Thrombin-activatable fibrinolysis inhibitor (TAFI), also called procarboxypeptidase U (proCPU), is a plasma zymogen that can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The activated form of TAFI (TAFIa, CPU) removes C-terminal lysine residues of plasmin-modified fibrin (FN’) that mediate positive feedback in plasminogen (Pg) activation, thereby attenuating fibrinolysis. The plasma concentration of TAFI is ~75nM. Since the half-maximal effect of TAFIa occurs at 1nM, only about 1.3% of TAFI needs to be activated to exert an effect on clot lysis. The first objective of this work was to design a novel assay that can measure functional TAFIa levels in plasma. We have successfully developed a reliable TAFIa assay that is sensitive to TAFIa concentrations as low as 12pM. This assay is not confounded by endogenous levels of Pg or by a naturally occurring polymorphism at position 325 that affects the half-life of TAFIa. The second objective was to measure TAFI activation in vivo in primates in response to thrombin generation, E. coli-induced sepsis, or post-surgical stress. It was found that chimpanzees generated substantial amounts of TAFIa in a transient manner which had a half-life of ~8 to 10 minutes in response to thrombin generation. Baboons generated very high levels of TAFIa transiently in response to sublethal or lethal doses of E. coli. Interestingly, baboons generated less TAFIa when infused with thrombin followed by the blocking of endothelial protein C receptor (EPCR) than when the baboons were infused with only thrombin. Human patients that underwent hip replacement surgery showed sustained, elevated levels of TAFIa for up to 42 days after surgery. The third objective was to determine the kinetics of TAFIa-catalyzed release of bound plasminogen from soluble fibrin degradation products. The kinetics conformed to the Michaelis-Menten equation, which allowed the determination of Km, kcat and Vmax values for varying concentrations of TAFIa. In addition, the enzymatic activity of TAFIa appeared to saturate, probably because at a high concentration of TAFIa, the kinetics of plasminogen release were rate limiting. / Thesis (Master, Biochemistry) -- Queen's University, 2007-09-27 13:08:29.462

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細胞為本生物檢測法（CBB）是指任何一種以細胞系統及其身上發生的生物反應為基礎原之直接檢測方法。在這篇文中，有關CBB 的工作大致分為個主要章節，描述如下： / 第一章節探討以可調控式納米孔為基礎的電阻脈衝感應檢測法（RPS）進人血紅細胞的毒學研究。我們成功檢測到血紅細胞在低滲環境下的體型大小變化，並以式細胞儀及激光共焦掃描顯微鏡驗證上述實驗結果。此外，我們亦使用重皂甙（PD）及四溴雙酚A（TBBPA）種化合物以進毒試驗。在此章節，我們展示以RPS 在人體細胞系統的首次應用。這種RPS 無疑成為一種創新及低成本的技術，有助快速研究與血液有關的疾病。 / 第二章節探討以細胞為本的納米毒學研究。由於納米子已被廣泛應用於生物醫學上，它的應用同時帶出有關其毒性及其帶出的健康問題。在這章節，我們主要研究種同塗層的納米條（Au-NRs），其塗層分別為十烷基三甲基溴化氨（CTAB）及聚乙二醇（PEG）。透過一在人嗜鹼性細胞KU812 的毒實驗，我們展示Au-NRs 的生物相容性取決於它的塗層。CTAB 塗層的Au-NRs 會引發KU812 細胞死亡及其過敏性反應，而PEG 塗層的Au-NRs 則會引發以上反應。 / Cell-based Bioassay (CBB) refers to any kind of assay which detection principle is based on direct monitoring of biological events in living cell systems.The work in this thesis is divided to two main chapters described as follows. / The first focus was the characterization of human erythrocytes toxicology using tunable nanopore-based resistive pulse sensing (RPS). We successfully monitored the size changes of the erythrocytes in hypotonic environment. Results were confirmed with flow cytometry and confocal laser scanning microscopy (CLSM). Furthermore, drug assays were performed, in which the erythrocytes were treated with polyphyllin D (PD) or tetrabromobisphenol A (TBBPA). Here we reported the first application of the tunable nanopore-based RPS on human live cell system. This RPS system served as a novel and low-cost technique for rapid analysis of bloodrelated diseases at point-of-care. / Another focus was cell-based bioassays in nanotoxicology. As nanoparticles have been widely applied in biomedical and biological aspects, health issues have been raised and the toxicity of nanoparticles is much concerned. In this study, we investigated the cytotoxicity of CTAB- and PEG-coated gold nanorods (Au-NRs). Through a series of toxicological studies and using human basophils cell line KU812, we demonstrated the biocompatibility of coating of Au-NRs was critical to the cytotoxicity to cells. CTAB-coated Au-NRs, rather than PEG-coated Au-NRs, were able to induce cell death and allergic response in KU812 cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Ka Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references. / Abstracts also in Chinese. / Members of Examination Committee --- p.i / Declaration --- p.ii / Publications --- p.iii / Abstract --- p.iv / Acknowledgements --- p.viii / List of Figures --- p.xi / Table of Content --- p.xii / General Introduction --- p.1 / Chapter Chapter 1 --- Cell-based Bioassays using Resistive Pulse Sensing --- p.13 / Chapter 1.1 --- Introduction --- p.14 / Chapter 1.2 --- Materials and Methods --- p.26 / Chapter 1.2.1 --- Preparation of human erythrocytes --- p.26 / Chapter 1.2.2 --- Resistive pulse sensing (RPS) --- p.26 / Chapter 1.2.3 --- Hemolysis assay --- p.27 / Chapter 1.2.4 --- Osmolarity assay --- p.28 / Chapter 1.2.5 --- Confocal laser scanning microscopy --- p.28 / Chapter 1.2.6 --- Flow cytometry --- p.29 / Chapter 1.2.7 --- Polyphyllin D (PD) assay --- p.29 / Chapter 1.2.8 --- Tetrabromobisphenol A (TBBPA) assay --- p.30 / Chapter 1.2.9 --- Statistical analysis --- p.30 / Chapter 1.3 --- Results --- p.31 / Chapter 1.3.1 --- Measurement of human erythrocytes using RPS --- p.31 / Chapter 1.3.2 --- RPS measurement of human erythrocytes in hypotonic enviornment --- p.36 / Chapter 1.3.3 --- RPS measurement of PD-treated human erythrocytes --- p.48 / Chapter 1.3.4 --- RPS measurement of TBBPA-treated human erythrocytes --- p.55 / Chapter 1.4 --- Discussions and Conclusion --- p.64 / Chapter 1.5 --- References --- p.68 / Chapter Chapter 2 --- Cell-based Bioassays on Human Basophils --- p.71 / Chapter 2.1 --- Introduction --- p.72 / Chapter 2.2 --- Materials and Methods --- p.74 / Chapter 2.2.1 --- Reagents --- p.74 / Chapter 2.2.2 --- Preparation of the CTAB-coated and PEG-coated nanorods --- p.74 / Chapter 2.2.3 --- Cell culture and preparation --- p.75 / Chapter 2.2.4 --- Alamar blue assay --- p.76 / Chapter 2.2.5 --- Calcein leakage assay and propidium iodide staining assays --- p.77 / Chapter 2.2.6 --- Histamine release assay --- p.78 / Chapter 2.2.7 --- Beta-hexosaminidase release assay --- p.79 / Chapter 2.2.8 --- Statistical analysis --- p.80 / Chapter 2.3 --- Results --- p.81 / Chapter 2.3.1 --- Physical and optical properties of the gold nanorods --- p.81 / Chapter 2.3.2 --- CTAB-, but not PEG-coated Au-NRs elicits allergic degranulation --- p.83 / Chapter 2.3.3 --- CTAB-coated Au-NRs are more cytotoxic than PEGcoated Au-NRs --- p.88 / Chapter 2.3.4 --- CTAB-, but not PEG-coated, Au-NRs cause plasma membrane permeabilization --- p.96 / Chapter 2.3.5 --- CTAB-, but not PEG-coated, Au-NRs disrupt plasma membrane asymmetry --- p.108 / Chapter 2.4 --- Discussions and Conclusion --- p.113 / Chapter 2.5 --- References --- p.116 / General Discussions and Conclusion --- p.118 / References --- p.124

No / BACKGROUND: The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring. METHODS: Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay. RESULTS: FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay. CONCLUSIONS: The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

Yes / The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors. / Yorkshire Cancer Research, Wellcome Trust

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