Algunos aprendizajes y postulados para la intervención social

Desde los años noventa, la intervención social soportada en ONGs y en empresas privadas, como parte de las políticas de responsabilidad social corporativa; ha estado experimentando una continua expansión, especialmente en el ámbito de los modelos de desarrollo local basados en la comunidad. En esa perspectiva, son muchos los casos y proyectos que han recibido apoyo, sobre todo en áreas marginales en las que el Estado tiene escasa presencia y el desarrollo del mercado es mínimo; sin embargo, son poco conocidas las evaluaciones hechas acerca de las bondades, deficiencias o fallas de esa propuesta y menos sobre su capacidad para resolver los problemas que la motivaron. Independiente del balance de bondades o falencias que pueda arrojar ese esquema, este documento reconoce su importancia, es consciente de su futuro crecimiento y se concentra por ello en abordar tres aspectos que pueden incidir notablemente en su eficiencia y positivos resultados, pues le suministran valiosas señales sobre factores considerados como los realmente determinantes del crecimiento y el desarrollo de una nación, una región o una localidad; sobre preconceptos y comportamientos que caracterizan la manera de ver y actuar tanto de promotores o ejecutores de los proyectos, como de los beneficiarios de los mismos; y sobre las restricciones y fragilidades de los procedimientos que se suelen seguir para la selección y evaluación de los proyectos. Palabras claves: Desarrollo, Local, Intervención Social, Comunidad, Proyectos, fundamentos, umbrales, sucesos estilizados /Abstract Since 1990s, social intervention in Colombia undertaken by NGO’s and private companies, as part of their corporate social responsibility policies, has been experiencing a continuous expansion, especially in the field of community based local development models . Particularly in marginal areas where the State has little presence and market development is minimal, there are many cases and projects that have received some kind of support. However, there are very few known assessments that had been made about the benefits, shortcomings or failures from such a proposal and less on their capability to solve the problems from which they arise. Beyond the balance on pros and cons of this proposal, this paper recognizes its importance, and being aware of its future growth focuses on three issues that can significantly affect its efficiency and positive results. Thus they provide valuable signals on factors considered the determinants of growth and development of a nation, a region or a locality; about beliefs and behaviors that characterize the way we view and act as both promoters and implementers of projects and beneficiaries as such, as well as regarding the limitations and weaknesses of the procedures usually followed for the selection and evaluation of projects

Effector-mediated suppression of plant defense against biotrophs through activation of antagonistic defense against necrotrophs

Plant hormones are small molecules involved in the regulation of plant growth, development, reproduction and stress responses. Salicylic acid (SA) and jasmonates (JA) are essential for the activation of defence responses against pathogens. SA signaling is involved in triggering immunity against biotrophic pathogens while JA activates resistance against necrotrophic pathogens. The SA and JA pathways are mostly antagonistic: elevated biotroph resistance correlates with increased necrotroph susceptibility, and vice versa. Using transcriptomics to look for a functional overlap between plant gene silencing and type III-mediated plant responses we found that genes associated to JA signaling were overrepresented in the overlapping set, more so than SA-related genes. We present here the results of the ensuing analysis, showing effector-mediated activation of the JA pathway as a virulence mechanism, and establishing a novel role for gene silencing in the regulation of the JA pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech - See more at: http://riuma.uma.es/xmlui/handle/10630/5393/submit/12176e8e39815b4d6112192a6815221718868c2f.continue#sthash.RYCQTkwy.dpu

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Suppresses Transcriptional Gene Silencing

Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation but the fact that plants might use methylation as a defense against geminiviruses and the impact that viral genome methylation may have during the infection, remain controversial. We have found that geminiviruses reduce the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species and we have identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3. The presence of Rep, suppresses transcriptional gene silencing (TGS) of an Arabidopsis transgene and of host loci whose expression is strongly controlled by MET1. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at certain loci at CG sites. The biological relevance of these findings and the role of Rep as a TGS suppressor will be discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO, FEDE

Análisis de la infección por geminivirus en plantas con la maquinaria de metilación del DNA alterada.

Los geminivirus son virus de plantas pertenecientes a la familia Geminiviridae. Poseen un genoma compuesto por una o dos moléculas de DNA circular de cadena simple y una cápside compuesta de 2 partes icosaédricas gemelas, de ahí el nombre de geminivirus. Uno de los geminivirus más estudiados es el virus del rizado amarillo de la hoja de tomate (TYLCV, Tomato yellow leaf curl virus), causante de importantes pérdidas en las cosechas en zonas templadas, subtropicales y tropicales. Este geminivirus codifica 6 proteínas, de las cuales sólo la proteína Rep es esencial para su replicación. La metilación del DNA es una marca epigenética que promueve el silenciamiento génico a nivel transcripcional (TGS) y juega un importante papel en el mantenimiento de la integridad del genoma mediante el silenciamiento de transposones. Diversos estudios sugieren que participa de manera relevante en la defensa de la planta frente a virus de DNA como los geminivirus (Raja et al, 2008; Yang et al, 2011, Zhang et al, 2011). Los geminivirus son capaces de interferir en el mecanismo de TGS propio de la planta; una de las proteínas implicadas en la supresión de dicho mecanismo es Rep, la cual induce una disminución en los niveles de expresión de las metiltransferasas de mantenimiento MET1 y CMT3 de Arabidopsis thaliana y Nicotiana benthamiana, revierte el silenciamiento génico transcripcional de loci endógenos y transgenes e induce la hipometilación de loci cuya metilación es esencialmente dependiente de MET1 (Rodríguez-Negrete et al., 2013). Considerando que los datos previos sugieren que los geminivirus suprimen el mecanismo de TGS como un mecanismo de contra-defensa, nos popusimos evaluar la importancia de la metilación del DNA como mecanismo de defensa frente a los geminivirus. Para ello se midieron los niveles del geminivus TYLCV-Mld (Tomato yellow leaf curl virus, aislado Mld) tras la infección de mutantes de A. thaliana deficientes en la maquinaria de metilación: met1-3 (mutante en MET1), ros1-4 (mutante en la desmetilasa ROS1) y ddc (triple mutante DRM1, DRM2 y CMT3, siendo DRM1 y DRM2 metiltransferasas de novo). Por otro lado se midieron niveles del mismo geminivirus en plantas infectadas de N. benthamiana que presentan reducidos niveles de expresión de MET1, CMT3 o ROS1; la reducción de la expresión de estos genes de N. benthamiana fue generada mediante silenciamiento génico inducido por virus (VIGS). Se presentarán y discutirán dichos resultados.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Análisis del solapamiento funcional entre la respuesta de la planta frente a efectores secretados por Pseudomonas syringae y el silenciamiento génico

Las plantas están continuamente sometidas a infecciones por diferentes patógenos. Los patógenos bacterianos usan un complejo sistema de secreción para translocar proteínas efectoras al interior del huésped y suprimir así las respuestas de defensas. En los últimos años, se ha demostrado la participación de ciertos microRNAs en respuestas de defensas de la planta frente a patógenos bacterianos, así como la actuación de erectores bacterianos en rutas reguladas por miRNAs. Sin embargo poco se sabe acerca de cómo la regulación por microRNAs afecta a las respuestas de defensas de la planta frente a patógenos bacterianos, y de hecho menos es conocido sobre el impacto de la supresión de defensas mediada por efectores sobre las rutas reguladas por microRNAs. En este estudio, hemos realizado comparaciones entre análisis de microarrays, con el objetivo de diferenciar genes expresados diferencialmente (DEGs) en Arabidopsis, comunes entre la respuesta de defensa frente a efectores de Pseudomonas syringae y mutantes afectados en la biogénesis de miRNAs. El análisis de la anotación funcional de los DEGS comunes identificados, seguidos de un análisis de expresión mediante RT-qPCR nos ha servido para identificar procesos biológicos regulados por rutas de miRNAs en la interacción planta-patógenoUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Junta de Andalucía, Proyectos de Excelencia P06-CVI-02088; MINECO, Plan Nacional BIO2012-3564

Study of the functional domains of the PTGS suppressor V2 from geminivirus Beet curly top virus (BCTV)

Geminiviruses constitute a group of plant viruses that infect vegetable crops all over the world. Among the Geminiviridae family, the genera Mastrevirus, Begomovirus and Curtovirus are the most abundant. Suppression of gene silencing is a key mechanism for viral infection in plants. In begomovirus, V2 is a strong posttranscriptional gene silencing suppressor. We recently showed that V2 from curtovirus Beet curly top virus (BCTV) is a PTGS suppressor by impairing the RDR6/SGS3 pathway, as V2 from begomovirus. In order to identify the domains involved in the suppression activity and viral pathogenicity, we performed an alignment of several begomovirus and curtovirus V2 proteins. A protein kinase C (PKC) phosphorylation motif essential for suppression activity in begomovirus (P1) was found in all analysed sequences. We also found similar hydrophobic profiles, with two hydrophobic domains (H1 and H2) followed by a long hydrophilic domain. Then we generated BCTV V2 mutant proteins and performed transient assays in Nicotiana benthamiana plants to test their suppression activity. We also expressed them from a Potato virus X-derived vector to check the symptoms produced. Additionally, their subcellular localization was determined. Finally, we produced BCTV viruses mutated in the different domains and N. benthamiana plants were infected, analysing virus levels and symptoms produced. The results showed that P1, H1 and H2 are involved in the suppression activity and viral pathogenicity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Implementación de un Sistema de Monitoreo a nivel de prototipo de signos vitales: pulso, temperatura y saturación de oxígeno para pacientes

The Internet of things is a concept of digital interconnection between everyday objects and the Internet, which is generally used to improve comfort in daily tasks and optimize business. Thus, it can be integrated into the health world to give it medical applications, being one of the typical application scenarios of the telemedicine service, such as remote medical care. In this way, this article describes the development of a monitoring system for vital signs: pulse, temperature, and oxygen saturation in the blood, which is based on the comparison and selection of components through the research methodology, and its validation through statistical analysis. This monitoring system seeks to be used in the care of patients with home treatments as a compact and low-cost system to provide timely medical information and implement future Cloud Computing and Internet of Things technologies to perform remote monitoring of objective and subjective patient data. In addition, the design and implementation of the mainboard, the selection of biomedical sensors, the preparation of supports and programming are detailed.El Internet de las cosas es un concepto de interconexión digital de objetos cotidianos con Internet, el cual se emplea generalmente para mejorar el nivel de comodidad en las tareas cotidianas y optimizar negocios. Así también, puede ser integrado en el mundo de la salud para darle aplicaciones médicas siendo uno de los escenarios típicos de aplicación es el servicio de telemedicina, como la práctica de la atención médica a distancia. Es así que este artículo describe el desarrollo de un sistema de monitoreo de signos vitales: pulso, temperatura y saturación de oxígeno en la sangre; el cual se basa en la comparación y selección de componentes por medio de la metodología de la investigación, y su validación a través de un análisis estadístico. Dicho sistema de monitoreo busca ser utilizado en el cuidado de pacientes con tratamientos domiciliarios, como un sistema compacto y de bajo costo, con la capacidad de brindar información médica oportuna e implementar a futuro tecnologías de Computación en la nube e Internet de las Cosas para realizar un monitoreo remoto de los datos objetivos y subjetivos del paciente. Además, se detalla el diseño e implementación de la tarjeta principal, la selección de los sensores biomédicos, elaboración de soportes y programación

Study of the PTGS suppressor activity of V2 protein from geminivirus Beet curly top virus

Suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress post- and transcriptional gene silencing (PTGS/TGS). In Begomovirus V2 has been shown to be the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only C2 protein has been described to inhibit PTGS and TGS. Objective: Our main goal is to test the PTGS suppressor activity of BCTV V2 and to study further its gene-silencing suppression mechanism. Material and methods: To determine whether BCTV V2 is also a gene silencing suppressor we carried out transient expression assays in Nicotiana benthamiana wild-type or the 16c GFP-expressing line: plant leaves were agroinfiltrated with binary constructs to express GFP (35S:GFP) and V2. Visual detection of GFP fluorescence was confirmed by western blot. Relative levels of the GFP-specific siRNAs were determined by northern blot. We also expressed the V2 ORF from a Potato virus X-derived vector in N. benthamiana plants. As an approach to identify a genetic target of V2 in the antiviral silencing pathway, we carried out a complementation analysis of BCTV-∆V2 in a series of Arabidopsis thaliana mutants deficient in specific components of this pathway involved in DNA virus siRNA production and amplification (DCL2, DCL3, DCL4, RDR6 and RDR2). Arabidopsis plants were infected by agroinoculation and the amount of viral DNA was measured by real-time qPCR. To gain more insight into the gene silencing suppression mechanism of V2, we generated Arabidopsis plants overexpressing the viral protein. Wild-type (Col-0) plants, as well as, plants containing the SUC-SUL hairpin or the AMPLICON (AMP) constructs were transformed with the same V2 expression cassette used for the gene silencing assays. Results: Like its begomoviral counterpart, BCTV V2 is a potent PTGS suppressor and produces an HR-like response in N. benthamiana plants when expressed from PVX. The molecular and genetic analysis of transgenic plants expressing V2 indicates that, as the begomoviral V2, BCTV V2 inhibits the RDR6/SGS3-dependent silencing pathway. Finally, infection assays in Arabidopsis mutants confirm the importance of the RDR6/SGS3 pathway in defence against curtoviruses, and reveal an additional RDR6/SGS3-independent gene-silencing suppression mechanism of V2. Conclusions: BCTV V2, as begomovirus V2 protein, suppresses PTGS by impairing the RDR6/SGS3 pathway. Keywords: Geminivirus, BCTV V2, RNA-silencing suppressor.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech