Introduction: The wide-ranging prognostic implications of a breast cancer diagnosis highlight the need to better enable women to make informed decisions regarding screening and treatment options. As several cancer susceptibility syndromes have been linked to germline mutations resulting in defective DNA repair, including the predisposition to breast cancer due to BRCA1 and BRCA2 mutations, more subtle defects in DNA repair capacity may contribute to the components driving differential susceptibility within the general population. Hence, understanding the role of DNA repair capacity in breast cancer onset may aid in the development of a more comprehensive risk profile, thereby furthering the effort to target relevant populations for early screening. In the studies undertaken for this dissertation, we employed various methodologies capturing endpoints across different repair pathways detectable in blood to both further elucidate the etiologic basis of breast cancer development and leverage the information into the potential development of a screening biomarker. Methods: For the phenotypic assessment of nucleotide excision repair (NER) capacity, we developed an ELISA-based method to determine benzo(a)pyrene diolepoxide (BPDE)-DNA adduct capacity in lymphoblastoid cell lines. Gene expression levels were assessed with pre-designed Taqman kits in RNA-derived cDNAs from mononuclear cells using a real-time PCR-based platform. Methylation analysis was conducted with in-house designed assays on bisulfite-converted DNA from mononuclear cells using a pyrosequencing platform. Finally, single nucleotide polymorphisms (SNP) genotyping was assessed in DNA derived from white blood cells with pre-designed Taqman SNP genotyping assays using a real-time PCR-based platform. All studies were conducted in sister-sets enrolled in the New York site within the Breast Cancer Family Registry and all statistical analysis was conducted using the R Foundation for Statistical Computing (2011). Results: We did not detect an association between the ELISA-based phenotypic assessment of NER capacity in the lymphoblastoid cells lines of the sister-sets (n=246, 114 sister-sets) and breast cancer risk (OR = 1.0, 95%CI=0.95, 1.04). Furthermore, we did not observe a correlation with previously determined NER capacity in the same population using an immunohistochemical-based method (r= -0.01, p=0.86). In our gene expression study (n=569, 218 sister-sets), women in the lowest tertile of ATM expression had a heightened risk of breast cancer compared to women in the highest tertile of expression, adjusted for age at blood draw and smoking status (OR=2.12, 95%CI=1.09, 4.12). This association was largely restricted to women with an extended family history of breast cancer (pinteraction = 0.06). Additionally, women in the lowest tertile of MSH2 expression also had a heightened risk of breast cancer compared to women in the highest tertile of expression, adjusted for age at blood draw and smoking status (OR=2.75, 95%CI=1.31, 5.79). The association observed between reductions in ATM expression level and breast cancer risk was lost upon incorporating previously determined end-joining capacity of EcoRI-generated sticky end substrates (OR=1.28, 95%CI=0.15, 11.2) and HincII-generated blunt end substrates (OR=1.55, 95%CI=0.15, 15.5) into the model, suggesting that the impact on risk due to reductions in ATM expression maybe partially driven by the reduction in double strand break repair capacity. In our study investigating breast cancer risk due to the impact of epigenetic modulation on DNA repair gene activity (n=569, 218 sister-sets), no association with risk was observed due to differential promoter methylation levels of BRCA1 (OR=1.09, 95%CI=0.98, 1.20), MLH1 (OR=1.19, 95%CI=0.91, 1.55) or MSH2 (OR=0.89, 95%CI=0.48, 1.64). Furthermore, no correlation between BRCA1 and expression (r=-0.05, p=0.39) or MSH2 methylation and expression (r=-0.04, p=0.39) was observed. Finally, our mismatch repair genotyping study (n=714, 313 sister-sets) indicated an association between the variant MutY_rs3219489 (OR=2.23, 95%CI=1.10, 4.52) and breast cancer risk, as well as a borderline association with risk due to the variant MSH2_rs2303428 (OR=1.71, 95%CI=0.99, 2.95). Furthermore, a protective effect was observed due to the variant MLH3_rs175080, restricted to women without an extended family history of breast cancer (pinteraction = 0.03). Conclusion: These studies suggest that the deregulation of targets spanning various DNA repair pathways contribute to the risk of familial breast cancer.

Liu, Yi. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 154-175). / Abstracts also in Chinese. / Title from PDF title page (viewed on 25, October, 2016).

The pituitary hormone prolactin (Prl) is essential for alveolar morphogenesis and plays a role in breast carcinogenesis, however the mechanism that underlies these actions remains to be defined. Alterations in serum Prl provide the primary endocrine signal regulating developmental events in the mammary gland in sexually mature mammals. Prl production and post-translational phosphorylation by the pituitary is regulated by the neuropeptide Galanin (Gal) in response to hypothalamic signals integrating neuronal and endocrine inputs. Prl exerts its effects on the mammary epithelium in two ways, indirectly by modulation of the systemic hormonal environment, for example the release of progesterone from the corpus luteum, and directly by binding to Prl receptors (Prlr) within the mammary epithelium. Prl binding to Prlr initiates signalling predominantly via activation of the Jak2/Stat5 pathway, leading to altered patterns of gene transcription. One of these target genes is the ets transcription factor Elf5, which is required by the epithelium for alveolar morphogenesis. This thesis aims to further our understanding of the mechanisms by which prolactin exerts its influence on the mammary gland during alveolar morphogenesis and carcinogenesis. Transcript profiling revealed a lactation signature of 35 genes in Prlr+/- mice, Gal-/- mice and mice treated with a Prl mutant (S179D) that mimics phosphorylated Prl. We discovered that the majority of changes in gene expression were produced by prolactin rather than by Gal. The action of Gal was predominantly via modulation of Prl phosphorylation and release, as its effects were very similar to that of S179D. Knockout of Elf5 phenocopied knockout of Prlr, resulting in failure of alveolar morphogenesis and reduced expression of milk and lipid synthesis genes. Forced Elf5 expression at puberty resulted in aberrant differentiation of the terminal end buds and milk protein synthesis during ductal morphogenesis. Re-expression of Elf5 in Prlr-/- mammary epithelial cells completely rescued alveolar morphogenesis. These observations indicate that Elf5 is a master regulator of alveolar morphogenesis downstream of the Prlr. Loss of mammary epithelial Prlr resulted in reduced proliferation of low-grade neoplastic lesions resulting in increased tumour latency in the C3(1)/SV40T model of mammary carcinogenesis. There was no change in the growth rate, proliferation nor the morphology of tumours in Prlr-/-/C3(1)/SV40T transplants, thus Prl acts early in carcinogenesis to drive the proliferation of pre-invasive lesions resulting in faster progression to cancer.

RNA-Sequencing (RNA-Seq) has become one of the most widely used techniques to interrogate the transcriptome of an organism since the advent of next generation sequencing technologies [1]. A plethora of tools have been developed to analyze and visualize the transcriptome data from RNA-Seq experiments, solving the problem of mapping reads back to the host organism's genome [2] [3]. This allows for analysis of most reads produced by the experiments, but these tools typically discard reads that do not match well with the reference genome. This additional information could reveal important insight into the experiment and possible contributing factors to the condition under consideration. We introduce PARSES, a pipeline constructed from existing sequence analysis tools, which allows the user to interrogate RNA-Sequencing experiments for possible biological contamination or the presence of exogenous sequences that may shed light on other factors influencing an organism's condition.

The function and role of PAK6, serine/threonone kinase, in cancer progressionhas not yet been clearly identified. Several studies reveal that PAK6 may participate in key changes contributing to cancer progression such as cell survival, cell motility, and invasiveness. Basedon the membrane localization of PAK6 in prostate and breast cancer cells,we speculated that PAK6 plays a rolein cancer progression cells by localizing on the membrane and modifying proteins linked to motility and proliferation. We isolated the raft domain of breast cancer cells expressing either wild type (WT), constitutively active (SN), or kinase dead PAK6 (KM) and found that PAK6 is a membrane associated kinase which translocates from the plasma membrane to the cytosol when activated. The downstream effects of PAK6 are unknown ; however, results from cell proliferation assays suggest a growth regulatory mechanism. / by JoAnn Roberts. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Yau, Ka Long. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Ng Wah. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-113). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENTS --- p.VII / LIST OF TABLES --- p.X / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Etiological Factors of Hepatocellualr Carcinoma (HCC) --- p.4 / Chapter 1.2.1 --- Dietary Aflatoxins --- p.4 / Chapter 1.2.2 --- Liver Cirrhosis --- p.5 / Chapter 1.2.3 --- Alcohol Abuse --- p.6 / Chapter 1.2.4 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3 --- Literature Review on the Investigations of HBV Integrants in HCC --- p.16 / Chapter 1.3.1 --- Affected Host Junctions --- p.17 / Chapter 1.3.2 --- Viral Junctions --- p.18 / Chapter 1.4 --- Restriction Site Polymerase Chain Reaction (RS-PCR) --- p.19 / Chapter 1.5 --- Aims of Thesis --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Chemicals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Cell Cultures --- p.24 / Chapter 2.1.4 --- Nucleic Acids --- p.24 / Chapter 2.1.5 --- Enzymes --- p.25 / Chapter 2.1.6 --- Equipment --- p.25 / Chapter 2.1.7 --- Software and Web Resources --- p.26 / Chapter 2.2 --- Methods --- p.27 / Chapter 2.2.1 --- DNA Extraction --- p.27 / Chapter 2.2.2 --- RS-PCR --- p.31 / Chapter 2.2.3 --- Sequencing --- p.37 / Chapter 2.2.4 --- Spectral Karyotyping (SKY) --- p.38 / Chapter 2.2.5 --- Fluorescence In situ hybridization --- p.39 / Chapter Chapter 3 --- Investigation of HBV Integration Sites in HCC Cell lines --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell Lines --- p.47 / Chapter 3.2.2 --- RS-PCR --- p.47 / Chapter 3.2.3 --- Spectral Karyotyping --- p.48 / Chapter 3.2.4 --- Tyramide Signal Amplification for HBV in FISH Analysis --- p.48 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Identification of HBV Integration Sites in Cell Lines --- p.51 / Chapter 3.3.2 --- Evaluation of RSO Primer Efficiency --- p.52 / Chapter 3.3.3 --- SKY and FISH Analysis --- p.53 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- HBV Insertions in HCC Cell Lines --- p.64 / Chapter 3.4.2 --- Efficacy of RSO Primers --- p.65 / Chapter 3.4.3 --- Investigation of HBV Integration on Chromosomal Rearrangement --- p.65 / Chapter Chapter 4 --- Investigation of Hepatitis B Virus Integration Sites in Primary HCC --- p.67 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Materials and Methods --- p.69 / Chapter 4.2.1 --- Patients --- p.69 / Chapter 4.2.2 --- RS-PCR --- p.70 / Chapter 4.3 --- Results --- p.72 / Chapter 4.3.1 --- HBV Integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.72 / Chapter 4.4 --- Discussion --- p.88 / Chapter 4.4.1 --- HBV integration Sites in Primary HCC Tumors and Adjacent Non- malignant Liver --- p.88 / Chapter 4.4.2 --- Summary on HBV Integrants Identified --- p.91 / Chapter Chapter 5 --- Proposed Future Studies --- p.98 / Chapter 5.1 --- Correlation of Structural Aberrations with HBV Integrations --- p.99 / Chapter 5.2 --- Transcriptional Expression Study on the Genes Interrupted by or Located near the Virus Host Junctions --- p.100 / Chapter Chapter 6 --- References --- p.101

As a master regulator of the epithelial state, p63 is a family member of the well-known tumor suppressor p53. It has previously been connected to a cancer-associated process, epithelial-to-mesenchymal transition (EMT), and here we find that it can be regulated by oncogenes involved in breast tumorigenesis. Specifically, activated forms of PIK3CA and H-RAS are able to strongly repress expression of ∆Np63α, which is the major p63 isoform in epithelial cells. In mammary epithelial lines, this oncogene downregulation occurs at the transcriptional level, and complete repression occurs over the course of several days. As p63 is repressed, the cells undergo EMT and acquire the ability to invade individually through a 3D collagen matrix. Strikingly, even when p63 is suppressed but no oncogene action is present, these cells undergo a mesenchymal shift, suggesting the importance of this gene in maintaining the epithelial state. Furthermore, it is particularly interesting that p63 protein and RNA levels are often low in breast tumors. By connecting H-RAS and PIK3CA signaling to p63, it is hypothesized that such oncogene suppression could account for tumor progression in cases where p63 levels are low. Here, it is proposed that p63 acts in a tumor-suppressive manner, although it can be overcome by oncogenes leading to changes in differentiation state and migratory capability, therefore drastically affecting breast carcinogenesis.

Traditionally, cancer epidemiologists have investigated the causes of disease under the premise that patients with a certain site of disease can be treated as a single entity. Then risk factors associated with the disease are identified through case-control or cohort studies for the disease as a whole. However, with the rise of molecular and genomic profiling, in recent years biologic subtypes have increasingly been identified. Once subtypes are known, it is natural to ask the question of whether they share a common etiology, or in fact arise from distinct sets of risk factors, a concept known as etiologic heterogeneity. This dissertation seeks to evaluate methods for the study of etiologic heterogeneity in the context of cancer research and with a focus on methods for case-control studies. First, a number of existing regression-based methods for the study of etiologic heterogeneity in the context of pre-defined subtypes are compared using a data example and simulation studies. This work found that a standard polytomous logistic regression approach performs at least as well as more complex methods, and is easy to implement in standard software. Next, simulation studies investigate the statistical properties of an approach that combines the search for the most etiologically distinct subtype solution from high dimensional tumor marker data with estimation of risk factor effects. The method performs well when appropriate up-front selection of tumor markers is performed, even when there is confounding structure or high-dimensional noise. And finally, an application to a breast cancer case-control study demonstrates the usefulness of the novel clustering approach to identify a more risk heterogeneous class solution in breast cancer based on a panel of gene expression data and known risk factors.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is prevalent in Southeast Asia and China, where hepatitis B viral (HBV) infection is the main etiologic factor. Despite a high incidence of HCC developing in patients with HBV-induced liver cirrhosis, the molecular events underlying the malignant liver progression remain largely unclear. In an effort to characterize the genetic abnormalities involved in the HBV-related liver carcinogenesis, genome-wide exploration by metaphase comparative genomic hybridization (CGH) was performed on 100 cirrhotic HCC tumors that were derived from chronic hepatitis B carriers. CGH analysis indicated chromosomal instability in both early and advanced stage tumors where common genomic copy gains on 1q, 8q and 17q, and deletions on 4q, 8p, 13q, 16q and 17p found in both groups are suggestive of early events in hepatocarcinogenesis. Nevertheless, a combined univariate and multivariate statistical analyses highlighted for the first time preferential regional 3q26-q28, 7q21-q22 and 7q34-q36 gains in association with advanced stage HCC. The novel aberrant gains identified here thus formed basis for further mapping analysis for causative genes related to HCC progression in this thesis. / Near 50% of the advanced stage HCC manifested copy gains of chr 7q21-q22. High resolution mapping analysis by cDNA microarray-based CGH nominated 13 amplified candidates within the region 7q21-q22 Analysis on the mRNA expresson levels of these genes in a cohort of primary HCC compared to paired adjacent non-tumorous liver tissues by quantitative RT-PCR (qRT-PCR) indicated the up-regulation of the PFTK1 (PFTAIRE protein kinase 1) gene as the only candidate that demonstrated a close association with advanced metastatic tumors. The effects of PFTK1 on cell proliferation, migration and invasive phenotypes were further studied to substantiate its role in HCC progression. Upon gene suppression of PFTK1 in vitro by RNA interference (RNAi), a significant reduction in chemotactic migration, cellular invasion and an inhibition on cell motility were indicated, albeit cell proliferation remained unaffected. / Sub-cellular localization study of translated PFTK1 protein indicated protein localization in both the nucleus and cytoplasm. This has led to the further investigations of potential PFTK1 function at both the transcriptional and protein levels. (Abstract shortened by UMI.) / Sy Ming Hui. / "July 2005." / Advisers: Winnie Yeo; Nathalie Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 124-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.