by Tat-ming Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1981. / Bibliography: leaves 288-329.

Thesis (Ph.D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Mary J. Cloninger. Includes bibliographical references (leaves 224-240).

Administration of Concanavalin-A (Con A) induces severe injury to the hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A induced hepatitis. Although immune cells have been identified as the causative agent of Con A-induced hepatitis, the exact mechanism of pathogenesis remains unclear. When Con A was injected into CD44 wild type (WT) mice, it induced hepatitis as evident from increased plasma aspartate aminotransferase (AST) levels accompanied by active infiltration of mononuclear cells in the liver and significant induction of apoptosis. Interestingly, Con A injected C57BL/6 CD44-knockout (KO) mice exhibited increased hepatitis with higher levels of apoptosis in the liver and increased plasma AST levels when compared to the CD44 WT mice. Also, transfer of T cells from Con A injected CD44-KO mice into CD44 WT mice induced higher levels of hepatitis when compared to transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44-KO mice was partially due to increased production of cytokines such as TNF-a, IL-2 and IFN-g, but not Fas or FasL. Also, it was not caused by altered presence of T cell subsets. The increased susceptibility of CD44 KO mice to hepatitis correlated with increased resistance of T cells from CD44 KO mice to undergo apoptosis when compared to the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis. / Master of Science

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20672号 / 医博第4282号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 野田 亮, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Virtually all extracellular proteins in humans are glycoproteins and likewise are many biopharmaceuticals. The glycosylation is directly correlated to biological function and stability of these proteins. The ability to isolate glycoproteins is thus of great importance in many applications. The most common isolation method for glycoproteins is affinity chromatography using lectins, a ubiquitous and versatile group of carbohydrate-binding proteins. The lectin Concanavalin A (ConA) has long been used for this purpose but suffers from undesired leakage into the eluate, causing an inquiry of alternative chromatography ligands or optimization of the ConA resin.In this study, a total of 20 different lectins, including ConA, were evaluated and compared in terms of suitability as ligands in affinity chromatography for glycoprotein isolation. The lectins’ binding to glycoproteins were studied, mainly through microtiter plate binding assays using a monoclonal IgG1 antibody and Conalbumin (Ovotransferrin). Further, sugar-specificities and potential eluting sugars for the lectins were examined through inhibition with eight different carbohydrates. Additionally, the glycoprotein binding and leakage of ConA columns were examined, and a potential leakagereducing treatment of ConA resin evaluated.ConA was found to be superior in binding to the investigated glycoproteins but exhibited a limited binding when immobilized to an agarose resin. This discrepancy is likely a consequence of structurally hidden glycans on the used glycoproteins and requirements of long residence time when used in a chromatographic setting. Binding competition with several sugars were investigated with a similar microtiter plate binding assay. This method displayed potential to predict the behaviour of sugars and their suitability as eluting agents in a chromatography column. The best eluting sugar for ConA was showed to be methylmannoside, ideally in combination with methylglucoside. Lastly, evaluation of ConA columns with a crosslinking glutaraldehyde-treatment showed that the ConA ligand leakage may be significantly reduced, although further studies and optimizations are needed.This study thus presents a repertoire of lectins and their differences in terms of glycoprotein-binding and sugar-specificity, as well as evaluations of ConA columns’ efficiency and potential leakage-prevention.

O polissacarídeo quitosana é usado em aplicações biológicas, tais como entrega de drogas e engenharia de tecidos como matriz para o crescimento celular, devido à sua biocompatibilidade e biodegradabilidade. Uma das suas utilizações mais frequentes é na forma de membranas obtidas por casting com poli (etileno glicol) (PEG). Neste trabalho, membranas de quitosana-PEG foram modificadas e otimizadas com filmes nanoestruturados de concanavalina A (Con A) e jacalina. O processo de purificação não afetou as propriedades da quitosana, tais como cristalinidade, tamanho de cristalitos, grupos funcionais e grau de acetilação. A única exceção foi a diminuição da massa molecular, provavelmente pela quebra de cadeias por adição de ácido acético à solução. As membranas fabricadas com mistura de quitosana e PEG exibiram superfície mais rugosa, porosa, com energia de superfície mais elevada do que aquelas com quitosana pura. Misturas com 20 e 30% de PEG foram testadas, sendo as que contêm 20% mais adequadas para a funcionalização, devido ao maior tamanho dos poros, de acordo com imagens de microscopia de força atômica. Na funcionalização das membranas de quitosana-PEG com proteínas, o objetivo é obter a cobertura mais uniforme com maior energia de superfície. No processo de otimização, a deposição do filme nanoestruturado de proteína foi confirmada com PM-IRRAS, espectroscopia de fluorescência e dicroísmo circular, e a energia de superfície foi calculada usando o modelo de Owens- Wendt-Rabel-Kaelble a partir dos ângulos de contato para diferentes líquidos. Para Con A e jacalina, propriedades otimizadas foram obtidas com a menor concentração de proteína testada, 0,1 mg/mL, para um tempo de adsorção de 90 minutos. Além disso, o filme de jacalina levou à maior energia de superfície, ou seja, 56,7 mJ/m², comparado com 55,9 mJ/m² para amostras modificadas com Con A. Além disso, sob essas condições de otimização, a atividade da proteína foi mantida por 4 semanas para membranas armazenadas a 4ºC. Portanto, as membranas funcionalizadas são promissoras para crescimento celular e aplicações de engenharia de tecidos / The polysaccharide chitosan is used in various biological applications such as drug delivery and especially in tissue engineering as a matrix for cell growth due to its biocompatibility and biodegradability. One of its most frequent uses is in the form of membranes made via casting blended with poly(ethylene glycol) (PEG). In this work, chitosan-PEG membranes were optimized and modified with nanostructured films of concanavalin A (Con A) and jacalin. The purification process did not affect the chitosan properties, such as crystallinity, crystallite size, functional groups and degree of acetylation. The only exception was a decrease in the molecular mass, probably owing to chain scission by addition of acetic acid to the solution. The membranes made with chitosan and PEG exhibited a rougher, porous surface, with higher surface energy than those with neat chitosan. Blends with 20 and 30% PEG were tested, and those with 20% were considered as more suitable for functionalization owing to the larger size of the pores, according to atomic force microscopy images. The functionalization of the chitosan-PEG membranes with the proteins is aimed at achieving the most uniform coverage with the highest surface energy. In the optimization procedure, the deposition of the protein nanostructured film was confirmed with PM-IRRAS, fluorescence spectroscopy and circular dichroism, while the surface energy was calculated using the Owens-Wendt-Rabel-Kaelble model and the measured contact angles for several liquids. For both Con A and jacalin, optimized properties were obtained with the lowest protein concentration tested, viz. 0.1 mg/mL, for an adsorption time of 90 min. Furthermore, the jacalin film led to the highest surface energy, namely 56.7 mJ/m², to be compared with 55.9 mJ/m² for samples modified with Con A. Under these optimized conditions, the protein activity was kept for ca. 4 weeks if the coated membranes were stored at 4ºC. Therefore, the functionalized membranes are promising for cell growth and tissue engineering applications

Electrospun, non-woven, fibers have high surface area compared to conventional cast films. The thesis reports on the modification of electrospun fibers to concentrate and/or detect biohazards. In one study, electrospun fibers with affinity for the lectins ricin/concanavalin A were fabricated using molecular imprinting or through binding to immobilized antibodies, aptamers or lectin specific sugars. Attempts to fabricate imprinted electrospun fibers through inclusion of the template during the spinning process proved unsuccessful. However, electrospun fibers with affinity towards biohazards were successfully produced by post-modification with antibodies, aptamers or lectin specific saccharides. With regards to the latter, dextran, mannose and chitosan were immobilized onto nylon electrospun fibers that were partially hydrolyzed or treated with cyanuric chloride. The sugar-modified fibers bound significantly higher amount of lectins. Electrospun fibers were also fabricated, post modified with antibodies to capture and detect Salmonella. The study has illustrated the utility of electrospun fibers for biohazard diagnostics. / The National Center for Food Protection and Defense. USA

O polissacarídeo quitosana é usado em aplicações biológicas, tais como entrega de drogas e engenharia de tecidos como matriz para o crescimento celular, devido à sua biocompatibilidade e biodegradabilidade. Uma das suas utilizações mais frequentes é na forma de membranas obtidas por casting com poli (etileno glicol) (PEG). Neste trabalho, membranas de quitosana-PEG foram modificadas e otimizadas com filmes nanoestruturados de concanavalina A (Con A) e jacalina. O processo de purificação não afetou as propriedades da quitosana, tais como cristalinidade, tamanho de cristalitos, grupos funcionais e grau de acetilação. A única exceção foi a diminuição da massa molecular, provavelmente pela quebra de cadeias por adição de ácido acético à solução. As membranas fabricadas com mistura de quitosana e PEG exibiram superfície mais rugosa, porosa, com energia de superfície mais elevada do que aquelas com quitosana pura. Misturas com 20 e 30% de PEG foram testadas, sendo as que contêm 20% mais adequadas para a funcionalização, devido ao maior tamanho dos poros, de acordo com imagens de microscopia de força atômica. Na funcionalização das membranas de quitosana-PEG com proteínas, o objetivo é obter a cobertura mais uniforme com maior energia de superfície. No processo de otimização, a deposição do filme nanoestruturado de proteína foi confirmada com PM-IRRAS, espectroscopia de fluorescência e dicroísmo circular, e a energia de superfície foi calculada usando o modelo de Owens- Wendt-Rabel-Kaelble a partir dos ângulos de contato para diferentes líquidos. Para Con A e jacalina, propriedades otimizadas foram obtidas com a menor concentração de proteína testada, 0,1 mg/mL, para um tempo de adsorção de 90 minutos. Além disso, o filme de jacalina levou à maior energia de superfície, ou seja, 56,7 mJ/m², comparado com 55,9 mJ/m² para amostras modificadas com Con A. Além disso, sob essas condições de otimização, a atividade da proteína foi mantida por 4 semanas para membranas armazenadas a 4ºC. Portanto, as membranas funcionalizadas são promissoras para crescimento celular e aplicações de engenharia de tecidos / The polysaccharide chitosan is used in various biological applications such as drug delivery and especially in tissue engineering as a matrix for cell growth due to its biocompatibility and biodegradability. One of its most frequent uses is in the form of membranes made via casting blended with poly(ethylene glycol) (PEG). In this work, chitosan-PEG membranes were optimized and modified with nanostructured films of concanavalin A (Con A) and jacalin. The purification process did not affect the chitosan properties, such as crystallinity, crystallite size, functional groups and degree of acetylation. The only exception was a decrease in the molecular mass, probably owing to chain scission by addition of acetic acid to the solution. The membranes made with chitosan and PEG exhibited a rougher, porous surface, with higher surface energy than those with neat chitosan. Blends with 20 and 30% PEG were tested, and those with 20% were considered as more suitable for functionalization owing to the larger size of the pores, according to atomic force microscopy images. The functionalization of the chitosan-PEG membranes with the proteins is aimed at achieving the most uniform coverage with the highest surface energy. In the optimization procedure, the deposition of the protein nanostructured film was confirmed with PM-IRRAS, fluorescence spectroscopy and circular dichroism, while the surface energy was calculated using the Owens-Wendt-Rabel-Kaelble model and the measured contact angles for several liquids. For both Con A and jacalin, optimized properties were obtained with the lowest protein concentration tested, viz. 0.1 mg/mL, for an adsorption time of 90 min. Furthermore, the jacalin film led to the highest surface energy, namely 56.7 mJ/m², to be compared with 55.9 mJ/m² for samples modified with Con A. Under these optimized conditions, the protein activity was kept for ca. 4 weeks if the coated membranes were stored at 4ºC. Therefore, the functionalized membranes are promising for cell growth and tissue engineering applications