published_or_final_version / Microbiology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 133-143).

published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (M.S.)--Florida State University, 2004. / Advisor: Dr. Yun-Hwa Peggy Hsieh, Florida State University, College of Human Sciences, Dept. of Nutrition, Food and Exercise Sciences. Title and description from dissertation home page (viewed Apr. 18, 2005). Document formatted into pages; contains ix, 70 pages. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 1994. / Includes bibliographical references (leaves 137-156).

Thesis (M.S. in chemical engineering)--Washington State University, December 2008. / Title from PDF title page (viewed on Mar. 4, 2009). "School of Chemical Engineering and Bioengineering." Includes bibliographical references.

Autoantibodies are antibodies made abnormally to self-antigens that are expressed on the surface, in the cytoplasm, or in the nucleus of the cell. These antibodies are produced in certain people whose immune system has failed to work properly. It is a puzzle, however, how the B cells that produce autoantibodies which target the vital intracellular autoantigens and with potential deleterious effects to the cell, are able to exist in nature and not succumb to suicide. We have found a suitable model to answer this question. This is a newly-constructed mouse hybridoma (#476), which produces an IgG1 monoclonal antibody (mAb 476) to an epitope in the catalytic subunit of telomerase (TERT). This mAb stains TERT very well in both mouse and human cells. The unusual thing about hybridoma 476 is that, although it can grow and produce the antibody normally in culture, however, it fails repeatedly to grow in the peritoneum of normal BALB/c mice primed with an adjuvant, unlike other hybridomas of similar backgrounds that we have used. Control cell lines used in the study include a hybridoma (Mab2) which makes an IgG1 antibody to phosphorylcholine, and the myeloma fusion partner (Sp2/0) used to construct hybridoma 476. / When the antibody specificity was abrogated by genetic manipulation or by natural selection, the 476 cells were able to grow and produce ascites in vivo normally. This shows that the problem was specific, due to binding of the mAb with its antigen, TERT. Telomerase is a housekeeping enzyme in cells essential for maintaining the stability of the telomere, the DNA repeats found at the ends of chromosomes, failing which the cell undergoes senescence and eventually dies. It appeared that the peritoneum, unlike the culture medium, contained factors that presumably up-regulated the antibody production in the cells, which consequently increased the complex formation between TERT and mAb 476 in these cells, and led to cell apoptosis. Indeed, examination of the culture supernatant ("soup") of the peritoneal cells harvested from the adjuvant-primed mice, revealed no cytotoxic cytokines or chemokines present, but an abundant amount of IL-6. In parental 476 cells in culture, both soup and purified recombinant IL-6 were found to up-regulate the production of the intracellular antibodies, but not the secreted antibodies. The effect of these reagents on the Mab2 cells was different; both the intracellular and secreted antibodies were increased. (Abstract shortened by UMI.) / Niu Haitao. / "April 2005." / Adviser: Pak-Leong Lim. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0169. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 199-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (Ph. D. in Chemical Engineering)--Vanderbilt University, Aug. 2005. / Title from title screen. Includes bibliographical references.

A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxina foi construída no vetor pComb3X a partir do RNA total do hibridoma anti-digoxina. Seis clones foram isolados, todos com sequência idêntica na cadeia pesada. A cadeia leve apresentou 2 clones idênticos, um pseudogene e um clone com um aminoácido distinto no CDR2. Quatro clones apresentando variações na sequência do framework1 da cadeia leve foram expressos como fragmentos Fab solúveis. Todos apresentaram ligação à digoxina-BSA por ELISA e Western blotting. A ligação específica do anticorpo também foi confirmada pelo BIAcore, que permitiu ranqueamento entre os clones. / Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is very narrow. To counteract the toxic effect, polyclonal anti-digoxin Fab fragments are commercially available. Our goal was to obtain variants of monoclonal anti-digoxin Fab fragments by phage display technology, which allows the generation of high affinity and specificity antibody fragments. Anti-digoxin Fab fragments combinatorial library was constructed into pComb3X vector from total RNA of anti-digoxin hybridoma. Six clones were isolated and the heavy chain presented the same sequence. For the light chain, 2 clones were identical, one was a pseudogene and other one presented a distinct amino acid in the CDR2. Four clones presenting variations in the framework 1 were induced to express soluble Fab fragments, all positive for anti-digoxin binding in ELISA assays and Western blotting. The specific binding of the antibody was further confirmed by BIAcore, which allowed ranking of the clones.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.