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Luminescent transition metal complexes have arisen as viable alternatives to organic dyes for sensory applications due to their notable advantages. This thesis aimed to synthesize different kinds of iridium(III) complexes, explore their interactions with DNAs and investigate their application for the construction of oligonucleotide-based sensing platforms for important biomarkers. A series of iridium(III) complexes incorporating a variety of C^N and N^N donor ligands were synthesized and were demonstrated to possess G-quadruplex-selective binding properties via emission titration, UV/vis titration, fluorescence resonance energy transfer melting and G-quadruplex fluorescent intercalator displacement experiments. These G-quadruplex-selective iridium(III) complexes were utilized as signal transducers to monitor the conformational changes of oligonucleotides in oligonucleotide-based luminescent detection platforms for protein tyrosine kinase-7, interferon-gamma, sialic acidbinding immunoglobulin-likelectin-5 and thymine DNA glycosylase. And these designed platforms could work effectively in the diluted cell extract as the results in this thesis indicated.

Wong, Man Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-146). / Abstracts in English and Chinese. / Abstract --- p.iii / 摘要 --- p.v / Acknowledgments --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / List of appendix --- p.xv / Abbreviations --- p.xvi / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Background of Lentinula edodes --- p.1 / Chapter 1.2 --- Life cycle and mating system of Lentinula edodes --- p.1 / Chapter 1.3 --- Breeding and strain improvement --- p.5 / Chapter 1.4 --- Application of molecular markers --- p.6 / Chapter 1.5 --- Objectives and long term significance --- p.9 / Chapter Chapter 2 --- Genome survey sequencing and preliminary analysis --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.1.1 --- Genome sequencing of basidiomycetes --- p.11 / Chapter 2.1.2 --- Polymerase chain reaction-single strand conformational polymorphism --- p.12 / Chapter 2.1.3 --- Sequencing chemistry --- p.13 / Chapter 2.2 --- Materials and methods --- p.15 / Chapter 2.2.1 --- Strain and DNA extraction --- p.15 / Chapter 2.2.2 --- PCR-SSCP analysis --- p.15 / Chapter 2.2.3 --- Shotgun sequencing and sequence assembly --- p.17 / Chapter 2.2.4 --- Comparison with 5 basidiomycetes --- p.17 / Chapter 2.3 --- Results --- p.19 / Chapter 2.3.1 --- PCR-SSCP --- p.19 / Chapter 2.3.2 --- Shotgun sequencing and assembly --- p.21 / Chapter 2.3.3 --- Comparison with 5 basidiomycetes --- p.22 / Chapter 2.4 --- Discussion --- p.30 / Chapter Chapter 3 --- Cloning of A mating-type locus of Lentinula edodes --- p.33 / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Materials and methods --- p.35 / Chapter 3.2.1 --- Genome sequencing and assembly --- p.35 / Chapter 3.2.2 --- Genomic screening of A-mating type genes --- p.35 / Chapter 3.2.3 --- Gap filling and sequence confirmation --- p.36 / Chapter 3.2.4 --- Alignment of overlapping sequences to give contiguous sequence --- p.37 / Chapter 3.2.5 --- Open reading frame prediction and protein homolog search --- p.37 / Chapter 3.2.6 --- Conserved domain search --- p.37 / Chapter 3.2.7 --- Testing for polymorphism --- p.38 / Chapter 3.3 --- Results --- p.39 / Chapter 3.3.1 --- Genomic screening of A-mating type genes --- p.39 / Chapter 3.3.2 --- Gap filling and sequence confirmation --- p.45 / Chapter 3.3.3 --- Protein homologs and putative protein domains --- p.48 / Chapter 3.3.4 --- Polymorphism of A mating-type genes --- p.53 / Chapter 3.4 --- Discussion --- p.55 / Chapter 3.4.1 --- Genome mining of the A mating-type locus of L. edodes --- p.55 / Chapter 3.4.2 --- Genomic structure of the A mating-type region in L. edodes --- p.55 / Chapter 3.4.3 --- Functional protein domains in A mating-type genes --- p.56 / Chapter 3.4.4 --- Polymorphism of A mating- type locus --- p.58 / Chapter 3.4.5 --- Conclusion and future perspectives --- p.59 / Chapter Chapter 4 --- Simple sequence repeat (SSR) markers development --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Materials and methods --- p.62 / Chapter 4.2.1 --- Strains --- p.62 / Chapter 4.2.2 --- Datasets for SSRs mining --- p.63 / Chapter 4.2.3 --- in silico detection of SSR motifs and primer design --- p.63 / Chapter 4.2.4 --- SSR amplification --- p.64 / Chapter 4.2.5 --- Cloning and sequencing of PCR products --- p.64 / Chapter 4.2.6 --- Testing for polymorphism --- p.65 / Chapter 4.3 --- Results --- p.66 / Chapter 4.3.1 --- in silico detection of SSR motifs and primer design --- p.66 / Chapter 4.3.2 --- SSR amplification --- p.69 / Chapter 4.3.3 --- SSR polymorphism --- p.83 / Chapter 4.4 --- Discussion --- p.86 / Chapter 4.4.1 --- Efficiency of in silico detection of SSR motifs and primer design --- p.86 / Chapter 4.4.2 --- Effectiveness and polymorphism of SSR primer pairs --- p.89 / Chapter 4.4.3 --- Conclusion and future perspectives --- p.90 / Chapter Chapter 5 --- High-throughput sequencing of AP-PCR amplicons for SCAR markers development and phylogenetic analysis --- p.91 / Chapter 5.1 --- Introduction --- p.91 / Chapter 5.2 --- Materials and methods --- p.94 / Chapter 5.2.1 --- Strains --- p.94 / Chapter 5.2.2 --- AP-PCR analysis --- p.94 / Chapter 5.2.3 --- Re-amplification of AP-PCR amplicons --- p.96 / Chapter 5.2.4 --- GS-FLX sequencing --- p.96 / Chapter 5.2.5 --- Strain-specific sequences identification --- p.97 / Chapter 5.2.6 --- SCAR marker analysis --- p.97 / Chapter 5.2.7 --- Phylogenetic analysis --- p.99 / Chapter 5.3 --- Results --- p.100 / Chapter 5.3.1 --- AP-PCR analysis --- p.100 / Chapter 5.3.2 --- Re-amplification of AP-PCR amplicons --- p.100 / Chapter 5.3.3 --- GS-FLX sequencing and strain-specific sequence identification --- p.103 / Chapter 5.3.4 --- SCAR marker analysis --- p.106 / Chapter 5.3.5 --- Phylogenetic analysis --- p.108 / Chapter 5.4 --- Discussion --- p.111 / Chapter 5.4.1 --- Sensitivity of band detection --- p.111 / Chapter 5.4.2 --- SCAR marker development --- p.111 / Chapter 5.4.3 --- Phylogenetic analysis --- p.113 / Chapter 5.4.4 --- Conclusion --- p.114 / Chapter Chapter 6 --- Concluding remarks --- p.115 / Chapter 6.1 --- Project summary --- p.115 / Chapter 6.2 --- Future perspectives --- p.119 / Appendix --- p.121 / References --- p.141

Conservation biologists are interested in maintaining genetic variation in small populations, with a view to maintaining fitness and the ability of the species to adapt to changing environmental conditions. The most important type of genetic variation is therefore that which affects fitness and reproduction, and is therefore subject to natural selection. Such fitness traits are often quantitative, i.e. are the result of a suite of loci, and are continuously variable. Microsatellite markers are a popular method of determining the level of variation present in a species??? genome. The assumption is made that microsatellites, which are neutral markers, behave in the same manner as quantitative traits. If this assumption were proved incorrect, then the use of neutral markers in conservation monitoring would have to be re-evaluated. In this study, experiments have been conducted using Drosophila melanogaster to test the assumption that variation in quantitative traits under stabilising selection declines at the same rate as heterozygosity in microsatellite markers, during a population bottleneck. Experimental population bottlenecks were of two effective population sizes (Ne), Ne=2 for one generation and Ne=60 for 35 generations. Based on the effective population size, we expected both types of bottlenecks to lose 25% of neutral genetic variation. Ten replicates of each bottleneck were maintained, along with four large control populations with Ne=320. In each population, heterozygosity (He) for eight microsatellite loci was compared with the heritability and additive genetic variance of two quantitative traits subject to balancing selection: fecundity and sternopleural bristle number. Microsatellite heterozygosity decreased in accordance with neutral predictions, whereas additive genetic variation in quantitative traits altered more than expected in both large and in bottlenecked populations relative to the initial sampling values, indicating that variation in quantitative traits was not being lost at the same rate as predicted by neutral theory. For most traits, the changes in additive genetic variance were congruent in all populations, large or bottlenecked. This congruence suggests that a common process was affecting all populations, such as adaptation. A mite infestation in early generations is a possible source of selective pressure. When bottlenecked populations were compared to the contemporaneous large populations (Ne = 320), the additive genetic variance of most traits was seen to have been lost in accordance with predictions from the loss of microsatellite heterozygosity. Loss of variation in microsatellites can thus be used to predict the loss of variation in quantitative traits due to bottlenecks, but not to predict the potentially much larger changes due to other processes such as adaptation. The effects of concurrent environmental stress and reduced population size were also evaluated. Endangered populations are often subject to environmental stress in addition to reduced population size, but the effect of stress on the additive genetic variance of fitness traits in organisms undergoing population bottlenecks is unknown. If the presence of stress alters the level of additive genetic variance in fitness traits, the viability of such populations could be substantially affected. The loss of microsatellite heterozygosity was not affected by the presence of a stress agent during a bottleneck. I found some significant effects of stress on the additive genetic variance of sternopleural bristles and fecundity; there was also a significant interaction between stress and the response to directional selection in sternopleural bristles. There was also an increase in the coefficient of variation of VA for sternopleural bristles. Stress may therefore affect the manner in which populations respond to selective pressures.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 121-150). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 157-170). Also available in print.

Thesis (M.S.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains v, 70 p. : ill. Includes abstract. Includes bibliographical references.

Thesis (M.A.)--Central Connecticut State University, 2003. / Thesis advisor: Thomas R. King. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaves 35-39). Also available via the World Wide Web.

Gene expression profiles have identified five major molecular breast cancer subtypes (Luminal A, Luminal B, Basal-like, HER2+/estrogen receptor− , and Normal Breast-like) that show significant differences in survival. The cost and complexity of gene expression technology has impeded its clinical implementation. By comparison, immunohistochemistry is an economical technique applicable to the standard formalin-fixed, paraffin-embedded material commonly used in hospital labs, and has the advantage of simultaneously interpretation with histomorphology. In this thesis, I hypothesize that a surrogate panel of immunohistochemical biomarkers can be developed to discriminate the breast cancer biological subtypes. The main study cohort consists of over 4000 primary invasive breast tumors, assembled into tissue microarrays. These patients were referred to the British Columbia Cancer Agency between 1986-1992 and have staging, pathology, treatment and follow-up information. In summary, our results demonstrate that (1) the rabbit monoclonal antibody, SP1, is an improved standard for immunohistochemiscal estrogen receptor assessment in breast cancer; (2) the transcription factor, GATA-3, is almost exclusively expressed among estrogen receptor positive tumors but does not seem to predict for tamoxifen response among estrogen receptor positive patients; (3) the proliferation marker, Ki-67, together with HER2 can segregate Luminal A from Luminal B subtypes, which carry distinct risks for breast cancer relapse and death; and (4) the inclusion of the basal markers EGFR and ck5/6 to “triple negative” breast cancers provides a more specific definition of basal-like breast cancer that better predicts patient survival. These results consistently demonstrate that an immunopanel of six biomarkers (estrogen receptor, progesterone receptor, HER2, Ki-67, epidermal growth factor receptor and cytokeratin 5/6) can be readily applied to standard pathology specimens to subtype breast cancer samples based on their underlying molecular biology. These findings have been considered sufficient to justify application of this panel onto NCIC (MA5, MA12) and CALGB (9341 and 9741) clinical trials specimens. This followup work which is underway and will determine if the six marker immunopanel can guide decisions about which patients need aggressive systemic drug treatment, and thereby ensure patients get the most effective, individualized adjuvant systemic therapy for their breast tumor.

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