Desarrollo y caracterización de aptámeros de RNA y DNA frente a la proteína ErbB3 binding protein-1 mediante procedimientos de selección y evolución in vitro

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-10-2016El principal objetivo de esta Tesis ha sido el desarrollo y caracterización de aptámeros específicos frente a la proteína ErbB3-binding protein 1 (Ebp1). Los aptámeros, de DNA y RNA, se obtuvieron mediante selección in vitro y evolución in vitro. Las poblaciones finales de los cuatro procesos se analizaron y compararon para detectar y caracterizar los mejores aptámeros, así como para evaluar las estrategias utilizables para la obtención de los mismos. Los aptámeros son moléculas de DNA de cadena sencilla o de RNA, seleccionadas a partir de poblaciones combinatoriales mediante un proceso conocido como SELEX (Systematic Evolution of Ligands by EXponential enrichment), que son capaces de unirse específicamente a un ligando determinado. En comparación con otras moléculas de unión (naturales o sintéticas) los aptámeros poseen propiedades químicas y bioquímicas únicas, entre las que destaca la posibilidad de ser seleccionados frente a cualquier tipo de diana, incluso en condiciones no fisiológicas. Desde la aparición de la tecnología SELEX, en 1990, se han obtenido numerosos aptámeros con alta especificidad y afinidad frente a un amplio rango de moléculas diana, entre ellas proteínas, ácidos nucleicos y complejos macromoleculares de gran relevancia en virología. En este trabajo se han desarrollado aptámeros frente a ErbB3-binding protein 1 (Ebp1), una proteína ITAF (IRES-trans acting factor) también conocida ITAF45 y PA2G4, que estimula la traducción de la poliproteína del virus de la fiebre aftosa (FMDV) mediante su unión al IRES viral. Los principales objetivos de esta Tesis fueron: I) Obtener aptámeros de ssDNA y de RNA frente a la proteína Ebp1, comparando los procesos de selección in vitro de ambos ácidos nucleicos (DS, RS) con los de evolución in vitro (DE, RE), realizados en paralelo; II) Combinar la especificidad de los aptámeros por sus dianas con la sensibilidad de la amplificación de ácidos nucleicos mediante PCR a tiempo real para conseguir un método de detección ultrasensible basado en el ELONA-qPCR; III) Aplicar el sistema de ELONA-qPCR desarrollado a la caracterización funcional (incluyendo la cuantificación de la constante de disociación, Kd, y la capacidad de unión máxima, Bmax) de los aptámeros obtenidos; IV) Identificar motivos cortos de secuencia/estructura presentes en los aptámeros de alta afinidad que podrían estar implicados en la interacción aptámero- Ebp1; y V) Realizar una caracterización preliminar de la estructura tridimensional del IRES de FMDV, de Ebp1 y de complejos IRES-Ebp1 mediante microscopía de fuerza atómica (AFM).This Thesis is aimed to the development and characterization of aptamers specific to the ErbB3-binding protein 1 (Ebp1). DNA and RNA aptamers have been developed by means of in vitro selection as well as by in vitro evolution. The final populations of the four processes were analyzed and compared in order to find the best aptamers, as well as to evaluate the strategies for aptamers development. Aptamers are single-stranded DNA or RNA molecules selected from large combinatorial libraries through a process referred to as SELEX (Systematic Evolution of Ligands by EXponential enrichment) that are able to specifically bind to a desired target molecule. As compared to other natural and synthetic binding molecules, aptamers possess unique chemical and biochemical features, including the ability to be selected against virtually any target, even in non-physiological conditions. Since the set up of SELEX technology in 1990, many aptamers have been developed with high specificity and affinity to a broad range of target molecules, some of them being proteins, nucleic acid regions and macromolecular assemblies highly relevant in virology. In this work, we have developed aptamers against the ErbB3-binding protein 1 (Ebp1), an IRES-trans acting factor also known as ITAF45 and PA2G4 that stimulates translation driven by the FMDV IRES. The main objectives of this Thesis were the following: I) To obtain DNA and RNA aptamers against the protein Ebp1, comparing in vitro selection methods (DS, RS) with in vitro evolution processes (DE, RE) performed in parallel; II) To combine the specificity of the aptamers for their target molecules with the sensitivity of real-time nucleic acid amplification to set up an ultrasensitive detection method based on ELONA-qPCR; III) To apply the developed ELONAqPCR system to the functional characterization (including the quantification of the dissociation constant, Kd, and maximum binding capacity, Bmax) of the obtained aptamers; IV) To identify short sequence/structure motifs present in the high affinity aptamers that could be involved in the aptamer-Ebp1 interaction; V) Finally, to perform a preliminary analysis of the three-dimensional structure of FMDV IRES, Ebp1 and IRES-Ebp1 complexes by means of atomic force microscopy (AFM)

Adaptive exposure estimation for high dynamic range imaging applied to natural scenes and daylight skies

Digital imaging of natural scenes and optical phenomena present on them (such as shadows, twilights, and crepuscular rays) can be a very challenging task because of the range spanned by the radiances impinging on the capture system. We propose a novel method for estimating the set of exposure times (bracketing set) needed to capture the full dynamic range of a scene with high dynamic range (HDR) content. The proposed method is adaptive to scene content and to any camera response and configuration, and it works on-line since the exposure times are estimated as the capturing process is ongoing. Besides, it requires no a priori information about scene content or radiance values. The resulting bracketing sets are minimal in the default method settings, but the user can set a tolerance for the maximum percentage of pixel population that is underexposed or saturated, which allows for a higher number of shots if a better signal-to-noise ratio (SNR) in the HDR scene is desired. This method is based on the use of the camera response function that is needed for building the HDR radiance map by stitching together several differently exposed low dynamic range images of the scene. The use of HDR imaging techniques converts our digital camera into a tool for measuring the relative radiance outgoing from each point of the scene, and for each color channel. This is important for accurate characterization of optical phenomena present in the atmosphere while not suffering any loss of information due to its HDR.We have compared our method with the most similar one developed so far [IEEE Trans. Image Process. 17, 1864 (2008)]. Results of the experiments carried out for 30 natural scenes show that our proposed method equals or outperforms the previously developed best approach, with less shots and shorter exposure times, thereby asserting the advantage of being adaptive to scene content for exposure time estimation. As we can also tune the balance between capturing time and the SNR in our method, we have compared its SNR performance against that of Barakat’s method as well as against a ground-truth HDR image of maximum SNR. Results confirm the success of the proposed method in exploiting its tunability to achieve the desired balance of total Δt and SNR

Evaluation of Cleaning Processes Using Colorimetric and Spectral Data for the Removal of Layers of Limewash from Medieval Plasterwork

In the cultural heritage preservation of medieval buildings, it is common to find plaster walls covered in lime, which previously were painted in polychromy. The conservation interventions usually try to remove the whitewash, whilst maintaining the original color of the painted wall as much as possible. However, there is no agreement on which cleaning technique best preserves the original appearance of the colored plaster. Different pigments found below the lime layer may behave differently depending on the cleaning technique used. Usually, colorimetric or photometric area-based measurements are carried out to study the color of the cleaned areas to compare with their original color, obtained from pre-made plaster probes. However, this methodology fails when the mean color difference is not enough to fully characterize the changes in texture and color appearance. This study presents a set of experiments carried out using two different pigments (cinnabar and malachite) covered with lime, and treated with nine different cleaning techniques on plaster probes prepared according to medieval techniques. We have studied the effect of the cleaning process on the color and the homogeneity of the samples using a hyperspectral imaging workflow. Four different analysis methods are presented and discussed. Our results show that the proposed analysis is able to provide a much more comprehensive and diversified characterization of the quality of the cleaning method compared to the commonly used colorimetric or photometric area-based measurements.Ministry of Economy and Competitiveness HAR2015-66139-PEuropean Union (EU) HAR2015-66139-

Colorimetric and spectral data analysis of consolidants used for preservation of medieval plasterwork

The incorporation of new methods of heritage analysis belonging to other branches of science is currently providing very useful tools for the examination of preservation products. This paper outlines the use of spectral images as an alternative to traditional colorimetry measurements carried out with a spectrophotometer for the evaluation of color changes produced by consolidation treatments applied on polychromed plasterwork. Thus, for this investigation we used a total of 18 plaster test specimens which reproduce the techniques and materials present in plasterwork from the medieval era, on which a selection of six currently-used consolidants was applied. By doing this, we have proved that the use of this method over traditional colorimetry has several advantages, such as the analysis of spatial homogeneity or obtaining colorimetric data of the entire scanned surface.Ministry of Economy andCompetitiveness and by the European Regional Development Fund(MINECO/ERDF, EU) within the framework of the Research ProjectHAR2015-66139-P, as well as by the Excellence Project of theAndalusian Regional Government P12-HUM-1941

Eight-Channel Multispectral Image Database for Saliency Prediction

Saliency prediction is a very important and challenging task within the computer vision community. Many models exist that try to predict the salient regions on a scene from its RGB image values. Several new models are developed, and spectral imaging techniques may potentially overcome the limitations found when using RGB images. However, the experimental study of such models based on spectral images is difficult because of the lack of available data to work with. This article presents the first eight-channel multispectral image database of outdoor urban scenes together with their gaze data recorded using an eyetracker over several observers performing different visualization tasks. Besides, the information from this database is used to study whether the complexity of the images has an impact on the saliency maps retrieved from the observers. Results show that more complex images do not correlate with higher differences in the saliency maps obtained.Spanish Ministry of Science, Innovation, and Universities (MICINN) RTI2018-094738-B-I00European Commissio

Tratamientos de aguas residuales industriales con materia orgánica no biodegradable

El proyecto consiste en presentar los distintos tratamientos para la eliminación o reducción de materia orgánica no biodegradable en aguas residuales ndustriales. Se estudia el consumo, uso y características de las aguas residuales industriales, así como los contaminantes que presentan, dependiendo del tipo de industrias de las que proceden, haciendo hincapié en las aguas residuales industriales con materia orgánica no biodegradable, objeto del proyecto. Se lleva a cabo una recopilación de los distintos tratamientos que se utilizan en la actualidad, y de las nuevas tecnologías que se están desarrollando. Se describen los procesos de funcionamiento, sus ventajas e inconvenientes y aplicaciones más importantes, indicando uno o varios ejemplos a nivel industrial. Finalmente se desarrolla un ejemplo aplicado a un vertido industrial, procedente de una planta perteneciente a la industria petroquímica, comparando las distintas técnicas estudiadas, y seleccionando la tecnología más adecuada

Participación y salud mental en primera persona: toma de decisiones en el proyecto vital propio y asociacionismo

La participación de personas con diagnóstico de salud mental está repleta de claroscuros, de intentos por fomentarla pero también de obstáculos que la frenan, herencia de un modelo de atención estigmatizante basado en lo sintomático, en lo enfermo, y no en la integralidad y el reconocimiento de la persona y su recuperación como objeto. Las propuestas de participación comprometida no se pueden permitir abarcar tan solo lo individual, de tal forma que el reto consiste en transmutar, en primera persona, por convicción y mediante acción directa, lo individual y lo fragmentado, las demandas asistencialistas, en colectivo y estructural, en reivindicaciones y lucha por los derechos de todas las personas que presentan un problema de salud mental. No obstante, ni las condiciones materiales ni las herramientas para construir y articular un movimiento social fuerte y combativo se crean solas, así que es tarea de los profesionales de lo social, desde su humilde parcela de intervención, repensar cómo contribuir a que esta necesidad se convierta en realidad.<br /

Characterization of the evolution of indigo blue by multispectral imaging

FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento, Grant/Award Number: A-HUM-164-UGR18; Ministry of Economy and Competitiveness of Spain, Grant/Award Number: DPI2015-64571-R; Spanish Ministry of Economy and Competitiveness, Grant/Award Number: Ramon y Cajal program; Ministry of Economy and Competitiveness; Junta de Andalucia; FEDER (European Regional Development Fund).Indigo blue is a natural organic color from the Indigofera tinctoria plant. It is insoluble in water and does not require mordant to fix the color; therefore, due to its nature it can be considered as a pigment or dye. The identification of indigo in cultural heritage is very difficult due to the complexity of its chemical formula and the changes that it can undergo in certain conservation and exposition conditions. For this reason, characterization of possible degradation, transformation, and reaction processes is essential for its identification. In this study, multispectral imaging has been used for the first time as noninvasive technique to characterize the aging state of indigo blue using samples aged in a controlled climate chamber under extreme conditions of humidity, temperature, and light. The technique has proven to be very promising for this application, together with classification techniques based on machine learning approaches.FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento A-HUM-164-UGR18Ministry of Economy and Competitiveness of Spain DPI2015-64571-RSpanish Ministry of Economy and CompetitivenessMinistry of Economy and CompetitivenessJunta de AndaluciaEuropean Commissio