Thesis (Ph.D.)--Ohio University, August, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (p. 253-288)

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 177-196). Also available in print.

published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Surgery / Master / Master of Philosophy

Chronic obstructive pulmonary disease (COPD) is one of the world’s most common chronic diseases, and consists of chronic bronchitis that involves chronic inflammation of the bronchi, or emphysema that involves destruction of lung alveoli. In COPD patients, the airways become narrowed, and the airflow is irreversibly obstructed. This leads to a limitation of the flow of air to and from the lungs, causing shortness of breath (dyspnea), as well as abnormal inflammatory response in the lung. Nowadays, COPD is often under-diagnosed, as spirometry was not performed until patient has significant symptoms of dyspnea, cough and sputum production. At that stage, the COPD patients may have reached an advanced stage with considerable loss of lung function. Thus, biomarkers are of great interest for research and clinical purposes in COPD, especially for early diagnosis of COPD. In this study, the relationship between plasma levels of different biomarkers, including monocyte chemoattractant protein-1 (MCP)-1 (a primary chemoattractant biomarker), matrix metalloproteinase nine (MMP)-9, vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) (injury and repair biomarkers), and growth differentiation factor 15 (GDF)-15 (a novel biomarker), in 29 healthy ever-smokers and 116 COPD patients was investigated using commercially available enzyme-linked immunosorbent assay (ELISA) kits. We also investigated the correlations between these biomarkers and lung function. There were significant increases in plasma MCP-1, MMP-9, HGF and GDF-15 in COPD patients compared to healthy smokers. Among ever-smokers with or without COPD, plasma MCP-1, MMP-9 and HGF levels were inversely correlated with force expiratory volume in one second![FEV1 (% predicted)] after adjustment for age, smoking status and packyears smoked. Correlation was also found between plasma MCP-1 and HGF, plasma MMP-9 and HGF or GDF-15, plasma HGF and GDF-15 after adjustment for age, smoking status and pack-years smoked. Further multiple linear regression analyses demonstrated that plasma MMP-9 level increased with the COPD GOLD stages. In conclusion, our findings suggest that MMP-9 might be as an important biomarker for COPD initiation and progression. As this study provides only evidence of association rather than of causation, prospective studies are required to assess biological significance of these associations between the plasma biomarkers. / published_or_final_version / Medicine / Master / Master of Medical Sciences

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Obesity is one of the leading causes of world-wide life-threatening diseases, such as type 2 diabetes mellitus, atherosclerosis and cardiovascular diseases. So far, there is no effective, promising and safe remedy. Development of cost-effective anti-obesity therapies and promising biomarkers for obesity-related diseases have become a demanding task. Lipocalins, such as LCN13, were recently identified as potential drug target because of its beneficial effects on glucose and fat metabolism in mouse. LCN14 is a putative lipocalin that share high degree of homology with LCN13. In this study, it is experimentally proved that LCN14 is a secretory protein that is mainly expressed in white adipose tissues. It is also demonstrated that serum LCN14 level was significantly increased in mice with HFD treatment, and it was significantly reduced in diet-induced obese and diabetic (db/db) mice. In addition, the degree of suppression of circulating LCN14 was greater than that of LCN13 in diet-induced obese and db/db mice. Therefore, serum LCN14 level could be a promising marker for risk prediction of obesity and its complications. Further investigation is urgently needed to unveil the important roles of LCN14 in metabolism. / published_or_final_version / Medicine / Master / Master of Medical Sciences

Due to the high affinity and selectivity of aptamer selected from DNA library towards target proteins, the use of specific protein biomarkers to assist early diagnosis, and the recent rapid development of piezoelectric quartz crystal (PQC) biosensor technology, the research reported is focused on developing PQC biosensor using aptamers selected from a DNA library as selective coating for the detection of albumin in urine, two protein biomarkers for malaria in sera, and the determination of trace amount of SCV helicase protein from SAR in sera. Three new aptamer-PQC biosensors are developed with research conducted on aptamer selection, optimization of coating methods for aptamers on PQC, and coupling paramagnetic nanoparticle technology with PQC biosensor in flow injection analysis (FIA) to enrich analyte and eliminate sample matrix interference. A new approach for aptamer selection is successfully attempted in the development of the first aptamer-PQC biosensor with an aim to offer an early detection of microalbuminuria enabled by an increase in detection sensitivity with aptamer selected by its actual binding with albumin-PQC biosensor. The aptamer-PQC sensor developed has shown to give a working range between 0.1 to 10 ug/mL, a detection limit (S/N = 3, n=3) of 0.048 μg/ml, repeatability of RSD = 6.8% (n=3), a response time of 1 minute and a throughput of 60 samples/hour. The developed piezoelectric aptamer sensor is shown to have sufficient sensitivity to detect microgram quantities of albumin in urine. Aptamer-PQC biosensors are developed for the detection of two protein biomarkers, PfLDH for general diagnosis and PfHRP-2 for a specific diagnosis of Plasmodium falciparum malaria parasitemia, in sera. The results show satisfactory working range from 10-100 ng/mL for both PfLDH and PfHRP-2 protein biomarker, low detection limit of 1.8 and 4.7 ng/mL and satisfactory repeatability (%RSD, n = 3) of 7.4% and 9.2% for PfHRP-2 and PfLDH respectively. It is first report for aptamer based PQC biosensor to detect malarial PfHRP-2 and PfLDH at ppb range to meet the requirement for their diagnosis. To meet the highly demanding challenge for detecting protein biomarkers at pg/mL level in high-protein sera sample, the coupling of paramagnetic nanoparticle technology with aptamer-PQC biosensor was attempted for developing aptamer-PQC biosensor for selective detection of SCV helicase protein produced from SARS CoV replication. The coupling of aptamer coated paramagnetic nanoparticles for sample pretreatment to aptamer-PQC biosensor has shown to detect helicase protein in one-minute assay with a detection limit of 350 pg/mL. The aptamer-coated crystal exhibits a frequency shift linearly proportional to the concentration of SARS helicase from 1 to 1000 ng/mL with a correlation coefficient of 0.9975 and a repeatability of 6.8% (%RSD, n=3). After the enrichment procedure, recoveries of 102% and 119% were achieved using samples spiked with SARS helicase at concentrations of 10 ng/mL and 1.0 ng/mL respectively. It is the first report to detect for SCV helicase protein using PQC biosensor at pg/mL level after magnetic bead enrichment. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Liver cancer is the fifth most commonly diagnosed and the second most lethal malignancies worldwide, in which hepatocellular carcinoma (HCC) represents the majority subtype. High mortality rate of HCC is due to lack of effective treatments and early detection methods. Activation of cadherin-17 (CDH17)/β-catenin axis is found by our team in HCC and targeting components of this axis associated with anti-tumorigenesis. With limited knowledge on this axis in HCC, I plan to study molecules related to this axis as a way to uncover the cellular mechanism of this axis in liver tumorigenesis. Gene profiling data was re-analyzed to search for CDH17-associated genes in HCC clinical samples. The patient cohort was segregated into CDH17-high and CDH17-low group according to tumor/adjacent non-tumor expression ratio of CDH17. Serine peptidase inhibitor, Kazal type 1 (SPINK1) was found highly expressed in CDH17-high cases and its over-expression accounted for 73 % of total studied cases. Gene manipulation and inhibitor study in HCC cell lines suggested SPINK1 as a downstream molecule of CDH17/β-catenin axis in HCC. Further in silico analysis predicted potential binding sites of two transcriptional factors downstream of CDH17/β-catenin axis, lymphoid enhancer-binding factor 1 (LEF1) and T-cell factor 7 (TCF7), on SPINK1 promoter. Deletion or mutation of their binding sites on SPINK1 promoter suppressed the transcription of SPINK1 gene, while transient suppression of these two transcriptional factors resulted in reduction of SPINK1 level. As the direct link between SPINK1 and CDH17/β-catenin axis was confirmed, SPINK1 was hypothesized to possess tumorigenic properties like its upstream molecule CDH17. Suppression of SPINK1 using RNA interference in PLC and MHCC97-H HCC cells hampered growth, migration and colony formation abilities of suppressed cells. These phenotypic alterations accompanied with an inactivation of tumorigenic c-Raf/MEK/ERK pathway. These findings demonstrate the tumorigenic properties of SPINK1 in HCC. Next, the therapeutic potential of targeting SPINK1 in HCC was tested by using purified monoclonal antibody raised against recombinant SPINK1 protein (C4). C4 was capable in suppressing SPINK1 level based on results of immunocytochemisty, enzyme-linked immunosorbent assay and immunoneutralization. Treatment of HCC cells using C4 suppressed growth, migration and colony formation ability of cells by inactivating MAPK pathway. In subcutaneous tumor xenograft study, treating tumor-bearing mice with C4 at 8 mg/kg three times weekly inhibited tumor growth by around 65 %. These findings demonstrate C4 is a potential therapeutic for counteracting liver tumorigenesis. In conclusion, I have demonstrated for the first time SPINK1 as a novel downstream molecule of CDH17/β-catenin axis involved in HCC progression via activating MAPK pathway. Targeting this molecule with its specific monoclonal antibody is a potential approach for cancer therapy. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Not available