Terapias innovadoras basadas en vesículas extracelulares derivadas de células madre mesenquimales modificadas genéticamente

[ES] Las células mesenquimales estromales (MSC) poseen una serie de cualidades inmunológicas, pro-angiogénicas y regenerativas que las convierten en un excelente candidato para el tratamiento de diversas patologías. A pesar de las pruebas contundentes obtenidas en modelos preclínicos que demuestran la actividad terapéutica de las MSC, los ensayos clínicos no han podido mostrar hasta ahora un beneficio consistente, probablemente debido a deficiencias metodológicas y a la falta de estandarización, así como a la variabilidad genética intrínseca a los estudios en humanos. Debido a ello, ha sido necesario profundizar en los mecanismos responsables del beneficio terapéutico y rediseñar las estrategias clínicas. En los últimos años, se ha observado que la reparación tisular mediada por las MSC se produce de forma paracrina, y se ha constatado que las vesículas extracelulares (EVs) secretadas por las MSC (EVMSC) son capaces de recapitular las propiedades inmunosupresoras de las células parentales. Además, las estrategias terapéuticas basadas en vesículas tienen grandes ventajas en términos de bioseguridad y producción en condiciones de grado clínico, reduciendo significativamente el coste de dichas terapias. Sin embargo, la dosis efectiva en grandes mamíferos, incluidos los humanos, es bastante elevada y la producción industrial de EVs se ve dificultada en parte, por la senescencia proliferativa que afecta a las MSC durante la expansión celular masiva. En este trabajo hemos intentado solventar los principales escollos de la terapia con EVs incrementando su potencial inmunosupresor y reduciendo por tanto la dosis efectiva. Este incremento se ha conseguido gracias a la sobreexpresión del factor inducible por hipoxia 1-alpha y al desarrollo de un medio de acondicionamiento en cultivo basado en citoquinas. Además, la inmortalización de las células secretoras mediante la transducción del gen de la telomerasa humana ha permitido tanto la estandarización del producto como su producción a gran escala. La eficacia de estas EVs ha sido testada en diferentes poblaciones celulares in vitro: linfocitos T, monocitos, células Natural Killer, macrófagos, células endoteliales y fibroblastos; y en dos modelos de ratón: hipersensibilidad retardada y colitis aguda inducida por TNBS. En conclusión, hemos desarrollado una fuente de EVs de larga duración que secreta grandes cantidades de vesículas con mayor capacidad inmunosupresora y antiinflamatoria, facilitando un producto terapéutico más estándar y fácil de producir para el tratamiento de enfermedades inflamatorias inmunomediadas.[CA] Les cèl·lules mesenquimals estromals (MSC) posseeixen una sèrie de qualitats immunològiques, pro-angiogèniques i regeneratives que les converteixen en un excel·lent candidat per al tractament de diverses patologies. Tot i les proves contundents obtingudes en models preclínics que demostren l'activitat terapèutica de les MSC, els assaigs clínics no han pogut mostrar fins ara un benefici consistent, probablement degut a deficiències metodològiques i a la manca d'estandardització, així com a la variabilitat genètica intrínseca als estudis en humans. A causa d'això, ha calgut aprofundir en els mecanismes responsables del benefici terapèutic i redissenyar les estratègies clíniques. En els últims anys, s'ha observat que la reparació tissular intervinguda per les MSC es produeix de forma paracrina, i s'ha constatat que les vesícules extracel·lulars (EVs) secretades per les MSC (EVMSC) són capaços de recapitular les propietats immunosupressores de les cèl·lules parentals. A més, les estratègies terapèutiques basades en vesícules tenen grans avantatges en termes de bioseguretat i producció en condicions de grau clínic, reduint significativament el cost d'aquestes teràpies. No obstant això, la dosi efectiva en grans mamífers, inclosos els humans, és bastant elevada i la producció industrial de les EVs es veu dificultada en part, per la senescència proliferativa que afecta les MSC durant l'expansió cel·lular massiva. En aquest treball hem intentat solucionar els principals esculls de la teràpia amb EVs incrementant el seu potencial immunosupressor i reduint per tant la dosi efectiva. Aquest increment s'ha aconseguit gràcies a la sobreexpressió del factor induïble per hipòxia 1-alpha i a el desenvolupament d'un mitjà de condicionament en cultiu basat en citoquines. A més, la immortalització de les cèl·lules secretores mitjançant la transducció del gen de la telomerasa humana ha permès tant l'estandardització del producte com la seva producció a gran escala. L'eficàcia d'aquestes EVs ha estat testada en diferents poblacions cel·lulars in vitro: limfòcits T, monòcits, cèl·lules Natural Killer, macròfags, cèl·lules endotelials i fibroblasts; i en dos models de ratolí: hipersensibilitat retardada i colitis aguda induïda per TNBS. En conclusió, hem desenvolupat una font de EVs de llarga durada que secreta grans quantitats de vesícules amb major capacitat immunosupressora i antiinflamatòria, facilitant un producte terapèutic més estàndard i fàcil de produir per al tractament de malalties inflamatòries inmunomediades.[EN] Mesenchymal stromal cells (MSC) possess several immunological, pro-angiogenic and regenerative qualities that make them an excellent candidate for the treatment of various pathologies. Despite compelling evidence from preclinical models demonstrating the therapeutic activity of MSCs, clinical trials have so far failed to show consistent benefit, probably due to methodological shortcomings and lack of standardisation, as well as the genetic variability intrinsic to human studies. As a result, it has been necessary to further investigate the mechanisms responsible for therapeutic benefit and to redesign clinical strategies. In recent years, it has been observed that MSC-mediated tissue repair occurs in a paracrine pathway, and it has been confirmed that extracellular vesicles (EVs) secreted by MSC (EVMSC) are able to recapitulate the immunosuppressive properties of the parental cells. Moreover, vesicle-based therapeutic strategies have great advantages in terms of biosafety and production under clinical-grade conditions, significantly reducing the cost of such therapies. However, the effective dose in large mammals, including humans, is quite high and the industrial production of EVs is hampered in part by the proliferative senescence that affects MSC during massive cell expansion. In this work, we have attempted to overcome the main challenges of EVs therapy by increasing their immunosuppressive potential and thus reducing the effective dose. This increase has been achieved by overexpression of hypoxia-inducible factor 1-alpha and the development of a cytokine-based culture conditioning medium. In addition, immortalization of secretory cells by transduction with the human telomerase gene has allowed both product standardisation and large-scale production. The efficacy of these EVs has been tested in different cell populations in vitro: T lymphocytes, monocytes, Natural Killer cells, macrophages, endothelial cells and fibroblasts; and in two mouse models: delayed-type hypersensitivity and TNBS-induced acute colitis. In conclusion, we have developed a long-lasting source of EVs that secretes large amounts of vesicles with enhanced immunosuppressive and anti-inflammatory capacity, providing a more standard and easier-to-produce therapeutic product for the treatment of immune-mediated inflammatory diseases.Gómez Ferrer, M. (2022). Terapias innovadoras basadas en vesículas extracelulares derivadas de células madre mesenquimales modificadas genéticamente [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182560TESI

Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson's Disease Post-Mortem Brain through a New AlphaScreen-Based Assay

Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity

Data driven tools to assess the location of photovoltaic facilities in urban areas

Urban sustainability is a significant factor in combating climate change. Replacing polluting by renewable energies is fundamental to reduce the emission of greenhouse gases. Photovoltaic (PV) facilities harnessing solar energy, and particularly self-consumption PV facilities, can be widely used in cities throughout most countries. Therefore, locating spaces where photovoltaic installations can be integrated into urban areas is essential to reduce climate change and improve urban sustainability. An open-source software (URSUS-PV) to aid decision-making regarding possible optimal locations for photovoltaic panel installations in cities is presented in this paper. URSUS-PV is the result of a data mining process, and it can extract the characteristics of the roofs (orientation, inclination, latitude, longitude, area) in the urban areas of interest. By combining this information with meteorological data and characteristics of the photovoltaic systems, the system can predict both the next-day hourly photovoltaic energy production and the long-term photovoltaic daily average energy production.This work has been supported by the project RTI2018-095097-B-I00 at the 2018 call for I+D+i Project of the Ministerio de Ciencia, Innovación y Universidades, Spain. Funding for open access charge: Universidad de Málaga/CBUA, Spain

Generation of enhanced synthetic off-line signatures based on real on-line data

Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes, creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works. M. Díaz-Cabrera, M. Gómez-Barrero, A. Morales, M. A. Ferrer, J. Galbally, "Generation of Enhanced Synthetic Off-Line Signatures Based on Real On-Line Data" in 14th International Conference on Frontiers in Handwriting Recognition (ICFHR), Heraklion (Greece), 2014, 482 - 487One of the main challenges of off-line signature verification is the absence of large databases. A possible alternative to overcome this problem is the generation of fully synthetic signature databases, not subject to legal or privacy concerns. In this paper we propose several approaches to the synthesis of off-line enhanced signatures from real dynamic information. These synthetic samples show a performance very similar to the one offered by real signatures, even increasing their discriminative power under the skilled forgeries scenario, one of the biggest challenges of handwriting recognition. Furthermore, the feasibility of synthetically increasing the enrolment sets is analysed, showing promising results.This work has been partially supported by projects: MICINN TEC2012-38630-C04-02, Contexts (S2009/TIC-1485) from CAM, Bio-Shield (TEC2012-34881) from Spanish MINECO, TABULA RASA (FP7-ICT-257289) and BEAT (FP7-SEC-284989) from EU, and Cátedra UAM-Telefónica

On-line signature recognition through the combination of real dynamic data and synthetically generated static data

This is the author’s version of a work that was accepted for publication in Pattern Recognition . Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Pattern Recognition , 48, 9 (2005) DOI: 10.1016/j.patcog.2015.03.019On-line signature verification still remains a challenging task within biometrics. Due to their behavioral nature (opposed to anatomic biometric traits), signatures present a notable variability even between successive realizations. This leads to higher error rates than other largely used modalities such as iris or fingerprints and is one of the main reasons for the relatively slow deployment of this technology. As a step towards the improvement of signature recognition accuracy, the present paper explores and evaluates a novel approach that takes advantage of the performance boost that can be reached through the fusion of on-line and off-line signatures. In order to exploit the complementarity of the two modalities, we propose a method for the generation of enhanced synthetic static samples from on-line data. Such synthetic off-line signatures are used on a new on-line signature recognition architecture based on the combination of both types of data: real on-line samples and artificial off-line signatures synthesized from the real data. The new on-line recognition approach is evaluated on a public benchmark containing both real versions (on-line and off-line) of the exact same signatures. Different findings and conclusions are drawn regarding the discriminative power of on-line and off-line signatures and of their potential combination both in the random and skilled impostors scenarios.M. D.-C. is supported by a PhD fellowship from the ULPGC and M.G.-B. is supported by a FPU fellowship from the Spanish MECD. This work has been partially supported by projects: MCINN TEC2012-38630- C04-02, Bio-Shield (TEC2012-34881) from Spanish MINECO, BEAT (FP7-SEC-284989) from EU, CECABANK and Cátedra UAM-Telefónic

A novel hand reconstruction approach and its application to vulnerability assessment

This is the author’s version of a work that was accepted for publication in Information Sciences. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Information Sciences, 238 (2014) DOI: 10.1016/j.ins.2013.06.015The present work proposes a novel probabilistic method to reconstruct a hand shape image from its template. We analyse the degree of similarity between the reconstructed images and the original samples in order to determine whether the synthetic hands are able to deceive hand recognition systems. This analysis is made through the estimation of the success chances of an attack carried out with the synthetic samples against an independent system. The experimental results show that there is a high chance of breaking a hand recognition system using this approach. Furthermore, since it is a probabilistic method, several synthetic images can be generated from each original sample, which increases the success chances of the attack.This work has been partially supported by projects Contexts (S2009/TIC-1485) from CAM, Bio-Challenge (TEC2009-11186), BIOSINT (TEC2012-38630-C04-02) and Bio-Shield (TEC2012-34881) from Spanish MINECO, TABULA RASA (FP7-ICT-257289) and BEAT (FP7-SEC-284989) from EU, and Cátedra UAM-Telefónica. Marta Gomez-Barrero is supported by a FPU Fellowship from Spanish MECD

Perspectiva de género en docencia STEM

La Universitat Politècnica de Catalunya, dentro del III Plan de Igualdad de Género 2016-2020, está llevando a cabo diferentes acciones para promover la transversalidad de género en la universidad. El presente artículo expone el trabajo del proyecto Género y Docencia, cuyo objetivo principal es la capacitación del personal académico para la incorporación de la perspectiva de género en la docencia. Este proyecto cuenta con la participación voluntaria de 41 docentes de 8 titulaciones STEM. Su duración, que coincide con el cuatrimestre de primavera de 2019, ha permitido llevar a cabo las estrategias propuestas a lo largo del curso. Los aspectos que se han tenido en cuenta incluyen la relevancia social y de género de las asignaturas, la metodología inclusiva, la gestión del aula y la evaluación. Se ha tenido especial cuidado en la elaboración de indicadores para poder realizar la correcta evaluación del proyecto a su término. Se ha elaborado una herramienta de autoevaluación para el equipo docente y un cuestionario para analizar la sensibilización del alumnado, así como su percepción respecto a la falta de igualdad de género en el ámbito docente.Postprint (published version

Early-Onset Molecular Derangements in the Olfactory Bulb of Tg2576 Mice: Novel Insights Into the Stress-Responsive Olfactory Kinase Dynamics in Alzheimer’s Disease

The olfactory bulb (OB) is the first processing station in the olfactory pathway. Despite smell impairment, which is considered an early event in Alzheimer's disease (AD), little is known about the initial molecular disturbances that accompany the AD development at olfactory level. We have interrogated the time-dependent OB molecular landscape in Tg2576 AD mice prior to the appearance of neuropathological amyloid plaques (2-, and 6-month-old), using combinatorial omics analysis. The metabolic modulation induced by overproduction of human mutated amyloid precursor protein (APP) clearly differs between both time points. Besides the progressive perturbation of the APP interactome, functional network analysis unveiled an inverse regulation of downstream extracellular signal-regulated kinase (ERK1/2), and p38 mitogen-activated protein kinase (MAPK) routes in 2-month-old Tg2576 mice with respect to wild-type (WT) mice. In contrast, Akt and MAPK kinase 4 (SEK1)/ stress-activated protein kinase (SAPK) axis were parallel activated in the OB of 6-months-old-Tg2576 mice. Furthermore, a survival kinome profiling performed during the aging process (2-, 6-, and 18-month-old) revealed that olfactory APP overexpression leads to changes in the activation dynamics of protein kinase A (PKA), and SEK1/MKK4-SAPK/JNK between 6 and 18 months of age, when memory deficits appear and AD pathology is well established in transgenic mice. Interestingly, both olfactory pathways were differentially activated in a stage-dependent manner in human sporadic AD subjects with different neuropathological grading. Taken together, our data reflect the early impact of mutated APP on the OB molecular homeostasis, highlighting the progressive modulation of specific signaling pathways during the olfactory amyloidogenic pathology

All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting

Decreased kynurenine pathway potentiate resilience to social defeat effect on cocaine rewa

The kynurenine (KYN) pathway of tryptophan (TRP) degradation is activated by stress and inflammatory factors. It is now well established that social stress induces the activation of the immune system, with central inflammation and KYN metabolism being two of the main factors linking stress with depression. The aim of the present study was to evaluate the long-lasting changes in the KYN pathway induced by social defeat (SD) associated with the resilience or susceptibility to an increase in the conditioned rewarding effects of cocaine. Mice were exposed to repeated SD and 3 weeks later, a conditioned place preference (CPP) induced by a subthreshold dose of cocaine (1.5 mg/kg) was developed. KYN levels in plasma, cerebellum, hippocampus, striatum and limbic forebrain were studied at the end of the CPP procedure. Changes in the KYN pathway after exposure to pharmacological (oxytocin and indomethacin) and environmental interventions (environmental enrichment) were also evaluated. Our results showed that defeated susceptible (SD-S) mice had higher conditioning scores than resilient mice (SD-R). In addition, although KYN concentration was elevated in all defeated mice, SD-R mice showed smaller increases in KYN concentration in the cerebellum than SD-S mice. Oxytocin or Indomethacin treatment before SD normalized cocaine-induced CPP, although the increase in the KYN pathway was maintained. However, environmental enrichment before SD normalized cocaine-induced CPP and prevented the increase in the KYN pathway. The present study highlights the role of the KYN pathway and anti-inflammatory drugs acting on TRP metabolism as pharmacological targets to potentiate resilience to social stress effects