Approximately 15% of human cancers utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain the lengths of their telomeres. The Fanconi anaemia protein FANCJ localizes to telomeric foci in human ALT cells, but not in telomerase-positive or primary cells. Telomere-associated FANCJ frequently localizes with FANCD2 and BRCA1, and primarily localizes to ALT-associated PML nuclear bodies. Depletion of FANCJ in human ALT cells causes the loss of BRCA1 at telomeric foci and a decrease in telomeric repeat DNA content primarily as a result of the loss of the brightest telomeric repeat DNA foci. In contrast, depletion of the FANCD2 results in increased telomeric repeat DNA synthesis and this is suppressed upon the codepletion of FANCJ. Together, data from this study suggest that FANCJ is required for telomeric repeat DNA synthesis in human ALT cells, which may or may not be dependent on BRCA1, and FANCD2 restrains this synthesis.

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.

In addition to previously identified roles for RNA, several new ways in which RNA serves as a regulator of gene expression have recently been described. RNA molecules are involved in both transcriptional and post-transcriptional forms of regulation, sometimes heritably affecting gene activity. Whereas most previously characterized regulatory roles of RNA involve downregulation, I describe a role for maternal transcripts of a gene in promoting zygotic activity of that gene, which I term the licensing of genetic activity. This regulation occurs in the germ line, a tissue notable for its abundance of genetic surveillance mechanisms. The maternal-effect regulation described here was identified using alleles of a sex-determining gene in Caenorhabditis elegans called fem-1. Females homozygous for fem-1 deletions produce heterozygous offspring that exhibit germ-line feminization and have reduced fem-1 activity and transcript accumulation. This phenotype can be rescued by injecting fem-1 RNA into the maternal germ line. The reduction in activity of the zygotic fem-1 locus is heritable, suggesting that the gene is becoming epigenetically silenced. Thus the maternal fem-1 RNA licenses the activity of the zygotic locus by preventing its silencing. By restricting germ-line activity to genes that were expressed in the germ line of the previous generation, this process may contribute to protecting the identity and integrity of the germ line. I performed an RNAi screen of candidate genes to ask whether they are required for maternal-effect silencing or licensing. Several enhancers and suppressors of germ-line feminization in the descendants of fem-1 deficiency homozygotes were identified. Chromatin regulation may be involved, and small-RNA pathways are important for both the silencing and licensing components of fem-1 regulation. Based on my characterization of this phenomenon, I proposed models of how maternal-effect regulation of fem-1 may be mediated. To test predictions of certain models, I examined whether specific characteristics of fem-1 make it susceptible to this silencing. Results of these experiments limit the possible models of maternal-effect regulation and suggest directions for future investigation.

The transition from cross-fertilization to predominant self-fertilization is considered the most common evolutionary transition in flowering plants. This change in mating system has profound influences on the amounts and patterns of genetic diversity within and among populations, and on key genetic and demographic processes. The main goal of my thesis is to determine the molecular population genetic consequences of this transition in the annual neotropical aquatic plant Eichhornia paniculata (Pontederiaceae) using DNA sequence from individuals sampled from throughout the species’ geographic range. Populations exhibit a wide range of mating patterns associated the evolutionary breakdown of tristyly facilitating specific contrasts between outcrossing and selfing populations. Analysis of molecular variation supported the hypothesis of multiple origins of selfing, including the evolution of two morphologically distinct selfing variants from Central America and the Caribbean. A survey of 10 nuclear loci from 225 individuals sampled from 25 populations demonstrated the joint influence of mating system, population size and demographic bottlenecks in affecting patterns of nucleotide variation. Small selfing populations exhibited significantly lower genetic diversity compared with larger outcrossing and mixed mating populations. There was also evidence for higher population differentiation and a slower decay of linkage disequilibrium in predominately selfing populations from the Caribbean region. Coalescent simulations of the sequence data indicated a bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years ago. To investigate the consequences of transitions from outcrossing to selfing across the genome, I used high-throughput, short-read sequencing to assemble ~27,000 ESTs representing ∼24Mbp of sequence. Characterization of floral transcriptomes from this dataset identified 269 genes associated with floral development, 22 of which were differentially expressed in three independently derived selfing lineages compared to an outcrossing genotype. Evidence for relaxed selection in selfing lineages was obtained from an analysis of a subset of ~8000 orthologous sequences from each genotype, as predicted by theory. Selfing genomes showed an increase in the proportion of nonsynonymous to synonymous changes and relaxation of selection for codon usage bias. My thesis represents the most detailed investigation to date of the molecular population genetic consequences of intraspecific variation in the mating systems of plants.

Plk4 is the most divergent member of the family of polo like kinases (Plks). Plk4-/- embryos arrest at approximately day 7.5 p.c. but Plk4+/- mice are viable and fertile. However, 50% of Plk4+/- mice develop spontaneous tumours of the liver, lung and soft tissues by 2 years of age. Here I investigate the mechanisms that underlie Plk4-related tumourigenesis. Plk4+/- murine embryonic fibroblasts (MEFs) spontaneously become immortal in vitro with increasing passage number and are tumourigenic in vivo when injected into NOD SCID mice. Cytogenetic analysis showed that Plk4 deficient cells are chromosomally unstable with a large number of chromosomal aberrations and increased ploidy. These results demonstrate that early loss of a single Plk4 allele is sufficient to drive cell immortalization, chromosomal instability and tumourigenicity in vivo. In two independent expression array analyses, gene expression patterns that would decrease cell migration were overrepresented in Plk4+/- MEFs. A series of spreading and migration assays functionally validated these results, supporting the hypothesis that Plk4 regulates cell motility. Endogenous Plk4 localized to filopodia and lamellipodia in motile cells and to protrusions of spreading cells; the latter localization was transient and it disappeared by 4h after cell seeding, at which point Plk4 was located in the centrosomes, as typically observed in interphase cells. Transient transfection with Flag-Plk4 enhanced spreading and migration, as well as actin remodeling. Taken together, these data demonstrate temporal regulation of Plk4 in relation to the process of membrane remodeling, and a functional role for Plk4 in cell motility. Plk4 is haploinsufficient for tumour suppression in mice. Plk4 is located at human chromosome 4q28, a region often deleted in primary liver cancer specimens. Here I show that loss-of-heterozygosity (LOH) occurs at the Plk4 locus in ≈50% of human hepatocellular carcinomas (HCC) as well as in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumours, but not with mutations in the remaining allele. These results implicate Plk4 as a potential haploinsufficient tumour suppressor in the genesis of human HCC. With continuing high rates of the predisposing conditions Hepatitis B and non-alcoholic steatohepatitis, and delayed diagnosis, HCC is a global health issue and carries a grave prognosis. A better understanding of genetic predisposition will help guide future screening programs.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.