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Over-Expression, Purification and Crystallization of the DNA Binding and Dimerization Region of Epstein-Barr Nuclear Antigen-1 / Over-Expression, Purification and Crystallization of Epstein-Barr Nuclear Antigen-1Barwell, Jean 04 1900 (has links)
EBV episomes replicate once per cellular S phase, during latent infection of host cells. Only one viral protein, Epstein-Barr Nuclear Antigen-1 (EBNA-1) is required for replication; the rest of the replication machinery is provided by the cell. EBNA-1 is an excellent model to study the molecular events required for DNA replication and its regulation because viral replication is limited to once per cell cycle. EBNA-1 is a member of a special class of DNA binding proteins called origin binding proteins (OBPs). These specialized proteins bind to distinct DNA sequences in the genome called origins of replication, where DNA replication is initiated. Origin binding proteins may serve to distort the DNA at the origin and may also attract the cellular replication machinery. Structural studies of the DNA binding and dimerization region of EBNA-1 using X-ray crystallography were undertaken in order to better understand how OBPs bind to origin DNA sequences and facilitate the assembly of the cellular replication apparatus. Six truncation mutants of EBNA-1, all containing the DNA binding and dimerization region of EBNA-1, were cloned, over-expressed in bacteria and purified to apparent homogeneity. Four of these clones were crystallized using the method of hanging-drop vapour-diffusion. Two fragments, EBNA₄₇₀₋₆₁₉ and EBNA₄₇₀₋₆₀₇, formed well-ordered crystals that diffracted beyond 2.5 Å resolution. In addition , this study also demonstrates the value of finding the most suitable piece of the protein for crystallization. This piece should fold into a compact domain for efficient packing into a crystal. Finding the optimal piece of the protein reduces the time spent searching for crystallization conditions. / Thesis / Master of Science (MS)
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Estudo comparativo \'in vitro\' entre preparações de imunoglobulina \'G\', para uso intravenoso, obtidas de plasma humano de variadas procedências e processadas por diferentes técnicas de separação / \"In vitro\" comparative study between imunoglobulin G preparations, intravenous use, human plasma derived from different plasma sources and different separation techniquesBarna, Geny Aparecida de Oliveira 26 June 2001 (has links)
Os efeitos protetores da imunidade humoral são medidas por uma família de glicoproteínas chamadas anticorpos ou imunoglobulinas. As preparações de imunoglobulina G (IgG) utilizadas em nosso país são importantes. No Brasil, a primeira preparação de IgG foi obtida na Fundação Pró-Sangue Hemocentro de São Paulo em 1993. O presente estudo avaliou preparações de IgG obtidas de misturas de plasma humano de variadas procedências, inclusive a preparação obtida no Brasil. Foram avaliados os seguintes parâmetros: concentração protéica, distribuição das subclasses da IgG, atividade de anticorpos específicos e segurança quanto a agentes patogênicos transmissíveis pelo sangue. Em algumas preparações, a concentração protéica de IgG e a distribuição das suas subclasses estavam fora das especificações. As preparações apresentaram atividade de anticorpos específicos contra os vírus das hepatites A e B, do herpes simples, da rubéola, citomegalovírus; contra a bactéria Streptococcus pyogenes β-hemolítico do grupo A e contra o parasita Toxoplasma gondii. A qualidade de matéria-prima utilizada em algumas das preparações de IgG não foi adequada em função de reações positivas para anticorpos contra alguns agentes infecciosos, tais como HTLV I/II, HAV, HBV, HCV e Treponema pallidum. Esse estudo também mostrou a necessidade de se implantar urgente um programa abrangente para avalição das preparações de IgG a serem consumidas pela população brasileira. / A family of glicoproteins, which are called antibodies or immunoglobulins (IgG), mediates the protective effects of humoral immunity. In Brazil, the IgG for intravenous use are imported from other countries. The first Brazilian immunoglobulin G for therapheutic use was obtained from human plasma at the Fundação Pró-Sangue Hemocentro de São Paulo. The present study was carried out to evaluate different preparations of IgG, human plasmad-derived, include the preparation from Brazil. The protein concentration, IgG subclass distribution, specific antibody activities and safety regarding the main blood transmitted infectious diseases were analyzed. In some preparations, IgG protein concentration and subclass distribution were different from their specifications. Some preparations showed specific antibody activity against the following antigens: A and B hepatitis virus, rubella, herpes simplex virus, citomegalovirus, measles virus, Streptococcus pyogenes β-hemolytic group A and Toxoplasma gondii. The presence of antibodies against antigens such as HTLV I/II, HAV, HBV, HCV and Treponema pallidum has compromissed the quality guaranty of the material-source (plasma) used in some preparations. This study has also showed that a complete and effective program for the quality evaluation of IgG preparations used in Brazil is needed and should be urgently established
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Estudo comparativo \'in vitro\' entre preparações de imunoglobulina \'G\', para uso intravenoso, obtidas de plasma humano de variadas procedências e processadas por diferentes técnicas de separação / \"In vitro\" comparative study between imunoglobulin G preparations, intravenous use, human plasma derived from different plasma sources and different separation techniquesGeny Aparecida de Oliveira Barna 26 June 2001 (has links)
Os efeitos protetores da imunidade humoral são medidas por uma família de glicoproteínas chamadas anticorpos ou imunoglobulinas. As preparações de imunoglobulina G (IgG) utilizadas em nosso país são importantes. No Brasil, a primeira preparação de IgG foi obtida na Fundação Pró-Sangue Hemocentro de São Paulo em 1993. O presente estudo avaliou preparações de IgG obtidas de misturas de plasma humano de variadas procedências, inclusive a preparação obtida no Brasil. Foram avaliados os seguintes parâmetros: concentração protéica, distribuição das subclasses da IgG, atividade de anticorpos específicos e segurança quanto a agentes patogênicos transmissíveis pelo sangue. Em algumas preparações, a concentração protéica de IgG e a distribuição das suas subclasses estavam fora das especificações. As preparações apresentaram atividade de anticorpos específicos contra os vírus das hepatites A e B, do herpes simples, da rubéola, citomegalovírus; contra a bactéria Streptococcus pyogenes β-hemolítico do grupo A e contra o parasita Toxoplasma gondii. A qualidade de matéria-prima utilizada em algumas das preparações de IgG não foi adequada em função de reações positivas para anticorpos contra alguns agentes infecciosos, tais como HTLV I/II, HAV, HBV, HCV e Treponema pallidum. Esse estudo também mostrou a necessidade de se implantar urgente um programa abrangente para avalição das preparações de IgG a serem consumidas pela população brasileira. / A family of glicoproteins, which are called antibodies or immunoglobulins (IgG), mediates the protective effects of humoral immunity. In Brazil, the IgG for intravenous use are imported from other countries. The first Brazilian immunoglobulin G for therapheutic use was obtained from human plasma at the Fundação Pró-Sangue Hemocentro de São Paulo. The present study was carried out to evaluate different preparations of IgG, human plasmad-derived, include the preparation from Brazil. The protein concentration, IgG subclass distribution, specific antibody activities and safety regarding the main blood transmitted infectious diseases were analyzed. In some preparations, IgG protein concentration and subclass distribution were different from their specifications. Some preparations showed specific antibody activity against the following antigens: A and B hepatitis virus, rubella, herpes simplex virus, citomegalovirus, measles virus, Streptococcus pyogenes β-hemolytic group A and Toxoplasma gondii. The presence of antibodies against antigens such as HTLV I/II, HAV, HBV, HCV and Treponema pallidum has compromissed the quality guaranty of the material-source (plasma) used in some preparations. This study has also showed that a complete and effective program for the quality evaluation of IgG preparations used in Brazil is needed and should be urgently established
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