Made available in DSpace on 2014-10-09T12:33:11Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:13Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:54:30Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:46Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:54:30Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:46Z (GMT). No. of bitstreams: 0 / Endostatina, um fragmento do colágeno XVIII de 20 kDa, é um potente inibidor de angiogênese e crescimento tumoral. Foi previamente demonstrado que a administração contínua de endostatina em modelos animais melhorou a eficácia e potência da terapia antitumoral, comparada com a administração subcutânea diária por injeções de endostatina. A liberação contínua da proteína antiangiogênica endostatina para a circulação sistêmica poderia ser um tratamento antiangiogênico ideal. O sistema Theracyte é um sistema de membranas de politetrafluoretileno semi-permeáveis para macro-encapsulamento e implante de células geneticamente modificadas para liberação de proteínas terapêuticas in vivo e que não requer a imunossupressão do hospedeiro. Com a finalidade de demonstrar a utilidade deste sistema, células CHO expressando (his)6-met-endostatina foram injetadas em dispositivos de imunoisolamento Theracyte, que foram imediatamente implantados em camundongos imunodeficientes (SCID). Em outro modelo de implante de dispositivos de imunoisolamento, os dispositivos Theracyte foram implantados em animais e depois do tempo de cicatrização (17 dias), as células expressando endostatina foram injetadas dentro dos dispositivos. Níveis altos e constantes de endostatina de até 3,7 g/ml foram detectados no plasma durante os dois meses de duração do estudo em ambos os modelos de implante dos dispositivos de imunoisolamento. Níveis mais altos de endostatina (até 6,7 g/ml) foram detectados no plasma de animais implantados com o mesmo número de células livres. Análise histológica de cortes corados por hematoxilina/eosina dos dispositivos retirados dos animais mostraram que haviam células aparentemente viáveis dentro dos dispositivos. A análise imuno-histoquímica utilizando anticorpo anti-endostatina mostrou a existência de reação nas células dentro do dispositivo e também do lado de fora, demonstrando que a endostatina, secretada pelas células recombinantes confinadas, extravasou da membrana, atingindo os tecidos ao redor. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN/CNEN-SP

Eleven nickel compounds representing a range of solubilities and biological activities were tested for toxicity, mutagenicity, and cytosolic and nuclear nickel uptake in AS52 cells. values ranging LC50 from 2-130 ug Ni/ml for particulates and 120-150 ug Ni/ml for the water soluble salts (NiCl2, NiS04, Ni(CH3C00)2) were determined. The Ni(OH)2, NiC03 , and nickel sulphides (Ni3S2 , Ni 7S6 , amorphous NiS) exhibited similar toxicities (LC50's of 2.0, 5.8, 4.1, 8.2, 4.1 ~g Ni/ml respectively), while the nickel oxides were less toxic and showed large variations between the black, Li 2Ni 8010 , and green NiO forms (LC50's of 18.1, 75, 130 ug Ni/ml). Concentrations reducing survival to the range 20-80% were tested for mutagenicity and degree of nickel uptake. Although nickel compounds have been reported to be only weak or equivocal mutagens, the results indicate a low but significant increase in mutation rate at the gpt locus induced by all the nickel compounds tested. The majority of compounds displayed nuclear to cytoplasmic nickel ratios of ≈ 1:4 to 1:2, though this was ≈ 1:20 for nickel salts. NiC03 appeared to be intermediate in behaviour with a ratio of ≈ 1:12. Comparison of the eleven compounds at the same toxicity level (LC50) showed a 75-fold difference in exposure levels but about a 10-fold difference in cytoplasmic and nuclear nickel levels. There appears to be a very good correspondence between previously reported dissolution half times (T50's) of the compounds tested and the cytosolic nickel levels at a given toxicity level. For the water-soluble salts, previous reports have shown that cellular distribution varies from that of particulates due to differences in the manner of uptake. The present work confirms this and suggests that the compounds can be divided into three classes: watersoluble salts producing very low nuclear levels and high cytosolic levels, inert nickel oxides (green NiO and lithium nickel oxide) with relatively low nuclear and cytosolic nickel levels, and the remaining compounds (the major class) with relatively high cytosolic levels and nuclear nickel levels. Overall , the data supports the N i eke 1-Ion Hypothesis which suggests that the Ni 2+ ion is the active agent in nickel toxicity and mutagenicity, and that, as a first approximation, its intracellular concentration is responsible for the observed effects, irrespective of the nickel compound. / Thesis / Master of Science (MS)

Most glyco-engineering approaches used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We investigated whether the over expression of nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT), would be a means to improve the sialylation process in CHO cells. We hypothesized that increasing the expression of the CMP-SAT in the cells would increase the transport of the CMP-sialic acid in the Golgi lumen, hence increasing the intra-lumenal CMP-sialic acid pool, and resulting in a possible increase in sialylation extent of proteins being produced. We report the construction of a CMP-SAT expression vector which was used for transfection into CHO-IFNγ, a CHO cell line producing human IFNγ. This resulted in approximately 2 to 5 times increase in total CMP-SAT expression in some of the positive clones as compared to untransfected CHO-IFNγ, as determined using real-time PCR analysis. This in turn concurred with a 9.6% to 16.3% percent increase in site sialylation. This engineering approach has thus been identified as a novel means of improving sialylation in recombinant glycoprotein therapeutics. This strategy can be utilized feasibly on its own, or in combination with existing sialylation improvement strategies. It is believed that such multi-prong approaches are required to effectively manipulate the complex sialylation process, so as to bring us closer to the goal of producing recombinant glycoproteins of high and consistent sialylation from mammalian cells. / Singapore-MIT Alliance (SMA)

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 110-125). Also available online via the World Wide Web.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 141-157). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/23). Includes bibliographical references.

Made available in DSpace on 2014-10-09T12:36:04Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:22Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP