This suggests that PCDH10 has characteristics of a tumour suppressor in medulloblastoma.