Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 103-111). Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D.)--City University of Hong Kong, 2009. / "Submitted to Department of Biology and Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy." Includes bibliographical references (leaves 133-147)

Thesis (Ph. D.)--University of California, San Diego, 2003. / Vita. Includes bibliographical references (leaves 353-374).

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 190-204). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

The development of microdevices for applications related to bioanalysis is described. There are two types of microdevices involved in this study: DNA (or RNA) microarrays and bead-based microfluidic devices. First, a new method to fabricate DNA microarrays is developed: replication of DNA microarrays. It was shown that oligonucleotides immobilized on a glass master can hybridize with their biotin-modified complements, and then the complements can be transferred to a streptavidinfunctionalized replica surface. This results in replication of the master DNA array. Several innovative aspects of replication are discussed. First, the zip code approach allows fabrication of replica DNA arrays having any configuration using a single, universal master array. It is demonstrated that this approach can be used to replicate master arrays having three different sequences (spot feature sizes as small as 100 [mu]m) and that master arrays can be used to prepare multiple replicas. Second, it is shown that a surface T4 DNA polymerase reaction improves the DNA microarray replication method by removing the requirement for using presynthesizd oligonucleotides. This in-situ, enzymatic synthesis approach is used to replicate DNA master arrays consisting of 2304 spots and arrays consisting of different oligonucleotide sequences. Importantly, multiple replica arrays prepared from a single master show consistent functionality to hybridization-based application. It is also shown that RNA microarrays can be fabricated utilizing a surface T4 DNA ligase reaction, which eliminates the requirement of modified RNA in conventional fabrication schemes. This aspect of the work shows that the replication approach may be broadly applicable to bioarray technologies. A different but related aspect of this project focuses on biosensors consisting of microfluidic devices packed with microbeads conjugated to DNA capture probes. The focus here is on understanding the parameters affecting the hybridization of DNA onto the probeconjugated microbeads under microfluidic flow conditions. These parameters include the surface concentration of the probe, the flow rate of the solution, and the concentration of the target. The simple microfluidic device packed with probe-conjugated microbeads exhibits efficient target capture resulting from the inherently high surface-area-to-volume ratio of the beads, optimized capture-probe surface density, and good mass-transfer characteristics. Furthermore, the bead-based microchip is integrated with a hydrogel preconcentrator enhancing the local concentration of DNA in a icrochannel. The integration of the preconcentrator into the bead-based capture chip allows significantly lower limit of detection level (~10-fold enhancement in the sensitivity of the microbeadbased DNA detection). / text

published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy

No description available.

DNA is both the oldest and newest of targets for cancer therapy. While it is already being targeted by many anticancer drugs in the clinic, the development of sequence-specific DNA binders has brought it back to the limelight as a valuable anticancer drug target.My studies on DNA interacting agents was initiated with the DNA intercalator campotothecin, and also included topoisomerase I enzyme. I have evaluated the structure of topoisomerase I C-terminal domain that consists of the active site tyrosine. My data indicate that this domain exists in a molten globule conformation with a fluctuating tertiary structure. These fluctuations are suggested to be important in interaction with the topoisomerase I core domain and DNA. I have also evaluated the DNA interactions of the camptothecin analogue homocamptothecin and have determined that homocamptothecin intercalate with DNA in the absence of topoisomerase I, and that such intercalation results in its lactone stabilization. Subsequently, the mechanism of topoisomerase I mediated inhibition of HIF-1 by camptothecin was explored. I have shown that camptothecin stimulate topoisomerase I cleavage complex formation in the HIF-1 binding site, which is suggested to prevent the DNA binding of HIF-1.The second part of this study was focused on understanding the mechanism of action of another DNA binder, XR5944. Designed as a dual topoisomerase inhibitor, XR5944 was subsequently shown to have a different mechanism of action - inhibition of trancription. The NMR structural analysis, in our lab, of the drug-DNA complex showed that XR5944 bis-intercalate with DNA, while binding in the DNA major groove. Driven by these combined interaction modes, XR5944 is shown to inhibit the DNA binding and the subsequent transcriptional activity of specific transcription factors such as estrogen receptors and AP-1, which are overexpressed in certain cancers.Finally, I have analyzed G-quadruplex structures formed by telomeric DNA. The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase. Thus the telomeric DNA G-quadruplex has been considered as an attractive anticancer drug target. Telomeric DNA forms multiple G-quadruplex conformations, and my data reveal the conformations of the major G-quadruplexes formed by human telomeres.

No description available.