Yiu Wai Han. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 146-156) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In addition to a central circadian clock in the suprachiasmatic nucleus (SCN), zebrafish (Danio rerio) have local clock systems in their peripheral tissues. These peripheral tissues express a complement of clock genes that can be synchronized with the 24 h light/dark cycle and thus may be entrained by light. To date, teleost multiple tissue (tmt) opsin identified from Fugu rubripes and Danio rerio is the only opsin that has been proposed as a candidate to mediate this cellular photoentrainment (Moutsaki et al., 2003). Here we report the discovery of a multigene family of tmt opsins found not only in the teleost fishes, but in vertebrates,including amphibians, birds, reptiles, and some mammals. Phylogenetic analysis demonstrated that this gene family consists of three main classes, tmtI, tmtII and tmtIII, with each duplicating further to give two paralogues in the zebrafish genome. Their predicted amino acid sequences contain most of the characteristic features for the function of a photopigment opsin, as well as seven transmembrane segments indicative of a G protein coupled receptor (GPCR) superfamily. Significantly, reverse transcription polymerase chain reaction (RT-PCR) reveals that the tmt opsin genes in zebrafish are both temporally and spatially regulated. To investigate if these tmt photopigments mediate light-activated currents in cells, each opsin was expressed in vitro and the responses characterised by calcium imaging, whole-cell patch clamp electrophysiology, UV-Vis spectrophotometric analysis, and bioluminescence reporter assay. Collectively, these data suggest that some of the opsin photoproteins signal via Gi-type G protein pathway. Interestingly, the spectral analysis obtained shows that most tmt opsins tested are UV-sensitive when reconstituted in vitro with 11-cis and all-trans retinal, indicating an intrinsic bistable dynamics. Using site directed mutagenesis on one of the tmt opsins, tmt10, the potential spectral tuning sites involved in UV detection were tested. As part of this study, tmt opsin cDNAs were isolated from three populations of Mexican tetra (Astyanax mexicanus): surface, Pachon and Steinhardt. This allowed for a direct comparison between the tmt opsins present in the dark adapted species (cavefish) versus those of the light adapted species (zebrafish). It is hoped that the findings from this project will contribute to our understanding of non-visual light detection in fish and the evolution of their non-image forming photoreception.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.