Iroquois (Irx) homeobox genes have important and redundant functions during embryogenesis. Irx3/5 double knock out (Irx3/5 KO) mouse embryos exhibit severe hindlimb phenotypes. In these mutant hindlimbs, digit 1 and tibia are absent, moreover femur and pelvis are hypoplastic. Here, we demonstrate that Irx3/5 are expressed in the hindlimb field prior to limb bud initiation, and are required at this early stage for the pattern formation along the anteroposterior axis. Their early function is involved in prepatterning and positioning the Shh expression domain. In addition, Irx3/5 KO mutant hindlimb buds have a mild outgrowth defect and increased cell death at early stages of limb development, which may explain the small hindlimb bud size in these mutant embryos. To examine whether Irx3/5-expressing cells are the origin of lost and affected structures in Irx3/5 KO mutant hindlimbs, targeting vectors with Cre genes inserted into the Irx5 locus have been generated.

Reactive oxygen species (ROS) are a by-product of aerobic metabolism and have been implicated in cancer, arthrosclerosis, diabetes and aging. Antioxidant enzymes, such as superoxide dismutase (SOD), work to neutralize ROS and oxidative stress occurs when the antioxidant capacity of the cell is overwhelmed. Using a Drosophila mutant with defective cytoplasmic SOD function (cSODn108), we are able to study the consequences of excess ROS on gene expression. Microarray experiments indicate gene expression changes associated with immune response, heat shock, detoxification, proteolysis, carbohydrate metabolism, lipid metabolism and behaviour. Behavioural and physiological assays investigated possible phenotypes predicted by changes in gene expression. We found that cSODn108 mutants feed less yet demonstrate a remarkable resistance to starvation. In addition, cSODn108 mutants show a reduced response to sucrose, odorants and decreased locomotor activity. These phenotypes correlate with observed gene expression changes and suggest a potentially altered energy metabolism in response to chronic oxidative stress.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with no cure. TAR-DNA binding protein 43 (TDP-43) is the major component of the cytoplasmic inclusions characteristic of ALS. Transgenic Drosophila lines expressing wild-type, mutant and splice variants of human TDP-43 were generated. I find that ubiquitous expression of all TDP-43 transgenes, except for TDP-43∆C-term, is sufficient to cause lethality. I also show that eye-specific expression of a TDP-43∆N-term splice variant, which localizes diffusely to the cytosol, results in increased cell toxicity suggesting an association between cytosolic localization and toxicity. Consistent with this model, I find that the TDP-43∆N-term splice variant is capable of recruiting full length TDP-43 into the cytoplasm, and I suggest this may represent an initiating event in TDP-43-linked ALS. Altogether, my results seem to indicate that exclusion of TDP-43 from the nucleus rather than its presence in aggregates is linked to increased cytotoxicity and lethality in ALS.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

Pulmonary metastasis is the most frequent cause of osteosarcoma (OS) mortality. The aim of this study was to discover and characterize genetic networks differentially expressed in metastatic OS. Supervised network analysis of OS expression profiles was performed to discover genetic networks differentially activated or organized in metastatic OS. Broad trends among the profiles of metastatic tumours included aberrant activity of intracellular organization and translation networks, as well as disorganization of metabolic networks. The differentially activated PRKCε-RASGRP3-GNB2 network, which interacts with the disorganized DLG2 hub, was additionally found to be differentially expressed among in vitro models of human OS metastasis. PRKCε transcript was more abundant in some metastatic OS tumours; however the difference was not significant overall. In functional studies, PRKCε was not found to be involved in migration of M132 OS cells, but its protein expression was induced in M112 OS cells following IGF-1 stimulation.

Reading disabilities (RD) have a significant genetic basis and chromosomes 3p12-q13 and 15q15-21 have shown replicated linkage to RD or reading measures. This study evaluated two RD candidate genes within these regions: DYX1C1 on chromosome 15q21 and ROBO1 on chromosome 3p12. DYX1C1 was tested for association using a family-based analysis of two independent samples. No statistically significant association was observed between the 10 tested DYX1C1 single nucleotide polymorphisms (SNPs) and RD or any of the quantitative traits. A review and meta-analysis of the potentially functional SNPs at the -3G/A and 1249G/T positions did not find strong support for these alleles as risk alleles for RD. ROBO1 was also evaluated in this study using SNPs that previously showed association with memory and reading measures in a population-based sample. None of the SNPs showed significant association with RD or any of the quantitative traits after correction for multiple testing.

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.

The role of variation in the TAS2R50 bitter taste receptor gene is unknown, but may influence taste perception and dietary habits. Individuals (n=1171) aged 20 to 29, from the Toronto Nutrigenomics and Health Study, completed a food preference checklist and a semi-quantitative food frequency questionnaire to assess their preference and intake of potentially bitter foods and beverages. DNA was isolated from blood and genotyped for 3 polymorphisms in the TAS2R50 gene (rs2900554 A>C; rs10772397 A>G; rs1376251 A>G). Taste intensity was examined using taste strips infused with 3µg of naringin. The rs2900554 SNP was associated with naringin taste intensity, grapefruit preference and grapefruit intake in females. Homozygotes for the C allele reported the highest frequency of experiencing a high naringin taste intensity, disliking grapefruit and not consuming grapefruit. The rs10772397 and rs1376251 SNPs were associated with disliking grapefruit. These results suggest that naringin may be a ligand for the T2R50 receptor.

Eukaryotic organisms utilize post-translational modifications of highly conserved histone proteins to control gene expression programs. Methylation of lysine 4 on histone H3 (H3K4me) in particular, is thought to be associated with actively transcribed DNA. Paradoxically, recent evidence has suggested that H3K4me has a repressive function as well. Pho23, a member of the highly conserved ING family of tumour suppressor proteins, binds H3K4me and is a component of the gene repressive complex, Rpd3L. My genetic analysis suggests that Pho23 controls transcriptional repression via H3K4me and that Pho23 is itself regulated by the sequence-specific DNA-binding protein Ume6. Moreover, this Ume6-regulated function appears to be governed by Ume6 phosphorylation by Mck1, an evolutionarily conserved kinase. Finally, while Ume6/Pho23 are known to function together with the histone deacteylase Rpd3, my findings suggest the existence of an Rpd3-independent function for Pho23.