RESUMO
Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional "housekeeping" genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.
Assuntos
Cobre/farmacologia , Grupo dos Citocromos c/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zinco/farmacologia , Linhagem Celular , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/normas , Perfilação da Expressão Gênica , Humanos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de ReferênciaRESUMO
Copper is an essential cofactor of complex IV of the electron transfer chain, and it is directly involved in the generation of mitochondrial membrane potential. Its deficiency induces the formation of ROS, large mitochondria and anemia. Thus, there is a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis. Copper depletion might end in cellular apoptosis or necrosis. However, before entering into those irreversible processes, mitochondria may execute a series of adaptive responses. Mitochondrial adaptive responses (MAR) may involve multiple and diverse mechanisms for preserving cell life, such as mitochondrial dynamics, OXPHOS remodeling and bioenergetics output. In this study, a mild copper deficiency was produced in an animal model through intraperitoneal injections of bathocuproine disulfonate in order to study the MAR. Under these conditions, a new type of mitochondrial morphology was discovered in the liver. Termed the "butternut squash" mitochondria, it coexisted with normal and swollen mitochondria. Western blot analyses of mitochondrial dynamics proteins showed an up-regulation of MFN-2 and OPA1 fusion proteins. Furthermore, isolated liver mitochondria displayed OXPHOS remodeling through a decrease in supercomplex activity with a concomitant increase at an individual level of complexes I and IV, higher respiratory rates at complex I and II levels, higher oligomycin-insensitive respiration, and lower respiratory control ratio values when compared to the control group. As expected, total ATP and ATP/ADP values were not significantly different, since animal's health was not compromised. As a whole, these results describe a compensatory and adaptive response of metabolism and bioenergetics under copper deprivation.
Assuntos
Adaptação Fisiológica/fisiologia , Cobre/deficiência , Metabolismo Energético/fisiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Animais , Quelantes/farmacologia , Cobre/metabolismo , Masculino , Camundongos , Fenantrolinas/farmacologia , Espécies Reativas de OxigênioRESUMO
El presente artículo surge de la revisión documental del concepto violencias escolares en bases de datos, artículos de revista, bibliotecas y demás medios de información, dentro de la investigación: "Violencias escolares presentes en las ins tituciones educativas católicas. Una aproximación a los discursos y prácticas en el Colegio Parroquial Emaús de la ciudad de Medellín", de la Maestría en Intervenciones Psicosociales de la Fundación Universitaria Luis Amigó. La búsqueda documental arrojó tres categorías: Bullying, violencias de los escolares y conflicto escolar, que alimentaron la construcción en torno a la violencia escolar, evidenciando la necesidad de pluralizar el concepto, más allá de tipificarlo en el bullying, para abarcar sus diversas manifestaciones.
This article comes from the review of documents in databases, journal articles, libraries and other ways, in research "School Violence present in Catholic educational institutions. An approach to the discourses and practices in Emaús Parish School of Medellin", within the Maestría en Intervenciones Psicosociales de la Fundación Universitaria Luis Amigó. During the search we found three theoretical framework: Bullying, School Violence, and Conflict School, which fueled construction around school violence evidencing the need to pluralize the concept beyond typify in Bullying, to cover its various manifestations.
Assuntos
Humanos , Bullying , Estudantes/psicologia , Psicologia do Adolescente , Agressão/éticaRESUMO
Copper is an essential micronutrient that functions as an enzymatic cofactor in a wide range of cellular processes. Although adequate Cu levels are essential for normal metabolism, excess Cu can be toxic to cells. Cellular responses to copper deficiency and overload involve changes in the expression of genes directly and indirectly involved in copper metabolism. However little is known on the effect of physiological copper concentration on gene expression changes. In the current study we aimed to establish whether the expression of genes encoding enzymes related to cholesterol (hmgcs1, hmgcr, fdft) and fatty acid biosynthesis and LDL receptor can be induced by an iso-physiological copper concentration. The iso-physiological copper concentration was determined as the bioavailable plasmatic copper in a healthy adult population. In doing so, two blood cell lines (Jurkat and THP-1) were exposed for 6 or 24 h to iso- or supraphysiological copper concentrations. Our results indicated that in cells exposed to an iso-physiological copper concentration the early induction of genes involved in lipid metabolism was not mediated by copper itself but by the modification of the cellular redox status. Thus our results contributed to understand the involvement of copper in the regulation of cholesterol metabolism under physiological conditions.