Liu , Huan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 43-45). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Gel electrophoresis of proteins --- p.1 / Chapter 1.1.1 --- Principles of gel electrophoresis --- p.1 / Chapter 1.1.2 --- Polyacrylamide gel --- p.3 / Chapter 1.1.3 --- Buffer systems --- p.5 / Chapter 1.1.4 --- Capillary electrophoresis --- p.6 / Chapter 1.2 --- Methods to transfer proteins from gel to membrane --- p.7 / Chapter 1.2.1 --- Simple diffusion --- p.8 / Chapter 1.2.2 --- Ultrasound transfer --- p.8 / Chapter 1.2.3 --- Tank transfer --- p.9 / Chapter 1.2.4 --- Semidry transfer --- p.9 / Chapter 1.2.5 --- Transfer efficiency improvements --- p.10 / Chapter 1.3 --- Visualizing immunoblots --- p.11 / Chapter 1.3.1 --- Membrane staining --- p.11 / Chapter 1.3.2 --- Radiometric detection in blotting --- p.12 / Chapter 1.3.3 --- Bioluminescence-enhanced detection --- p.13 / Chapter 1.3.4 --- Chemiluminescence-enhanced detection --- p.14 / Chapter 1.4 --- Miniaturized electrophoresis and blotting methods --- p.15 / Chapter 1.5 --- Objective of the project --- p.18 / Chapter Chapter 2 --- Dip-pen gel electrophoresis in capillaries --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Experimental section --- p.21 / Chapter 2.2.1 --- Materials and reagents --- p.21 / Chapter 2.2.2 --- PA gel fabrication in capillary --- p.22 / Chapter 2.2.3 --- Setup for electrophoresis in capillary --- p.23 / Chapter 2.3 --- Results and discussion --- p.24 / Chapter 2.3.1 --- PA gel polymerization quality at tip --- p.24 / Chapter 2.3.2 --- Separation efficiency in capillary --- p.25 / Chapter 2.4 --- Conclusions --- p.27 / Chapter Chapter 3 --- Western blotting by dip-pen electrophoresis --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Experimental section --- p.30 / Chapter 3.2.1 --- Materials and reagents --- p.30 / Chapter 3.2.2 --- Protein sample preparation --- p.31 / Chapter 3.2.3 --- Dip-pen electrophoresis based Western blot --- p.31 / Chapter 3.2.4 --- Detection on membrane --- p.32 / Chapter 3.3 --- Results and discussion --- p.33 / Chapter 3.3.1 --- Separation performance on nitrocellulose membrane --- p.33 / Chapter 3.3.2 --- Comparison among different %T PA gel --- p.34 / Chapter 3.3.3 --- SDS-protein complexes capture and immunoblotting --- p.38 / Chapter 3.4 --- Conclusions --- p.39 / Chapter Chapter 4 --- Conclusions --- p.40 / Chapter 4.1 --- Summary --- p.40 / Chapter 4.2 --- Future perspective --- p.41 / References --- p.43

Monoclonal antibodies (MCA) produced against infectious laryngotracheitis virus (ILTV) of chickens reacted in western blotting experiments with several different ILTV protein bands in the absence of tunicamycin which inhibits carbohydrate synthesis. Most of the MCA lost their reactivity in western blotting experiments when extracts of tunicamycin-treated ILTV CELC were used, suggesting their specificity for carbohydrate-based epitopes. In an indirect immunofluorescence test most of the MCA bound primarily to cytoplasmic antigens except some MCA which bound primarily to nuclear antigens. Additivity ELISA was also performed to study whether MCA are against the same epitope or different epitopes. The polymerase chain reaction (PCR) was developed as a diagnostic technique for detection of ILTV using primers made from a portion of the ILTV thymidine kinase gene. The 647-basepair amplified ILTV PCR product was labeled to create a non-radioactive, biotinylated DNA probe. Hybridization was performed using the probe to detect ILTV. Both PCR and hybridization detected ILTV, and neither hybridization nor PCR gave positive results with any other pathogen. Hybridization was specific for ILTV, However, slight hybridization occurred with CELC DNA when relatively relaxed conditions were used. In another experiment, diagnostic tests to detect ILTV in tracheas of experimentally-infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, PCR, and hybridization, were performed and compared. Using virus isolation as a reference, the sensitivity and specificity of the tests were calculated. The IP test and IFAT performed better than any other test used in this study. / Graduation date: 1995

Indiana University-Purdue University Indianapolis (IUPUI) / 17-β estradiol (E2) exerts protective effects in hypoxia-induced pulmonary hypertension (HPH) via endothelial cell estrogen receptor (ER)-dependent mechanisms. However, the effects of hypoxia on ER expression in the pulmonary-right ventricle (RV) axis remain unknown. Based on previous data suggesting a role of ER-β in mediating E2 protection, we hypothesized that hypoxia selectively up-regulates ER-β in the lung and pulmonary endothelial cells. In our Male Sprague-Dawley rat model, chronic hypoxia exposure (10% FiO2) resulted in a robust HPH phenotype associated with significant increases in ER- β but not ER-α protein in the lung via western blotting. More importantly, this hypoxia-induced ER-β increase was not replicated in the RV, left ventricle (LV) or in the liver. Hence, hypoxia-induced ER-β up-regulation appears to be lung-specific. Ex vivo, hypoxia exposure time-dependently up-regulated ER-β but not ER-α in cultured primary rat pulmonary artery endothelial cells (RPAECs) exposed to hypoxia (1% O2) for 4, 24 or 72h. Furthermore, the hypoxia induced ER-β protein abundance, while not accompanied by increases in its own transcript, was associated with ER-β nuclear translocation, suggesting increase in activity as well as post-transcriptional up-regulation of ER-β. Indeed, the requirement for ER-β activation was indicated in hypoxic ER-βKO mice where administration of E2 failed to inhibit hypoxia-induced pro-proliferative ERK1/2 signaling. Interestingly, HIF-1α accumulation was noted in lung tissue of hypoxic ER-βKO mice; consistent with previously reported negative feedback of ER-β on HIF-1α protein and transcriptional activation. In RAPECs, HIF-1 stabilization and overexpression did not replicate the effects of ER- β up-regulation seen in gas hypoxia; suggestive that HIF-1α is not sufficient for ER-β up- regulation. Similarly, HIF-1 inhibition with chetomin did not result in ER-β down-regulation. HIF-1α knockdown in RPAECs in hypoxic conditions is currently being investigated. Hypoxia increases ER- β, but not ER-α in the lung and lung vascular cells. Interpreted in context of beneficial effects of E2 on hypoxic PA and RV remodeling, our data suggest a protective role for ER-β in HPH. The mechanisms by which hypoxia increases ER-β appears to be post-transcriptional and HIF-1α independent. Elucidating hypoxia-related ER-β signaling pathways in PAECs may reveal novel therapeutic targets in HPH.

Indiana University-Purdue University Indianapolis (IUPUI) / Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.