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20. Desenvolvimento de processo de recuperação de aprotinina a partir de efluente industrial centrado em cromatografia em quimotripsina imobilizada

Author

Genaro, Ana Carolina Barros de, Miranda, Everson Alves, 1959, Bueno, Sonia Maria Alves, Silva, João Batista de Almeida e, Universidade Estadual de Campinas. Faculdade de Engenharia Química, Programa de Pós-Graduação em Engenharia Química, and UNIVERSIDADE ESTADUAL DE CAMPINAS

Subjects
Hidroxiapatita, Inibidores de proteases, Proteínas - Purificação, Cromatografia de afinidade
Abstract

Orientador: Everson Alves Miranda Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica Resumo: A aprotinina é um inibidor de serino-proteases presente em órgãos bovinos, utilizado como composto farmacêutico em operações cirúrgicas e em cultura de células. Atualmente a aprotinina não é comercialmente produzida no Brasil. O objetivo deste trabalho foi desenvolver um processo de recuperação de aprotinina a partir de efluente industrial da produção de insulina bovina (SPI), centrado em cromatografia por quimotripsina imobilizada. Foram estudadas três estratégias: a) cromatografia em quimotripsina imobilizada; b) cromatografia em hidroxiapatita seguida de cromatografia em quimotripsina imobilizada e c) cromatografia em quimotripsina imobilizada seguida de cromatografia de afinidade por íon metálico imobilizado (IMAC) utilizando cobre como ligante. A eficiência do processo foi analisada através ensaios de inibição de tripsina e quimotripsina, "dot blot" e eletroforese das frações cromatográficas. A primeira estratégia resultou na recuperação e purificação de aprotinina, da coluna em quimotripsina imobilizada, principalmente nos valores de pH 4,0 e 3,0, quando realizada dessorção em degrau de pH. Na segunda estratégia inicialmente estudou-se o efeito da influência do pH e concentração de NaCI na adsorção de aprotinina de alta pureza na hidroxiapatita. Um planejamento estatístico experimental indicou como condição de alta eficiência tampão fosfato 1 mM, pH 6,5 e 18 mM de NaCI. Dentre 11 soluções estudadas, cloreto de cálcio 3 mM foi a que apresentou melhores índices de dessorção. Entretanto esta segunda estratégia não foi eficiente quando o SPI foi aplicado ao processamento, provavelmente devido a baixa especificidade da hidroxiapatita frente a aprotinina no meio. A terceira estratégia foi a melhor opção estudada para a recuperação e purificação da aprotinina. IMAC foi eficiente como etapa final da purificação depois da recuperação em quimotripsina imobilizada: fatores de purificação global foram tão altos quanto 952 Abstract: Aprotinin is a pharmaceutical compound used in surgeries and tissue cultures, currently not commercially produced in Brazil. It is a serine-protease inhibitor present in bovine organs. The objective of this work was to develop a process for the recovery of aprotinin from industrial effluent of bovine insulin production (SPI), based on affinity chromatography onto immobilized chymotrypsin. Three approaches were used: a) chromatography onto immobilized chymotrypsin; b) chromatography onto hydroxyapatite followed by chromatography onto immobilized chymotrypsin; and c) chromatography on immobilized chymotrypsin followed by immobilized metal ion affinity chromatography (IMAC) with cupper as the ligant. Efficiency was analysed based on trypsin and chymotrypsin inhibitions, dot blotting, and electrophoresis assays of the chromatographic fractions. The first approach resulted in aprotinin recovery and purification from the chymotrypsin column with pH step elution gradient, mainly at pH 4.0 and 3.0. The second approach was initiated by studying the effects of phosphate buffer solution pH and NaCI concentration on high purity aprotinin adsorption from buffer solutions onto hydroxyapatite. This study was carried out according to a factorial experimental design and indicated 18 mM NaCI and a pH of 6.5 as a high efficiency adsorption condition. Among 11 solutions tested, 3 mM calcium chloride was selected as the best eluent. However, this second approach was not efficient when applied to the processing of SPI probably due to the low specificity of hydroxyapatite towards aprotinin in this medium. The third approach was the best option studied for aprotinin recovery and purification. IMAC was efficient in further purifying aprotinin after its recovery and purification with the chymotrypsin chromatography: overall purification factors were as high as 952 Mestrado Desenvolvimento de Processos Biotecnológicos Mestre em Engenharia Química

Published
2021
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29. Influence of prenatal stress on metabolic abnormalities induced by postnatal intake of a high-fat diet in BALB/c mice

Author

Juárez, Yamila R., Quiroga, Sofía, Prochnik, Andrés, Wald, Miriam Ruth, Tellechea, Mariana L., Genaro, Ana María, and Burgueño, Adriana

Subjects
STRESS PRENATAL, GENES, OBESIDAD, METABOLISMO
Abstract

Fil: Juarez, Yamila. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Quiroga, Sofía. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Prochnik, Andrés. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Wald, Miriam Ruth. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tellechea, Mariana L. Hospital de Niños Ricardo Gutiérrez. División de Endocrinología. Fundación de Endocrinología Infantil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones Endocrinológicas “Dr. César Bergadá”; Argentina Fil: Genaro, Ana María. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Genaro, Ana María. Universidad de Buenos Aires. Facultad de Ciencias Médicas. Departamento de Farmacología; Argentina Fil: Burgueño, Adriana Laura. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Abstract: Prenatal insults during fetal development result in increased likelihood of developing chronic disease. Obesity, the biggest risk factor for the development of metabolic disease, is affected by several genetic and environmental factors. High-fat diet (HFD) consumption is usually linked with the development of obesity. The main goal of this study was to analyze the impact of the exposure to a HFD in prenatally stressed animals. For this purpose, we subjected pregnant BALB/c mice to restraint stress for 2 h a day between gestational day (GD) 14 and GD 21. Prenatally stressed and control offspring of both sexes were postnatally exposed to a HFD for 24 weeks. We found that prenatal stress (PS) per se produced disturbances in males such as increased total blood cholesterol and triglycerides, with a decrease in mRNA expression of sirtuin-1.When these animals were fed a HFD, we observed a rise in glucose and insulin levels and an increase in visceral adipose tissue gene expression of leptin, resistin, and interleukin-1 beta. Although females proved to be more resilient to PS consequences, when they were fed a HFD, they showed significant metabolic impairment. In addition to the changes observed in males, females also presented an increase in body weight and adiposity and a rise in cholesterol levels.

Published
2020

30. Thyroid hormones increase inducible nitric oxide synthase gene expression downstream from PKC-[zeta] in murine tumor T lymphocytes

Author

Barreiro Arcos, Maria Laura, Gorelik, Gabriela, Klecha, Alicia, Genaro, Ana Maria, and Cremaschi, Graciela A.

Subjects
Thyroid hormones -- Properties, Cell proliferation -- Research, Gene expression -- Research, Protein kinases -- Research, Thyroxine -- Properties, T cells -- Physiological aspects, Biological sciences
Abstract

Regulation of cell proliferation by thyroid hormone (TH) has been demonstrated, but the effect of THs and the mechanisms involved in lymphocyte activity have not been elucidated. Differential expression of PKC isoenzymes and high nitric oxide synthase (NOS) activity have been described in tumor T lymphocytes. We have analyzed the direct actions of TH on normal T lymphocytes and BW5147 T lymphoma cells in relation to PKC and NOS activities. THs increased tumor and mitogen-induced normal T lymphocyte proliferation. PKC isoenzyme-selective blockers impaired these effects in both cell types, indicating the participation of [Ca.sup.2+]-dependent and -independent isoenzymes in normal and tumor cells, respectively. TH actions were blunted by extra- and intracellular [Ca.sup.2+] blockers only in normal T lymphocytes, whereas NOS blockers impaired TH-induced proliferation in T lymphoma cells. Incubation for 24 h with TH induced a rise in total and membrane-associated PKC activities in both cell types and led to a rapid and transient effect only in tumor cells. THs increased atypical PKC-[zeta] expression in BW5147 cells and classical PKC isoenzymes in mitogen-stimulated normal T cells. TH augmented NOS activity and inducible NOS protein and gene expression only in tumor cells. Blockade of PKC and the atypical PKC-[zeta] isoform inhibited TH-mediated stimulation of inducible NOS and cell proliferation. These results show, for the first time, that differential intracellular signals are involved in TH modulation of lymphocyte physiology and pathophysiology. 3,5,3'-l-triiodothyronine; thyroxine; T lymphoma; protein kinase C isoenzymes doi:10.1152/ajpcell.00316.2005

Published
2006

45. Influence of prenatal stress on metabolic abnormalities induced by postnatal intake of a high-fat diet in BALB/c mice

Author

Juárez, Yamila Raquel, Quiroga, Sofía, Prochnik, Andrés, Wald, Miriam, Tellechea, Mariana Lorena, Genaro, Ana María, and Burgueño, Adriana Laura

Abstract

AbstractPrenatal insults during fetal development result in increased likelihood of developing chronic disease. Obesity, the biggest risk factor for the development of metabolic disease, is affected by several genetic and environmental factors. High-fat diet (HFD) consumption is usually linked with the development of obesity. The main goal of this study was to analyze the impact of the exposure to a HFD in prenatally stressed animals. For this purpose, we subjected pregnant BALB/c mice to restraint stress for 2 h a day between gestational day (GD) 14 and GD 21. Prenatally stressed and control offspring of both sexes were postnatally exposed to a HFD for 24 weeks. We found that prenatal stress (PS) per seproduced disturbances in males such as increased total blood cholesterol and triglycerides, with a decrease in mRNA expression of sirtuin-1. When these animals were fed a HFD, we observed a rise in glucose and insulin levels and an increase in visceral adipose tissue gene expression of leptin, resistin, and interleukin-1 beta. Although females proved to be more resilient to PS consequences, when they were fed a HFD, they showed significant metabolic impairment. In addition to the changes observed in males, females also presented an increase in body weight and adiposity and a rise in cholesterol levels.

Published
2021
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