published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

A digoxigenin-labeled nonradioactive detection system was used to screen a zebrafish cDNA library for p53-like and ras-like genes. One clone was isolated and identified as an incomplete p53-like gene. The insert size of this clone is 1777 bp, which encodes part of evolutionarily conserved region II and all of regions III, IV, and V. A magnetically enriched whole zebrafish cDNA library was constructed to enhance possible recovery of ras-like genes in zebrafish. One clone, termed Zras-Bl, carried an insert of 2592 bp with an open reading frame encoding a 188 amino acid residue ras p21 protein. Based on total protein sequence, this expressed zebrafish ras p21 is most closely related to human N-ras (91% homology), with lesser homology to Ha-ras (84%) and Ki-ras (85%). Preliminary partial sequence data obtained by genomic and reverase transcriptasepolymerase chain reaction (RT-PCR) screening indicate the presence of at least one additional expressed ras gene in zebrafish. The tumorigenicity and Ki-ras mutational properties of dietary 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DBP) were compared in rainbow trout. Both chemicals elicited predominantly 12(1)G->A and 12(2)G->T mutations in trout liver tumors. Two {12(1)G->T and 12(2)G->T} and one {12(1)G->A and 12(2)G->T} double mutation were also observed in DBP livers tumors, but not in DMBA liver tumors. Some stomach tumors from both chemicals exhibited so much DNA degradation that routine PCR amplification was not possible. Among sixteen DMBA stomach tumors with intact DNA, no Ki-ras mutations were found. Of sixteen DBP stomach tumors examined, one had 12(1)G->A and two had 13(1)G->C mutations. The observed G->T transversions are compatible with apurinic mutagenesis driven by unstable DNA adducts arising from one-electron oxidation, but this is not true for the major G->A transitions or G->C transversions and rare double mutations found in this study. The low sensitivity of direct sequencing may limit the frequency of ras mutant detection in this study. / Graduation date: 1996

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Leung Chun-on, Paul. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 149-167). / Content Page / Acknowledgments --- p.7 / Chapter / Chapter Chapter1 --- Introduction --- p.8 / Chapter Chapter2 --- Literature Review --- p.13 / Chapter 2.1 --- Anatomy of the cervix --- p.13 / Chapter 2.2 --- Classification --- p.14 / Chapter 2.2.1 --- Cervical intraepithelial neoplasia (CIN) --- p.14 / Chapter 2.2.2 --- Cervical cancer --- p.17 / Chapter 2.2.3 --- Incidence and screening --- p.21 / Chapter 2.2.4 --- Etiology / Chapter 2.2.4.1 --- Sexual and reproductive factors --- p.23 / Chapter 2.2.4.2 --- Smoking as a risk factor --- p.23 / Chapter 2.2.4.3 --- Male partner contribution --- p.24 / Chapter 2.2.4.4 --- Human papillomaviruses and cervical cancer --- p.24 / Chapter 2.2.4.5 --- Oral contraceptive pills --- p.27 / Chapter 2.2.4.6 --- Oncogenes and tumour suppresser genes --- p.28 / Chapter 2.2.4.7 --- Oncogenes and cervical cancer --- p.35 / Chapter 2.3 --- Immunohistochemical technique in cancer study / Chapter 2.3.1 --- Principle of immunostaining --- p.39 / Chapter 2.3.2 --- Fixation --- p.40 / Chapter 2.3.3 --- Section preparation --- p.41 / Chapter 2.3.4 --- The choice of antibodies --- p.41 / Chapter 2.3.5 --- Enzyme labels --- p.42 / Chapter 2.3.6 --- Blocking endogenous enzymes --- p.43 / Chapter 2.3.7 --- Blocking background staining --- p.43 / Chapter 2.3.8 --- Dilution preparation --- p.44 / Chapter 2.3.9 --- The Avidin-Biotin technique --- p.44 / Chapter 2.3.10 --- Control --- p.47 / Chapter 2.3.11 --- Antigen retrieval --- p.47 / Chapter 2.3.12 --- Cell counting and scoring --- p.49 / Chapter 2.4 --- The application of Polymerase Chain Reaction Single-Strand Conformation Polymorphism(PCR-SSCP) in cancer study --- p.52 / Chapter Chapter3 --- Materials and Methods --- p.56 / Chapter 3.1 --- Materials --- p.56 / Chapter 3.2 --- Methods --- p.61 / Chapter 3.2.1 --- Specimens collection --- p.61 / Chapter 3.2.2 --- Antibodies preparation --- p.63 / Chapter 3.2.3 --- Immunohistochemical staining and antigen retrieval procedures --- p.63 / Chapter 3.2.4 --- Cell counting and scoring --- p.68 / Chapter 3.2.5 --- PCR-SSCP analysis for myc gene mutation --- p.70 / Chapter 3.2.5.1 --- DNA extraction --- p.70 / Chapter 3.2.5.2 --- PCR --- p.72 / Chapter 3.2.5.3 --- Preparing the single strand DNA --- p.73 / Chapter 3.2.5.4 --- Electrophoresis --- p.73 / Chapter 3.2.5.5 --- Gel drying and scanning --- p.77 / Chapter 3.2.6 --- Statistical analysis --- p.77 / Chapter Chapter 4 --- Result --- p.78 / Chapter Chapter 5 --- Discussion --- p.126 / Chapter Chapter 6 --- Conclusions --- p.144 / Reference --- p.148

There is growing evidence that RNAs compete for binding and regulation by a finite pool of microRNAs (miRs), thus regulating each other through a competing endogenous RNA (ceRNA) mechanism. My dissertation work focused on systematically studying ceRNA interactions in cancer by reverse-engineering context-specific miR-RNA interactions and ceRNA regulatory interactions across multiple tumor types and study the effects of these interactions in cancer. I attempted to use ceRNA interactions to explain how genetic and epigenetic alterations are propagated to target established drivers of tumorigenesis. Using bioinformatics analysis of primary tumor samples and experimental validation in cell lines, I have investigated the roles that mRNAs and noncoding RNAs can play in tumorigenesis via ceRNA interactions. Specifically, I studied how RNAs target tumor-suppressors and oncogenes as ceRNAs, and attempted to accounting for some of the missing genomic variability in tumors.