Uso de microfluorescencia de rayos X y otras técnicas nucleares no destructivas para el estudio de joyas y metales arqueológicos

Hoy en día, el estudio de objetos antiguos de metal incluye el análisis por medio de métodos físico-químicos no destructivos desarrollados en las décadas pasadas. Uno de los parámetros más importantes que deben conocerse de una muestra es su composición química, ya que esta información contribuye significativamente a la determinación de varias suposiciones y a la respuesta de muchas preguntas que surgen mientras se estudia un objeto de gran valor histórico. La fluorescencia de rayos X es una técnica bien conocida y muy empleada en el análisis elemental de objetos antiguos de metal y el nuevo desarrollo del microhaz focalizado de rayos X descrito en este texto incrementa su adecuación para el estudio de técnicas antiguas de fabricación y decoración. Esta técnica de análisis superficial se complementa con la técnica de transmisión de rayos gamma, que ha sido utilizada para la estimación de la composición en el interior de monedas de plata-cobre y objetos realizados en otras aleaciones del cobre. Describimos también el sistema de microfluorescencia confocal portátil desarrollado por nuestro Grupo para la obtención de perfiles en profundidad de forma no destructiva.The study of ancient metal artefacts includes nowadays the non-destructive analysis by means of chemical-physical methods developed in the past decades. One of the most important parameters to be known from a sample is its chemical composition, as this information significantly contributes to the determination of several assumptions and to the response of many questions arisen from the study of a valuable heritage object. X-ray fluorescence is a well-known technique very much employed for the elemental analysis of ancient metal artefacts and the new implementation of a microfocused X-ray beam described in this text increases its applicability for the study of manufacture and decorative ancient techniques. This surface analytical method is complemented with the Gamma Ray Transmission technique, which has been employed in the bulk composition estimation of silver-copper coins and other copper based alloy samples. We also describe a portable confocal microfluorescence system developed by our Group to obtain non-destructive depth profiles.Ministerio de Economía y Competitividad (España) HAR2012-33002Ministerio de Economía y Competitividad (España) HAR2015-67113-PJunta de Andalucía P09-HUM454

Caracterización de un vidrio rojo medieval procedente de las vidrieras del Monasterio de las Huelgas de Burgos

En el año 2007 se culminó el proceso de restauración integral de las vidrieras que cierran los ventanales de la Sala Capitular del Claustro de S. Fernando del Real Monasterio de Santa María la Real de las Huelgas de Burgos. Un vidrio rojo, de características peculiares, extraído durante el proceso de conservación-restauración de una de las vidrieras, se ha caracterizado mediante las técnicas de Microscopía óptica de luz reflejada (MOLR), Microscopía electrónica de emisión de campo (MEEC), Fluorescencia de rayos X (FRX), Emisión de rayos X inducidos por partículas (PIXE) y Microscopía electrónica de transmisión (MET) para conocer su composición química y su estructura. Los análisis realizados por las técnicas de FRX y PIXE permiten comprobar la buena concordancia que existe entre los resultados. El vidrio analizado presenta una sección transversal muy original, formada por un vidrio soporte ligeramente verdoso y múltiples capas de color rojo, característico de los siglos XIII y XIV. El análisis mediante micro-PIXE en cámara de vacío ha permitido obtener información sobre la distribución en profundidad de los elementos que lo componen.In the year 2007 the process of conservation-restoration of the windows of the apostles that close the windows of the Chapter Hall of Cloister of S. Fernando del Real Monasterio de Santa María la Real de las Huelgas de Burgos was completed the process. A red glass obtained from the process of conservation and restoration has been characterized by Optical microscopy by light reflected (OMLR), Field emission scanning electron microscopy (FE-SEM), X ray fluorescence espectrpmetry (XRF), Particle induced X ray emission (PIXE), Transmission electron microscopy (TEM) to identify the chemical composition and structure. Chemical analys by XRF and PIXE are in the good agreement. The studied glass has a very original cross section, consisting of a lightly greenish glass holder and multilayers of red glass, characteristic of the thirteenth and fourteenth centuries. A elemental mapping of the cross-section showing the distribution of elements in the glass has been obtained by micro PIXE analysis in a vacuum chamber

Nuevo sistema portátil de micro-fluorescencia de rayos X basado en óptica de policapilares para aplicaciones en Patrimonio Cultural

Ministerio de Ciencia e Innovación (España) HAR2009-07449Junta de Andalucía P09-HUM454

Desmetilación activa del ADN: un mecanismo epigenético para la reactivación de genes silenciados

Los mecanismos de control epigenético son esenciales para una regulación estable de los patrones de expresión génica y desempeñan un papel central en los ciclos de vida de animales y plantas. La metilación de la citosina en el carbono 5 del anillo de pirimidina (5-meC) es una marca epigenética estable, pero reversible, que promueve el silenciamiento génico transcripcional. Comprender cómo se regula el estado de metilación del genoma a nivel global o local requiere una definición de los procesos enzimáticos que metilan y desmetilan el ADN. Sin embargo, aunque las enzimas responsables del establecimiento y mantenimiento de la metilación de ADN han sido bien caracterizadas, los mecanismos de desmetilación no se conocen con exactitud. Nuestro grupo, junto con otros, ha obtenido datos genéticos y bioquímicos que sugieren que dos proteínas de Arabidopsis con dominio ADN glicosilasa (ROS1 y DME) actúan como ADN desmetilasas capaces de activar la expresión de genes previamente silenciados. Nuestros resultados previos indican que ROS1 y DME catalizan la liberación de 5-meC del ADN mediante un mecanismo ADN glicosilasa. Estos resultados sugieren que una de las funciones de ROS1 y DME es iniciar el borrado de 5-meC mediante un proceso de escisión de bases y proporcionan una importante evidencia bioquímica a favor de la existencia de una ruta de desmetilación activa en plantas. En la actualidad, nuestro grupo de investigación se concentra en caracterizar funcionalmente este novedoso mecanismo de control epigenético mediante una aproximación multidisciplinar que combina metodologías del campo de la bioquímica, la genética y la biofísica. Este estudio suministrará una información esencial para entender los mecanismos responsables de la reprogramación epigenética en el núcleo celular, con aplicaciones potenciales en biotecnología y biomedicin

The life of the peroxisome: from birth to death

Peroxisomes are dynamic and metabolically plastic organelles. Their multiplicity of functions impacts on many aspects of plant development and survival. New functions for plant peroxisomes such as in the synthesis of biotin, ubiquinone and phylloquinone are being uncovered and their role in generating reactive oxygen species (ROS) and reactive nitrogen species (RNS) as signalling hubs in defence and development is becoming appreciated. Understanding of the biogenesis of peroxisomes, mechanisms of import and turnover of their protein complement, and the wholesale destruction of the organelle by specific autophagic processes is giving new insight into the ways that plants can adjust peroxisome function in response to changing needs

ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion

Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation

A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine

DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases

Epidemiología básica. Material docente para prácticas en ciencias de la salud

Material docente teórico práctico para apoyar la enseñanza de la epidemiología. Consta de 4 unidades, cada una de ellas con una introducción teórica y unos ejercicios basados en artículos publicados en inglés en revistas científicas

Antioxidant Systems are Regulated by Nitric Oxide-Mediated Post-translational Modifications (NO-PTMs)

Nitric oxide (NO) is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs) such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS), such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs) peroxiredoxins (Prx) and all the enzymatic components of the ascorbate-glutathione (Asa-GSH) cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance.JB-M would like to thank the Ministry of Science and Innovation for funding the Ph.D. fellowship (F.P.U.). This study was supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (project BIO2012-33904), Junta de Andalucía (P10-AGR-6038 and groups BIO286 and BIO192) and RECUPERA2020 in Spain.Peer reviewedPeer Reviewe

Single-nucleotide and long-patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell-free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′- or 3′-blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short-patch BER) or several nucleotides (long-patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways