The roundworm Caenorhabditis elegans possesses a single, conserved phosphatidylinositol 3-kinase (PI3K) signaling pathway that regulates somatic developmental decisions and lifespan through the Insulin-like receptor tyrosine kinase (RTK) DAF-2, the class I PI3K AGE-1 and the 3-phosphoinositide-dependent protein kinase 1 (PDK1) homologue PDK-1. This pathway ultimately controls the action of Akt homologues on the forkhead transcription factor DAF-16. The C. elegans Akt orthologue akt-1 also negatively regulates the DNA damage-dependent apoptosis of worm germ cells by indirectly interfering with activation of the key transcription factor CEP-1, the sole homologue of p53 in the worm. Because upstream regulation by RTK/PI3K signaling is known to couple with downstream Akt kinase activity, I hypothesized that the worm daf-2/age-1/pdk-1 pathway would function upstream of akt-1/Akt in response to DNA damage. Surprisingly, this was not the case: daf-2/InsR and pdk-1/PDK1 do not function upstream of akt-1/Akt and instead promote DNA damage-induced germ cell apoptosis independently of CEP-1/p53 by regulating the B cell lymphoma (Bcl2) homologue CED-9 and the Apoptotic protease-activating factor 1 (Apaf1)-like adapter protein CED-4, respectively. Furthermore, PDK-1/PDK1 promotes germ cell apoptosis by a mechanism that does not include changes in the subcellular localization or absolute levels of CED-4/Apaf1, but does require the presence of CEP-1/p53. Therefore, daf-2/RTK, pdk-1/PDK1, and cep-1/p53 co-operate from independent pathways to drive germ cell death. The separation of worm Akt function from canonical RTK/PI3K regulation is consistent with the ability of AKT-1 to function without major changes in phosphorylation at threonine 350, a site homologous to Thr308 in mammals. Since this modification is an essential step in the activation of Akt proteins by PDK1, it is likely that damage-dependent germline activity of AKT-1 is controlled by a novel mechanism that does not involve phosphorylation by PDK-1 on key regulatory sites. These data argue that C. elegans re-arranges single homologous components of a signalling pathway to respond to different stimuli in vivo. Finally, I present data identifying the C. elegans ataxia and telangectasia and Rad3-related (ATR) kinase homologue ATL-1 as a potential target of AKT-1. Collectively, my work has uncovered a novel DNA damage-dependent pathway that allows AKT-1 to control CEP-1/p53-dependent apoptosis.

The Hedgehog (Hh) signaling pathway plays a critical role in modulating various developmental processes that requires fine tuning of the Hh signal, such that dysregulation can lead to cellular events involved in cancer. To elucidate the factors responsible for aberrant Hh activation and subsequent tumorigenesis, I investigated three distinct modulators of Hh signaling: (1) p53 tumour suppressor (2) primary cilia (3) PTHLH. During chondrocyte development, abnormal Hh signalling can result in benign cartilage tumours, called Enchondroma. As precursor lesions, enchondroma may progress to malignant neoplasia, collectively known as chondrosarcoma. Although the molecular events involved in this progression are poorly understood, inactivation of the p53 tumour suppressor has been identified in approximately one-third of chondrosarcoma. Using an enchondroma mouse model, I showed that p53 deficiency can cause chondrosarcoma to develop. The combined inhibitory effects of Hh and p53 pathways on the pro-apoptotic factor, IGFBP-3, suppressed apoptosis and was demonstrated to play a critical role in the progression to chondrosarcoma. The primary cilium is an organelle that serves as a signaling centre for the Hh pathway to allow for greater control of the signal output. Loss of primary cilia results in abnormal Hh signaling that is associated with cancer and various developmental defects. I observed a depletion of primary cilia in both human Chondrosarcoma and Enchondroma tumours compared to normal cartilage. Analysis of cilia-deficient mice revealed that defective ciliogenesis alone could lead to the formation of benign cartilage tumours. Furthermore, loss of primary cilia potentiated the effect of Hh signaling activation, revealing a novel role in cartilage tumorigenesis. Parathyroid-like hormone (PTHLH) is an essential inhibitor of the Hh pathway during chondrocyte development, however its function as a regulator of Hh in other tissue types are largely unknown. Through activation of PKA, PTHLH suppresses the activation of Gli transcription factors; downstream effectors of the Hh pathway. Using irradiated Ptch+/- mice that exhibit a high incidence of skin and brain tumours, I demonstrated that treatment with PTHLH agonist, PTH (1-34), results in inhibition of the Hh pathway, increased survival and a reduction in tumour incidence and size. Thus, PTH (1-34) may have therapeutic potential for Hhrelated cancers, especially given its known clinical safety in treating osteoporosis.

The fundamental repeating unit of all eukaryotic chromatin is the 147bp DNA:histone complex known as the nucleosome. Genome-wide studies have demonstrated that nucleosomes are organized with the 5’ promoter being nucleosome depleted and the transcribed region is occupied by a periodic array of positioned nucleosomes. While this organization is well described, the determinants, particularly trans-acting factors that contribute to this architecture are only partly described with gene expression, however, while the connection between chromatin and the various facets of gene expression regulation, especially in evolution, is apparent the detailed mechanisms remain to be described. In this thesis, I describe 1) The role of nucleosomes in gene expression evolution in closely related yeast species 2) The role of trans-acting factors (particularly transcription factors and co-factors) in determining the nucleosome depleted region of promoters and 3) The role of trans-acting factors in nucleosome spacing within genes.

Differential gene expression between sexes is the main contributor of the morphological and behavioral differences observed between them. Studying the signatures of these differences at the genetic level will help us understand the forces acting on them. The existence of androdioecious and gonochoristic species in the genus Caenorhabditis makes it suitable for sex-biased gene expression studies. In this thesis, I have assembled the transcriptome of C. sp. 9 and C. sp. 5 using de novo and reference-based techniques. Evolutionary analysis of the assembled contigs showed that genes with male-biased expression evolve faster than those with a female bias, as observed in other taxa. Furthermore, I found a positive correlation between gene expression and codon usage bias.

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.

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal. / La pollinisation et la fécondation induisent, dans le pistil, l'expression de nombreux messagers. Certains de ceux-ci sont nouvellement produits tandis que d'autres voient leur expression augmenter. Ce mémoire expose les résultats obtenus pour trois de ces gènes induits par la pollinisation et/ou la fécondation chez Solanum chacoense Bitt., une pomme de terre sauvage. Le premier, SPP2, est une désoxygénase de fonction inconnue de la famille des 2-oxoglutarate dépendantes, que les identités de séquence rapprochent d'enzymes impliquées dans les voies de biosynthèse d'alcaloïdes. Ce clone fut isolé simultanémment de deux banques d'ADNc construites à partir de pistils pollinisés depuis 48 heures (banque PP48) et 96 heures (banque 0v96), par la méthode d'hybridation soustractive. Le second gène, l'ACC oxydase qui est l'enzyme responsable de transformer l'ACC en éthylène, fut isolé dans la banque d'ADNc PP48 par la méthode du criblage différentiel. Le corps de ce mémoire traite principalement de ces deux premiers gènes, le troisième nommé SPP30 étant présenté en annexe sous forme d'article manuscrit, afin de mieux cerner la discussion. Ce troisième gène, SPP30, est très fortement homologue à un antigène de surface de Plasmodium falciparum, l'agent causal de la malaria, et fut isolé dans la banque d'ADNc PP48 par hybridation soustractive. L'ACC oxydase est un gène qui a déjà été caractérisé chez un grand nombre d'espèces de plantes. Depuis longtemps déjà il est établi que l'ACC oxydase est une importante enzyme de la voie de biosynthèse de l'éthylène, et que ce dernier est connu pour ses effets au niveau de la sénescence des pièces florales, sénescence qu'induisent les événements de pollinisation et de fécondation chez les végétaux. Pour cette raison, nous n'avons pas poussé très avant sa caractérisation. Une hybridation d'ARN de type northern démontre tout de même que, chez Solanum chacoense, l'expression de l'ACC oxydase augmente fortement dans le pistil suite à une pollinisation de 48 heures. L'intérêt du clone SPP2, quant à lui, nous est apparu grandissant au fur et à mesure que sa caractérisation progressait. Nous avons réussi, par le biais de nombreuses expérimentations, a démontrer que les ARNm de la désoxygénase SPP2 sont non seulement induits dans les ovaires matures par la pollinisation (compatible ou non), mais également par la fécondation et la blessure du style. On ne peut détecter les ARNm de SPP2 dans le style des fleurs matures, car son patron d'expression régresse de l'extrémité du style jusqu'à l'ovaire, et cela en fonction du développement floral. On constate aussi que l'augmentation de l'expression de SPP2 que l'on peut observer dans les ovaires matures de S. chacoense suite à la pollinisation et la fécondation, n'est pas le résultat du développement floral normal des fleurs. De plus, la période durant laquelle l'expression de SPP2 est maximale, correspond en fait à la période de réceptivité de la fleur à la fécondation. Finalement, le patron d'expression de SPP2 coïncide avec la zone d'abcission du style. Le patron d'expression de SPP2 nous laisse penser que cette enzyme pourrait être impliquée dans la voie de biosynthèse de métabolites secondaires pouvant jouer un rôle dans le guidage et la croissance des tubes polliniques vers les ovules. Aussi, la forme que prend l'expression des ARNm de SPP2 au niveau de la base du style, nous laisse penser que SPP2 pourrait également jouer un rôle de marqueur cellulaire entre le style et l'ovaire. Finalement, on sait que SPP2 est principalement exprimée au niveau du pistil, des feuilles et des fruits, et que son expression coïncide (dans les fleurs) avec la période pendant laquelle les fleurs de S. chacoense sont réceptives à la pollinisation et à la fécondation, alors que sa diminution coïncide avec la période à partir de laquelle la fécondation ne peut plus avoir lieu chez les fleurs. Tout ceci nous laisse penser que notre désoxygénase (présente de façon constitutive et induite par la pollinisation, la fécondation ou la blessure), pourrait être impliquée dans la voie de biosynthèse d'un métabolite secondaire tels qu'un alcaloïde possédant des propriétés anti-microbiennes ou encore, anti-herbivores.

Previous studies of genes associated with retroviral-induced neoplasia have provided the foundation for much of our current knowledge of both tumor suppressor and oncogenes, and have contributed to our understanding of both gene function and malignant transformation. The study of Friend virus-induced erythroleukemia, a well-studied example of multistage malignancy, has led to the identification of several oncogenes, including the Ets transcription factor, fli-1. Fli-1 plays a vital role in hematopoiesis, and vasculogenesis through the transcriptional regulation of its target genes, some of which are critical for the control of cellular proliferation, differentiation, and survival. The aberrant regulation of Fli-1 is associated with a number of cancers and human diseases, including erythroleukemia, Ewing’s sarcoma, lupus, and Jacobsen or Paris Trousseau syndrome. The essential goal set out to be achieved by the research presented herein is to establish a better understanding of both the oncogenic and developmental roles of Fli-1 by investigating the molecular basis by which its deregulated expression leads to fundamental aberration in the fine balance between proliferation and differentiation.

One percent of children in North America may be affected by fetal alcohol spectrum disorder (FASD). FASD remains difficult to diagnose because confirmation of maternal alcohol use is a diagnostic criterion, and women consuming alcohol during pregnancy are reluctant to divulge this information for fear of stigmatization and losing custody of the child. Consequently, using a biomarker to assess alcohol exposure would provide a tremendous advantage. Recently, the measurement of fatty acid ethyl ester (FAEE) in hair has provided a powerful tool for assessing alcohol exposure. My thesis fills a translational gap of research between the development of the FAEE hair test and its application in the context of FASD. The guinea pig has been a critical model for FASD research, in which FAEE hair analysis has previously distinguished ethanol-exposed dams/offspring from controls. My first study, reports a positive dose-concentration relationship between alcohol exposure and hair FAEE, in the human, and the guinea pig. Humans also displayed over an order of magnitude higher FAEE incorporation per equivalent alcohol exporsure, suggesting that the test will be a sensitive clinical marker of fetal alcohol exposure. My second study utilized multi-coloured rats to investigate the potential of a hair-colour bias, as has been reported for other clinical hair assays; no evidence of bias is reported here. My third study is the first to examine the clinical use of the FAEE hair test in parents at high risk of having children with FASD. Over one third of parents tested positive for excessive alcohol use. Parents were investigated by social workers working for child protection services, and my fourth study reports that hair FAEE results agree with social worker reports. Individuals highly suspected of abusing alcohol were at a significantly greater risk of testing positive, whereas individuals tested based on other reasons (such as to cover all bases) were negatively associated with testing positive. The last study of my thesis, confirmed an association between alcohol and drug use by parents at high risk for having children with FASD, posing an added risk to children. This work helps bridge a gap in translational research, suggesting that the FAEE hair test has potential for use in FASD diagnosis and research.