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dc.contributor.authorLee, Michael
dc.date.accessioned2020-02-14T03:09:10Z
dc.date.available2020-02-14T03:09:10Z
dc.date.submitted2019
dc.identifier.urihttps://hdl.handle.net/2123/21844
dc.description.abstractTelomeres are regions of repetitive DNA at the ends of human chromosomes that function to maintan the integrity of the genome. Telomere attrition is associated with celluar ageing, whilst telomere maintenance is a prerequisite for replicative immortality in cancer. There are two telomere maintenance mechanisms (TMM) that cancer cells can utilize, the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These two mechanisms synthesise telomeres in very distinct ways leading to differences in their telomere sequence composition and length. The molecular pathways involved with the selective activation of one TMM over the other remain unclear. In the last decade, whole genome sequencing (WGS) has proven to be an invaluable tool for the study of cancer, leading to the discovery of novel gene mutations that either drive the disease or confer an increased risk of developing it. The utility of this technique has led to the creation of vast cancer WGS data resources, in particular The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), that are available for cancer researchers worldwide to use. This provides an excellent resource from which we can better understand and associate genetic markers and telomere sequence content across cancers, as well as between tumours that utilise the telomere maintenance mechanisms telomerase and ALT. In order to utilise these available datasets, we require a WGS‐based approach to determine the TMM status of a tumour, as experimental validation requires obtaining cellular material. We propose that differences exist in telomere sequence composition and length between ALT and telomerase cancers that can be used to determine the TMM status of a tumour from WGS data. In this thesis, we first compared a range of WGS‐based telomere content measurement tools against the lab‐based technique q‐PCR, in order to assess their accuracy in quantitating telomere content, whilst simultaneously enriching for variant telomeric sequences. We then applied the best of these tools to two experimentally validated tumour datasets, pancreatic neuroendocrine tumours and melanomas, in order to directly analyse and compare the telomere sequence content between tumours that utilise ALT and those that do not. Finally, we exploited the differences in telomere sequence content in order to develop a classifier capable of determining the ALT status of a tumour from WGS data, and applied it to WGS data from 821 TCGA tumours, to identify the molecular pathways associated with the activation of ALT. We were able to demonstate that WGS‐based telomere content measurement tools perform well, producing comparable results to q‐PCR, with R2 = 0.9516. We have developed a methodology for the accuracte quantification of variant repeats within telomere sequences, identifying a number of differences in telomere sequence composition between ALT positive (+ve) and ALT negative (‐ve) tumours. We have demonstated the utility of this methodology to develop a WGS‐based classifier capable of predicting the ALT status of a tumour with 91.6% accuracy. Analysis of pathway mutations that were under‐represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways were involved in the survival of ALT tumours. Overall, we have demonstrated the capability and utility of WGS to investigate telomere sequence content, shown how telomere sequence content can be used to stratify cancers by TMM, and applied this to cancer WGS datasets to elucidate the genetic changes that associate with each TMM. This thesis provides a useful resource for future studies seeking to investigate the role of telomere sequence content in disease and overall health.en_AU
dc.publisherUniversity of Sydneyen_AU
dc.publisherFaculty of Medicine and Healthen_AU
dc.publisherChildren's Medical Research Instituteen_AU
dc.rightsThe author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.en_AU
dc.subjectTelomeresen_AU
dc.subjectTelomeraseen_AU
dc.subjectALTen_AU
dc.subjectWGSen_AU
dc.subjectCanceren_AU
dc.titleNext Generation Sequencing Strategies to Investigate Telomeres in Canceren_AU
dc.typePhD Doctorateen_AU
dc.type.pubtypeDoctor of Philosophy Ph.D.en_AU
dc.description.disclaimerAccess is restricted to staff and students of the University of Sydney . UniKey credentials are required. Non university access may be obtained by visiting the University of Sydney Library.en_AU


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