Actas de SABI2020

Los temas salientes incluyen un marcapasos pulmonar que promete complementar y eventualmente sustituir la conocida ventilación mecánica por presión positiva (intubación), el análisis de la marchaespontánea sin costosos equipamientos, las imágenes infrarrojas y la predicción de la salud cardiovascular en temprana edad por medio de la biomecánica arterial

Development of enabling technologies for single cell analysis with mass spectrometry

Mass spectrometry (MS) is an effective methodology for untargeted, label-free, highly multiplexed analyses of trace compounds based on their mass-to-charge ratios. For biological applications, these properties have generated interest in determining biomarkers of diseased states, detecting drug compounds and metabolites, and observing previously unknown chemical messengers. Recent developments in instrumentation have provided exquisite sensitivity with robust performance. A growing field of single cell chemical analysis has arisen around these figures of merit. While early reports utilized manual isolation and extraction, recent developments in high-throughput sampling have enabled the examination of large populations of cells. One such method includes the analysis of dispersed single cells on a flat surface. When cells are randomly seeded onto the surface, their locations have to be determined by optical imaging to direct acquisition of isolated cells efficiently. A variety of microprobe ionization sources are suitable for such analyses, though smaller probe footprints can utilize more densely seeded samples. This dissertation describes two technologies for performing single cell analysis with mass spectrometry. The first, synchronized desorption electrospray ionization (DESI), facilitates ambient ionization MS with high mass resolution, low duty cycle mass analyzers. The initial report utilized synchronized DESI for mass spectrometry imaging, but interrupting the desorption plume would be useful for profiling several locations on a surface in an arbitrary order for single cell analysis. The second methodology utilizes microscopy images to guide MS profiling. Specifically, image analysis software, called microMS, was developed to perform cell finding and correlate optical coordinates with the physical coordinates in a mass spectrometer. Since most of the functionality of microMS is decoupled from the mass spectrometer, the workflow can be easily extended to a variety of instruments. Using matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)-MS, rodent pancreatic islet cells were investigated and heterogeneous peptide processing was detected at the single cell level. With secondary ion mass spectrometry, disparate tissue from the mammalian nervous system was differentiated and further stratified into separate populations. A unique feature of such analyses is that only a fraction of the sample is consumed and the location of a cell is constant once the sample is dried. This property greatly simplifies sequential, follow-up analysis. As an example, MALDI-TOF-MS was utilized to rapidly screen a population of islet cells to select alpha and beta cell types. The locations of those cells were then targeted for liquid microjunction extraction in order to examine their metabolite profiles with capillary electrophoresis-MS. Finally, while microscopy-guided MS profiling is accurate enough to target single cells, the methodology is flexible enough to analyze much larger samples, including tissue sections or bacterial colonies. As an application, natural product mutant libraries were screened directly from E. coli colonies using microMS. The suite of technologies and protocols described increases the applicability of many mass spectrometers to characterize a range of cells, colonies and similar objects for their chemical composition

Estudio del armazón arquitectónico y del sistema vascular de los tumores neuroblásticos

Los pacientes con tumores neuroblásticos presentan una evolución clínica heterogénea, desde la regresión espontánea hasta una alta propensión para la diseminación metastática generalizada. Aunque la aplicación de una clasificación de riesgo pre-tratamiento bien definida tiene un papel central en la mejora de la supervivencia durante los últimos años, han de llevarse a cabo más avances para mejorar la superviencia de los pacientes en general y específicamente el subgrupo de pacientes de alto riesgo. El estudio morfológico del tejido tumoral está contribuyendo a dicha mejora. La categoría histológica o el porcentaje de estroma tumoral, así como el grado de diferenciación de las células neuroblásticas, determinadas por el patólogo con el microscopio óptico, son factores con un papel importante en el diagnóstico y el pronóstico de los pacientes. Actualmente, dada la relevancia de la matriz extracelular tumoral en la biotensegridad y la mecanotransducción, su arquitectura y la topología de sus elementos, así como su interacción están siendo cada vez más considerados. Su cuantificación y caracterización con técnicas de imagen microscópicas empiezan a ser utilizadas. Nuestra hipótesis es que el destino de una célula tumoral neuroblástica es complejo y entre otros factores, está determinado por las características de un grupo de elementos estructurales no celulares de la matriz extracelular. Además pensamos que aplicando los patrones derivados del análisis morfométrico de estos elementos y asociandolos al impacto de los factores pronósticos conocidos, se mejorará la supervivencia de los pacientes. Nuestro objetivo es el desarrollo de técnicas morfométricas para caracterizar distintos elementos del andamiaje de la matriz extracelular y de la vascularización con el fin de encontrar usos potenciales como nuevos marcadores con valor pronóstico para mejorar la estratificación de los pacientes, o como dianas terapéuticas para ser capaces de remodelar los elementos aberrantes del andamiaje tisular, incluyendo la microvascularización. Hemos construido 19 micromatrices de tejido incluyendo más de 500 neuroblastomas, que fueron teñidos con alzul alcián a pH 2,5, Gomori, tricrómico de Masson, orceína y anti-CD31 para glicosaminoglicanos, fibras de reticulina, fibras de colágeno tipo I, fibras elásticas y vasos sanguíneos, respectivamente. Las laminillas fueron digitalizadas con un escáner de preparaciones y distintos algoritmos de análisis de imagen fueron diseñados o personalizados para detectar y caracterizar la cantidad, el tamaño y la forma de los distintos elementos estudiados de la matriz extracelular. Estos parámetros se relacionaron con los distintos subgrupos de neuroblastoma, teniendo en cuenta varias características clínicas, histopatológicas y genéticas. Los resultados obtenidos mostraron que las fibras de reticulina eran los componentes mayoritarios del andamiaje fibroso y que la abundancia y arquitectura de la microvascularización era relevante para el pronóstico de los niños con neuroblastoma. Una matriz extracelular rígida y poco porosa con vasos sanguíneos con luces irregulares se detectó principalmente en tumores pertenecientes a pacientes con pronóstico desfavorable. Un subgrupo de la cohorte de alto riesgo con muy mala supervivencia pudo ser definido por variables morfométricas de las fibras de reticulina y de los vasos sanguíneos. Concretamente, las muestras con un mayores áreas ocupadas tanto por fibras de reticulina formando grandes redes entrecruzadas, ramificadas y de organización compleja, como por vasos sanguíneos, junto con capilares y vasos tipo sinusoide de forma irregular y vénulas y arteriolas dilatas, estaban asociadas a un pronóstico muy desfavorable. En esta cohorte, las células con amplificación del gen MYCN conllevaron cambios topológicos detectables en relación a las fibras de reticulina y los vasos sanguíneos. Podemos concluir que es possible y conveniente cuantificar la sustancia fundamental, caracterizar el andamiaje fibroso y el sistema vascular de los tumors neuroblásticos gracias al análisis morfométrico de imágenes microscópicas. Algunas de las características morfométricas relaciondas con los distintos elementos de la matriz extracelular estudiados podrían ser usadas como ayuda diagnóstica del grupo de pacientes con riesgo ultra alto, tras estudiar una mayor cohorte. Los resultados obtenidos sugieren la necesidad de realizar trabajos multidisciplinarios para integrar de estos estudios a nivel internacional y que la información morfométrica de los elementos de la matriz extracelular, incluyendo el sistema vascular, pueda ser utilizada para una terapia basada en la mecanotransducción.Neuroblastic tumor patients present an heterogeneous clinical evolution, from spontaneous regression to a high propensity for widespread metastatic dissemination. Although the application of a well-defined pre-treatment risk classification plays a central role in the improvement of survival during the last years, more efforts must be done to improve patient’s survival in general and specifically in the subgroup of high risk patients. The morphological study of the tumoral tissue is contributing to such improvement. The histological category or the percentage of tumoral stroma, as well as the degree of differentiation of neuroblastic cells, evaluated by the pathologist with light microscopy, are factors that play a role in the diagnosis and prognosis of the patients. Given the role of tumoral extracellular matrix in biotensegrity and mechanotransduction, its architecture and the topology of its elements, as well as their interaction are being increasingly considered. Its quantification and characterization with microscopic image techniques start to be used. We hypothesize that the destiny of a neuroblastic tumor cell is complex and, is in part directed by characteristics of a set of non-cellular extracellular matrix structural elements. Additionally, we think that the application of the patterns derived from the morphometric analysis of such elements and their association with the impact of the known prognostic factors, patient’s survival will be improved. We aim to develop morphometric techniques to characterize different extracellular matrix scaffolding and vascular elements to find out potential uses as new prognostic markers for a better pre-treatment stratification of the patients or as therapeutic targets to be able to remodel the aberrant elements of the tissue scaffolding, including microvascularization. We constructed 19 tissue microarrays including more than 500 neuroblastomas which were stained with alcian blue pH 2.5, Gomori, Masson’s trichrome, orcein and anti-CD31 for glycosaminoglycans, reticulin fibers, collagen type I fibers, elastic fibers and blood vessels, respectively. The slides were digitized with a whole-slide scanner and different image-analysis algorithms were designed or customized to specifically detect and characterize the amount, the size and the shape of the different extracellular matrix elements studied. These parameters were related to different neuroblastoma subgroups, taking into account several clinical, histopathological and genetic features. The results obtained showed that reticulin fibers were the main components of the fibrous scaffolding and that microvasculature amount and architecture were relevant in the prognosis of neuroblastoma patients. A stiff and poorly porous extracellular matrix with irregularly-shaped vascular lumens was mainly detected in tumors belonging to patients with unfavorable prognosis. A subgroup of the high risk cohort with very poor survival could be defined by morphometric variables of reticulin fibers and blood vessels. Specificallly, those samples with high stained areas occupied by reticulin fibers forming large, crosslinking, branching and disorganized networks and by blood vessels, as well as with irregularly-shaped capillaries and sinusoid-like vessels and dilated venules, presented a very unfavorable survival. In this cohort, cells with MYCN gene amplification led to detectable topological changes regarding reticulin fibers and bood vessels. We can conclude that it is possible and convenient to quantify the fundamental substance and characterize the architecture of the fibrous scaffolding and the vascular system of neuroblastic tumors by means of the morphometric analysis of microscopic images. Some of the morphometric features related to the different extracellular matrix elements studied could be used as a diagnostic support for the ultra-high risk group of patients, after studying a larger cohort. The obtained results suggest the need of developing multidisciplinary efforts for an international integration of these studies, and that the morphometric information of the elements of the extracellular matrix, including the vascular system, could be used for a therapy based on mechanotransduction

Actas de las VI Jornadas Nacionales (JNIC2021 LIVE)

Estas jornadas se han convertido en un foro de encuentro de los actores más relevantes en el ámbito de la ciberseguridad en España. En ellas, no sólo se presentan algunos de los trabajos científicos punteros en las diversas áreas de ciberseguridad, sino que se presta especial atención a la formación e innovación educativa en materia de ciberseguridad, y también a la conexión con la industria, a través de propuestas de transferencia de tecnología. Tanto es así que, este año se presentan en el Programa de Transferencia algunas modificaciones sobre su funcionamiento y desarrollo que han sido diseñadas con la intención de mejorarlo y hacerlo más valioso para toda la comunidad investigadora en ciberseguridad

Mapping Diffusion Properties in Living Cells

The function of living cells is based on chemical reactions. It has been shown that the velocity of these reactions is limited by the molecular transport in the cell. Therefore also the spatial organization of a cell plays a major role. In order to investigate such transport processes, fluorescence correlation spectroscopy (FCS) is often used in combination with fluorescently labeled proteins. In FCS a small subvolume of the cell (~1µm³) is observed with a laser-based microscope. The fluctuations of the fluorescence, emitted from this subvolume, are acquired. An autocorrelation analysis of these fluctuations reveals the concentrations and diffusion coefficients of the labeled particles. Usually, FCS is implemented using a confocal microscope, which can observe only a single spot at any time. For this thesis, FCS was extended to an imaging method, by combining it with light sheet fluorescence microscopy (SPIM). This relatively new widefield microscopy technique allows to observe an arbitrarily positionable, thin plane (diameter: 1-3µm) in the cell. By using a fast electron-multiplying charge-coupled device camera, the combination of SPIM and FCS allowed to map the motion also of relatively small autofluorescent proteins in living cells. At first, the setup of a light sheet microscope is described. This microscope was designed and optimized for SPIM-FCS measurements in living cells. Several test measurements show the applicability of SPIM-FCS to in vitro samples and to all larger compartments of a living cell (nucleus, cytoplasm, cellular membrane). Afterwards, the usability of several commercially available cameras as image sensor for SPIM-FCS measurements is assessed. At the time of writing, EM-CCD cameras offer the best trade-off between photosensitivity and achievable temporal resolution (~ 500µs). In addition to these linear cameras, also the use of single-photon avalanche diode (SPAD) arrays is investigated. These offer a significantly better temporal resolution (1-10µs) than current EM-CCD cameras, which would render them the ideal image sensor for SPIM-FCS. However, they do not yet reach the photo-sensitivity of EM-CCDs. Two different SPAD arrays were characterized in detail and first successful SPIM-FCS measurements of solute fluorescent molecules could be demonstrated. In a second step, SPIM-FCS was extended by a cross-correlation analysis (SPIM-FCCS), which allowed for the first time to map the interactions of differently labeled cytosolic molecules in living cells. For this purpose, the cross-correlation function between the fluorescence fluctuations from two different color channels is analyzed. A non-zero amplitude of this cross-correlation function is found only, if the differently labeled molecules interact and move together. Finally, the methods developed during this project were applied to different cellular systems. The mapping of the mobility of inert tracer molecules of different sizes allowed to measure the viscosity of the cytoplasm in different cells. A position-dependence of this mobility could only be found in the nucleoli. In addition, an important step in the remodelling cycle of the keratin intermediate filament system was investigated. As a third application, SPIM-F(C)CS measurements of different chromatin-associated proteins demonstrated the dynamics in the cellular nucleus. Mobility maps of labeled histone proteins revealed the organization of chromatin in interphase nuclei. In addition, the activity of the nuclear receptor RXR and a transcription factor were mapped

XXIV congreso anual de la sociedad española de ingeniería biomédica (CASEIB2016)

En la presente edición, más de 150 trabajos de alto nivel científico van a ser presentados en 18 sesiones paralelas y 3 sesiones de póster, que se centrarán en áreas relevantes de la Ingeniería Biomédica. Entre las sesiones paralelas se pueden destacar la sesión plenaria Premio José María Ferrero Corral y la sesión de Competición de alumnos de Grado en Ingeniería Biomédica, con la participación de 16 alumnos de los Grados en Ingeniería Biomédica a nivel nacional. El programa científico se complementa con dos ponencias invitadas de científicos reconocidos internacionalmente, dos mesas redondas con una importante participación de sociedades científicas médicas y de profesionales de la industria de tecnología médica, y dos actos sociales que permitirán a los participantes acercarse a la historia y cultura valenciana. Por primera vez, en colaboración con FENIN, seJane Campos, R. (2017). XXIV congreso anual de la sociedad española de ingeniería biomédica (CASEIB2016). Editorial Universitat Politècnica de València. http://hdl.handle.net/10251/79277EDITORIA

Dictionaries in the European Enlightenment: a testimony to the civilization of its time and the foundations of modern Europe

The text presents a plan for an international and multidisciplinary research project that is under preparation now and which is looking for collaborators from other universities or research centers. It aims to investigate the role that played monolingual, bilingual, and multilingual dictionaries published in the 18th century in the constitution of modern Europe as we know it now. It is well known that in the 18th century there appeared many dictionaries in various European countries. These dictionaries were mainly monolingual but there appeared many bilingual or plurilingual ones as well. They had a wide range of functions: linguistic (to write and understand texts), but also symbolic, representing the development and the level of civilization and prestige that a given language of culture had in times previous to 19th-century European linguistic nationalisms. Another aspect is text-oriented and text-based: the 18th-century dictionaries used to be built on relatively large sources of contemporary texts and they reflected the level of knowledge in various subjects. Therefore, they can be considered testimonies of contemporary linguistic thinking and the applied linguistics, but at the same time, they resume the development of science, legal thought, political science, etc., illustrating how knowledge spread in the Enlightenment at the international level. The project seeks to unite researchers dedicated to the linguistic historiography of philologies of European languages, historians of natural sciences, law, and social and political history, among other disciplines. It aims to offer a map of the intellectual and political globalization that began to take place in the 18th century as it is reflected in its dictionaries. The project currently counts with a small group of researchers from linguistic historiography of Romance languages. Researchers of the historiography of other European philologies are welcome and needed, and so are historians of natural and social sciences specialized in the 18th century. The main aim of the project is to stop working in parallel, horizontal and vertical, tunnels and to form a network, necessary for this type of transdisciplinary research

Calibración de un algoritmo de detección de anomalías marítimas basado en la fusión de datos satelitales

La fusión de diferentes fuentes de datos aporta una ayuda significativa en el proceso de toma de decisiones. El presente artículo describe el desarrollo de una plataforma que permite detectar anomalías marítimas por medio de la fusión de datos del Sistema de Información Automática (AIS) para seguimiento de buques y de imágenes satelitales de Radares de Apertura Sintética (SAR). Estas anomalías son presentadas al operador como un conjunto de detecciones que requieren ser monitoreadas para descubrir su naturaleza. El proceso de detección se lleva adelante primero identificando objetos dentro de las imágenes SAR a través de la aplicación de algoritmos CFAR, y luego correlacionando los objetos detectados con los datos reportados mediante el sistema AIS. En este trabajo reportamos las pruebas realizadas con diferentes configuraciones de los parámetros para los algoritmos de detección y asociación, analizamos la respuesta de la plataforma y reportamos la combinación de parámetros que reporta mejores resultados para las imágenes utilizadas. Este es un primer paso en nuestro objetivo futuro de desarrollar un sistema que ajuste los parámetros en forma dinámica dependiendo de las imágenes disponibles.XVI Workshop Computación Gráfica, Imágenes y Visualización (WCGIV)Red de Universidades con Carreras en Informática (RedUNCI