The evolution of prostate cancer from normal epithelium via the preneoplastic lesion of high-grade prostatic intraepithelial neoplasia to invasive carcinoma is characterised by a number of particular genomic abnormalities that are predominantly generated in the preneoplastic phase. Whilst there are numerous candidates for the cause of these alterations, telomere dysfunction is thought to be a major contributor. Telomeres are the terminal ends of human chromosomes, and when dysfunctional can lead to break-fusion-bridge cycles and multi-polar mitoses that generate numerical and structural chromosomal instability. The results presented reinforce the association of telomere dysfunction with the generation of certain markers of genomic instability such as abnormalities of the arms of chromosome 8. Furthermore, this work clarifies that the TMPRSSS2-ERG aberrations are not telomere related phenomena and are associated with a genomic deletion in a proportion of cases. Similarly, the PTEN microdeletions did not appear to have an association with telomere attrition. A previously unrecognised association between the telomere length in various types of prostatic epithelia and adjacent stroma is defined, suggesting evidence of a micro-environmental field effect in the generation of prostatic neoplasia. Finally, when examined retrospectively, it appears that telomere attrition, both in the HPIN epithelium and the stroma has independent prognostic value in the diagnosis of prostate cancer after a previous diagnosis of HPIN. Taken together, the research presented suggests important avenues for further research to determine the nature of barriers to the evolution of prostatic carcinogenesis such as oncogene- and telomere-induced senescence that may be exploited for therapeutic gain. These understandings may also help tailor management for prostate cancer such as risk stratification for men with HPIN and the use of targeted agents such as AKT inhibitors and telomerase inhibitors. In more advanced disease, translational application of this work has enabled a clinical trial of cytarabine in the treatment of metastatic hormone refractory prostate cancer.

The evolution of prostate cancer from normal epithelium via the preneoplastic lesion of high-grade prostatic intraepithelial neoplasia to invasive carcinoma is characterised by a number of particular genomic abnormalities that are predominantly generated in the preneoplastic phase. Whilst there are numerous candidates for the cause of these alterations, telomere dysfunction is thought to be a major contributor. Telomeres are the terminal ends of human chromosomes, and when dysfunctional can lead to break-fusion-bridge cycles and multi-polar mitoses that generate numerical and structural chromosomal instability. The results presented reinforce the association of telomere dysfunction with the generation of certain markers of genomic instability such as abnormalities of the arms of chromosome 8. Furthermore, this work clarifies that the TMPRSSS2-ERG aberrations are not telomere related phenomena and are associated with a genomic deletion in a proportion of cases. Similarly, the PTEN microdeletions did not appear to have an association with telomere attrition. A previously unrecognised association between the telomere length in various types of prostatic epithelia and adjacent stroma is defined, suggesting evidence of a micro-environmental field effect in the generation of prostatic neoplasia. Finally, when examined retrospectively, it appears that telomere attrition, both in the HPIN epithelium and the stroma has independent prognostic value in the diagnosis of prostate cancer after a previous diagnosis of HPIN. Taken together, the research presented suggests important avenues for further research to determine the nature of barriers to the evolution of prostatic carcinogenesis such as oncogene- and telomere-induced senescence that may be exploited for therapeutic gain. These understandings may also help tailor management for prostate cancer such as risk stratification for men with HPIN and the use of targeted agents such as AKT inhibitors and telomerase inhibitors. In more advanced disease, translational application of this work has enabled a clinical trial of cytarabine in the treatment of metastatic hormone refractory prostate cancer.

Cancer is a disease that involves genomic instability, to which c-Myc contributes during its initiation and progression. Over 70% of all human cancers show deregulated levels of c-Myc protein. The term genomic instability refers to genetic and/or epigenetic changes that alter the normal organization and function of genes and chromosomes. Genomic instability is a hallmark of cancer and often is associated with cancer. Deregulated c-Myc expression generates genomic instability by initiating intra- and extrachromosomally locus-specific gene amplification, gene rearrangements and karyotypic instability that includes translocations, fusions, insertions and deletions. Out of the several outlined pathways by which deregulated levels of c-Myc can lead to genomic instability, the work described in this thesis focuses on three with direct relevance to tumorigenesis; gene amplification (increase in gene copy number), remodeling of the chromosomal and telomeric structures in the interphase nucleus and comparing the effect of Myc to that of Epstein Bar virus (EBV) infection in remodeling the nuclear structure that may lead to genomic instability.

Metastasis is the spread of cancer cells from the primary tumor to distant sites. It is the most dangerous attribute of cancer, and also the principle cause of cancerrelated morbidity and mortality. Metastasis suppressor genes are a group of genes that suppress tumor metastasis without significant effect on tumorigenicity. NM23 was the first identified metastasis suppressor gene, and loss of its expression is a frequent hallmark of metastatic growth in multiple cancers (e.g. melanoma, carcinomas of breast, stomach and liver). NM23-H1 possesses at least three enzymatic activities, including nucleoside diphosphate kinase (NDPK), histidine kinase (hisK), and a more recently described 3f-5f exonuclease (EXO). While the hisK has been shown to be linked to the suppression of cell motility, the NDPK has been reported to be unrelated to the suppression of metastatic potential indirectly. Relevance of EXO has not been addressed. Other known 3f-5f exonuclease are closely associated with DNA repair functions, suggesting NM23-H1 may suppress mutations required for metastasis. As a transcription factor, NM23 has been shown to modestly downregulate the transcription on PDGF-A chain, a growth factor oncogene, either alone or in association with another transcriptional factor, Pur@. At the same time, identification of NM23-H1 as a 3f-5fexonuclease suggests the role of NM23-H1 in DNA repair. Etoposide and cisplatin elicited nuclear translocation of H1 within 4 h in HeLa and HepG2 cells, seen as accumulation of H1 in small intranuclear foci, strongly suggesting the DNA repair function of H1. To investigate the enzymatic function contributing to metastasis suppressor activity of H1, complementation system was used by transfecting NM23-H1 with individually disrupted enzymatic function into 2 melanoma cell lines, 1205LU and WM793. Overexpression of H1 in 1205LU suppressed lung metastasis in vivo without effect on indices of transformation (e.g. proliferation, soft agar colonization). EXO- deficient H1 and NDPK-deficient H1 lost suppression of lung metastasis, while hisK-deficient H1 maintained suppressor activity. Consistent with the results in 1205LU cells, EXO-deficient H1 and NDPKdeficient H1 lost suppression of the progression of WM793 cells in protein-free medium, while WT and hisK-deficient H1 prevented the progression. Taken together, these data suggest that the NDPK and/or 3f-5fEXO activity of H1 inhibits the progression of premetastatic cells to the metastatic phenotype, possibly via a DNA repair function or other structural transactions with DNA.

No description available.

No description available.

Cancer is a disease that involves genomic instability, to which c-Myc contributes during its initiation and progression. Over 70% of all human cancers show deregulated levels of c-Myc protein. The term genomic instability refers to genetic and/or epigenetic changes that alter the normal organization and function of genes and chromosomes. Genomic instability is a hallmark of cancer and often is associated with cancer. Deregulated c-Myc expression generates genomic instability by initiating intra- and extrachromosomally locus-specific gene amplification, gene rearrangements and karyotypic instability that includes translocations, fusions, insertions and deletions. Out of the several outlined pathways by which deregulated levels of c-Myc can lead to genomic instability, the work described in this thesis focuses on three with direct relevance to tumorigenesis; gene amplification (increase in gene copy number), remodeling of the chromosomal and telomeric structures in the interphase nucleus and comparing the effect of Myc to that of Epstein Bar virus (EBV) infection in remodeling the nuclear structure that may lead to genomic instability.

Medicago sativa (Alfalfa) is an important agricultural plant for animal forage and nitrogen fixation, and has potential value in ligno-cellulosic energy production. In the quest to understand the plant, I generated a draft genome sequence of M. sativa via two reduced representation sequencing approaches: methylation-dependent filtration, and high CoT filtration. Libraries created from each approach were sequenced on an Illumina next-generation sequencing platform yielding approximately 2.5Gb of raw data. A combination of reference-based genome assembly approaches using the closely related species, Medicago truncatula as a reference, and de novo genome assembly approaches were performed to assemble the draft genome. The reference-based assembly generated 312,011 contigs with weighted median contig length (N50) of 247 bases, whereas de novo assembly produced 547,304 contigs with N50 of 275 bases. The creation of the M. sativa draft genome is vital for downstream functional analyses such as genome wide gene mining and gene expression profiling.

Medicago sativa (Alfalfa) is an important agricultural plant for animal forage and nitrogen fixation, and has potential value in ligno-cellulosic energy production. In the quest to understand the plant, I generated a draft genome sequence of M. sativa via two reduced representation sequencing approaches: methylation-dependent filtration, and high CoT filtration. Libraries created from each approach were sequenced on an Illumina next-generation sequencing platform yielding approximately 2.5Gb of raw data. A combination of reference-based genome assembly approaches using the closely related species, Medicago truncatula as a reference, and de novo genome assembly approaches were performed to assemble the draft genome. The reference-based assembly generated 312,011 contigs with weighted median contig length (N50) of 247 bases, whereas de novo assembly produced 547,304 contigs with N50 of 275 bases. The creation of the M. sativa draft genome is vital for downstream functional analyses such as genome wide gene mining and gene expression profiling.

High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome. Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance. To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks. In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.