Determinación de la calidad interna del kiwi mediante técnicas no destructivas: impacto e imagen hiperspectral.

El kiwi es un fruto con epidermis pilosa de color pardo-verdoso cuyo aspecto externo no sufre cambios significativos a lo largo del proceso de maduración. El objetivo de este trabajo fue evaluar el potencial de distintas técnicas no destructivas para la determinación de la calidad interna de kiwi. Se trabajó con frutos de pulpa verde (Actinidia deliciosa) ?Hayward? y de pulpa amarilla (Actinidia chinensis) ?Hort16A? y tres estados de madurez. Se realizaron determinaciones mecánicas y acústicas con el impactador lateral LPF y el equipo comercial AWETA, ambos para la estimación no destructiva de la firmeza, y se adquirieron imágenes con cámara hiperespectral (rango 400-1000 nm), como herramienta para la estimación global del estado de madurez del fruto. Se encontró que existe una correlación positiva y significativa (0,94? rpearson ?0,97) entre las variables Fuerzamáx. (impactador) y Frecuenciaresonante máx. (AWETA) con la Fuerzamáx registrada en el ensayo de referencia ?compresión con bola? realizado sobre los mismos frutos con una máquina universal de ensayos. Las imágenes hiperespectrales permitieron evidenciar el distinto nivel de madurez de los frutos en evolución y dentro de cada lote e incluso en distintas zonas de un mismo fruto, apreciándose cierto patrón espacial común en la evolución de la madurez dentro de los frutos.

Compatibilidad de Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) y Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), importantes enemigos naturales de la mosca blanca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) en cultivos hortícolas, con nuevas barreras físicas selectivas y modernos plaguicidas

Los programas de Gestión Integrada de Plagas (GIP) promueven el uso de estrategias de control que sean respetuosas con el medio ambiente, sin embargo el uso de insecticidas en los cultivos hortícolas sigue siendo necesario para el control de determinadas plagas, como es el caso de la mosca blanca Bemisia tabaci (Gennadius). Por ello, el objetivo de esta tesis es el estudio de la integración de las tres estrategias de control más empleadas hoy en día para el control de plagas: el control biológico, el físico y el químico. Una primera parte de este trabajo ha consistido en el estudio de los efectos letales y subletales de once insecticidas, aplicados a la dosis máxima de campo, sobre los enemigos naturales Eretmocerus mundus Mercet y Amblyseius swirskii Athias-Henriot, mediante ensayos de laboratorio y persistencia (laboratorio extendido). Para la evaluación de la toxicidad de los insecticidas sobre los estados de vida más protegidos de estos enemigos naturales, se trataron bajo la Torre de Potter las pupas de E. mundus y los huevos de A. swirskii. Además, se llevaron a cabo ensayos de contacto residual para determinar los efectos letales y subletales de estos insecticidas sobre el estado adulto de ambas especies de enemigos naturales. Para ello, los pesticidas se aplicaron sobre placas de cristal (laboratorio) o sobre plantas (laboratorio extendido: persistencia). Los resultados mostraron que los insecticidas flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen y spirotetramat eran compatibles con el estado de pupa de E. mundus (OILB 1: Inocuos). Sin embargo, abamectina, deltametrina y emamectina fueron categorizadas como ligeramente tóxicas (OILB 2) al causar efectos deletéreos. Los dos pesticidas más tóxicos fueron spinosad y sulfoxaflor, los cuales redujeron significativamente la emergencia de las pupas tratadas (OILB 4: Tóxicos). Flonicamida, flubendiamida, metoxifenocida y spiromesifen fueron compatibles con el estado adulto de E. mundus (OILB 1: Inocuos). Abamectina, deltametrina, emamectina, metaflumizona y spiromesifen pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Al contrario, spinosad y sulfoxaflor no resultaron ser compatibles (OILB D: Persistentes), aunque la realización de ensayos adicionales es necesaria para ver los efectos de los mismos en campo. Todos los insecticidas estudiados, excepto el spirotetramat (OILB 2: Ligeramente tóxico), fueron selectivos para el estado de huevo de A. swirskii (OILB 1: Inocuos). Flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen, spirotetramat y sulfoxaflor, fueron compatibles con el estado adulto de A. swirskii (OILB 1: Inocuos). Abamectina, deltametrina, emamectina y spinosad pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Entre las nuevas estrategias de la GIP, los plásticos y mallas fotoselectivas han demostrado ser una herramienta importante para el control de plagas y enfermedades en cultivos hortícolas protegidos. Por ello, en una segunda parte de este trabajo, se estudiaron tanto los efectos directos, como la combinación de efectos directos y mediados por planta y plaga de ambientes pobres en luz UV, en presencia o ausencia del Virus del rizado amarillo del tomate (TYLCV), sobre E. mundus. En primer lugar, se realizó un ensayo al aire libre para la evaluación de la capacidad de vuelo de E. mundus en cajas tipo túnel (1 x 0,6 x 0,6 m) cubiertas con distintas barreras absorbentes de luz UV. Se detectó un efecto directo en la capacidad de orientación de E. mundus, debido a que este parasitoide utiliza estímulos visuales para localizar a sus huéspedes, únicamente en las barreras que bloqueaban más del 65% de la luz UV (malla G). En segundo lugar, bajo condiciones de invernadero, se evaluó la combinación de efectos directos y mediados por planta y plaga sobre E. mundus, usando plantas de tomate sanas o infectadas con el TYLCV y cajas (30 x 30 x 60 cm) cubiertas con los distintos plásticos fotoselectivos. En este caso, no se observó ningún efecto en la capacidad benéfica del parasitoide cuando este estaba en contacto con plantas de tomate infestadas con ninfas de B. tabaci, lo que demuestra que este insecto usa estímulos táctiles para encontrar a sus huéspedes a cortas distancias. Además, las diferentes condiciones de radiación UV estudiadas tuvieron cierto impacto en la morfología, fisiología y bioquímica de las plantas de tomate, infestadas o no con el virus de la cuchara, detectándose pequeñas alteraciones en alguno de los parámetros estudiados, como el peso fresco y seco, el contenido en H y el espesor de las cutículas y de las paredes celulares de la epidermis foliar. Por último, no se observaron efectos de la radiación UV mediados por planta, ni en B. tabaci ni en su parasitoide, E. mundus. En una tercera parte, se evaluaron los efectos de una malla tratada con bifentrin sobre ambos enemigos naturales, en ensayos de laboratorio, semicampo y campo. Las mallas tratadas fueron diseñadas originariamente para el control de mosquitos vectores de la malaria, y actualmente se está trabajando para su uso en agricultura, como una nueva estrategia de control de plagas. En ensayos de laboratorio, cuando adultos de E. mundus y A. swirskii se expusieron por contacto durante 72 horas con la malla tratada (cajas de 6 cm diámetro), se registró una alta mortalidad. Sin embargo, en el ensayo de preferencia, estos enemigos naturales no fueron capaces de detectar la presencia de bifentrin y, en aquellos individuos forzados a atravesar la malla tratada, no se observó mortalidad a corto plazo (72 horas). En estudios de semicampo, llevados a cabo bajo condiciones de invernadero en cajas de 25 x 25 x 60 cm de altura, la capacidad benéfica de E. mundus no se vio afectada. Finalmente, en ensayos de campo llevados a cabo en invernaderos comerciales (4000m2) en Almería, A. swirskii no se vio afectado por la presencia en el cultivo de la malla tratada con bifentrin y los niveles de infestación de B. tabaci y F. occidentalis detectados bajo dicha malla, fueron inferiores a los del control. Por último, se ha evaluado la composición de la microflora bacteriana de tres especies de parasitoides, E. mundus, Eretmocerus eremicus Rose & Zolnerowich y Encarsia formosa Gahan, y la influencia de la misma en su susceptibilidad a insecticidas. Se llevó a cabo una extracción total de ADN de los insectos y la región variable V4 del ARNr se amplificó usando cebadores universales bacterianos. Para identificar las secuencias de los géneros bacterianos presentes en los parasitoides, se realizó una Next Generation sequencing (Illumina sequencing). Una vez identificados los géneros bacterianos, el gen ADNr 16S de las Actinobacterias se amplificó del ADN extraído de los insectos, usando cebadores universales bacterianos y específicos de Actinobacterias, y los productos de la Nested PCR fueron clonados para identificar todas las especies del género Arthrobacter. Tres bacterias (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi y A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), próximas a las especies de Arthrobacter presentes en los parasitoides, se obtuvieron de la colección bacteriana del BCCMTM/LMG y se midió su actividad esterasa. Finalmente, se realizaron ensayos con antibióticos (tetraciclina) y de contacto residual con insecticidas (abamectina) para determinar la influencia de las especies de Arthrobacter en la susceptibilidad de E. mundus a insecticidas. Los resultados muestran que este género bacteriano puede afectar a la toxicidad de E. mundus a abamectina, mostrando la importancia de la comunidad microbiana en enemigos naturales, factor que debe ser considerado en los estudios de evaluación de los riesgos de los insecticidas. ABSTRACT Integrated Pest Management (IPM) programs promote the use of control strategies more respectful with the environment; however the use of insecticides in vegetable crops is still needed to control certain pests, such as the whitefly Bemisia tabaci (Gennadius). Therefore, the objective of this work is to study the integration of the three most commonly used pest control strategies nowadays: biological, physical and chemical control. Firstly, the lethal and sublethal effects of eleven insecticides, applied at their maximum field recommended concentration, on the parasitic wasp Eretmocerus mundus Mercet and the predator Amblyseius swirskii Athias-Henriot has been assessed in the laboratory and in persistence tests (extended laboratory). To test the effects of pesticides on the most protected life stage of these natural enemies, E. mundus pupae and A. swirskii eggs were sprayed under a Potter precision spray tower. Laboratory contact tests were therefore conducted to determine the lethal and sublethal effects of these pesticides on the adult stage of these natural enemies. In the residual contact tests the pesticides were applied on glass plates (laboratory) or plants (extended laboratory: persistence). The study showed that the insecticides flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen and spirotetramat were selective for E. mundus pupae (IOBC 1: Harmless). Nevertheless, abamectin, deltamethrin and emamectin were categorized as slightly harmful (IOBC 2) due to the deleterious effects caused. The two most harmful pesticides were spinosad and sulfoxaflor, which significantly reduced the adult emergence from treated pupae (IOBC 4: Harmful). Flonicamid, flubendiamide, methoxyfenozide and spiromesifen were compatible with E. mundus adults (IOBC 1: Harmless). Base on the duration of the harmful activity, abamectin, deltamethrin, emamectin, metaflumizone and spirotetramat could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. On the contrary, spinosad and sulfoxaflor were not compatible (IOBC D: persistent), although additional studies are required to determine their effects under field conditions. All the pesticides tested, except spirotetramat (IOBC 2: Slightly harmful), were selective for A. swirskii eggs (IOBC 1: Harmless). Flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen, spirotetramat and sulfoxaflor were compatible with A. swirskii adults (IOBC 1: Harmless). However, abamectin, deltamethrin, emamectin and spinosad could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. Among new IPM strategies, UV-absorbing photoselective plastic films and nets have been shown to be an important tool for the control of pests and diseases in horticultural protected crops. Because of that, we secondly studied the plant and pest insect-mediated and/or the direct effects on E. mundus under different UV radiation conditions, in presence or absence of the Tomato Yellow Leaf Curl Virus (TYLCV). In the first experiment, performed outdoors, the flight activity of E. mundus was studied in one-chamber tunnels (1 x 0.6 x 0.6 m) covered with different photoselective barriers. Because E. mundus uses visual cues for host location at a long distance, a direct effect on its host location ability was detected, but only in the UV-absorbing barriers blocking more than 65% of the UV light (G net). In a second experiment, the direct and plant and pest insect-mediated effects of different UV radiation conditions on E. mundus were studied, inside cages (30 x 30 x 60 cm) covered with the different UVplastic films and under greenhouse conditions, using healthy or TYLCV-virus infected tomato plants. In this case, not any effect on the beneficial capacity of this parasitoid was detected, proving that he uses tactile cues at a short distance of the host. Moreover, the different UV radiation conditions studied had a certain direct impact in the morphology, physiology and biochemistry of tomato plants infested or not with the TYLCV, and small alterations in some parameters such as fresh and dry weight, H percentage and cuticle and cell wall thickness of epidermal cells of the leaves, were detected. Finally, none plant-mediated UV effects neither in the whitefly B. tabaci nor in their parasitic wasp were found. Thirdly, the effects of a bifenthrin treated net were evaluated in different laboratory, semi-field and field experiments on the natural enemies studied. Treated nets were developed long time ago aiming at the control of the mosquitoes vectors of malaria, and nowadays, there is a great interest on assessing the possibility of their use in agriculture. In laboratory assays, a high mortality was recorded when E. mundus and A. swirskii adults were exposed by contact to the bifenthrin treated net for 72 hours in small cages (12 cm diameter). However, these natural enemies were not able to detect the presence of bifenthrin in a dual-choice test and no short-term mortality (72 hours) was recorded in those individuals that went through the treated net. In semi-field assays, performed under greenhouse conditions with cages of 25 x 25 x 60 cm high, the beneficial capacity of E. mundus was not affected. Finally, in field assays carried out in commercial multispan greenhouses (4000 m2) in Almería, A. swirskii was not affected by the presence of the bifenthrin treated net in the crop and the B. tabaci and F. occidentalis infestation levels were significantly lower than in the control. Finally, the composition of the microflora present in three species of parasitoids, E. mundus, Eretmocerus eremicus Rose & Zolnerowich and Encarsia formosa Gahan, and its influence in their susceptibility to insecticides, have been assessed. A total DNA extraction was performed on insects and universal bacterial primers were used to amplify the variable V4 region of the rRNA. A Next Generation sequencing (Illumina sequencing) was performed to identify the sequences of the bacterial genera present in the parasitic wasps. Once, the bacterial genera were identified, 16S rDNA gene of Actinobacteria were amplified from insects DNA extracts using the universal bacterial and actinobacterial primers, and the nested PCR products, were cloned to identify the Arthrobacter species. Three bacteria (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi and A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), having the closest match with the Arthrobacter species present in the parasitic wasps, were obtained from the BCCMTM/LMG bacteria collection and its esterase activity was measured. Finally, antibiotic and residual contact tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides (abamectin). The results suggest that this bacterial genus can affect the toxicity of E. mundus to abamectin, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides.

Pavimentos urbanos : criterios para su uso y diseño

La necesidad creciente del ciudadano por recuperar las relaciones con su entorno en busca de un cambio que mejore su calidad de vida, hace que nos replanteemos las ciudades y los elementos que la componen. La presente investigación se centra en los pavimentos urbanos como elemento principal de conexión entre el ciudadano y su entorno, de generación del paisaje urbano. El paisaje urbano nos ofrece el pavimento como parte exterior y visible de su piel, la epidermis, en la que se apoya y desarrolla toda la actividad de la ciudad, dando forma e influyendo en la calidad del espacio Los pavimentos urbanos deben permitir la utilización y el uso de la vía pública exterior con total seguridad y confort para el ciudadano. El objetivo principal de la presente investigación es poder establecer criterios de diseño y uso en los pavimentos urbanos, atendiendo a los parámetros funcionales (estudio de la marcha humana: normal y patológica, ergonomía, antropometría, biomecánica) y formales o de diseño (relacionados con la dimensión, la forma, el color, la textura o las juntas, la usabilidad, durabilidad, desgaste). Se añaden factores externos como contaminantes, lugar, usos, climatología. La normativa existente, tanto a nivel nacional como internacional, no establece un nivel de seguridad para los pavimentos exteriores. La situación se agrava con la existencia de una variedad de métodos de ensayos y de directivas europeas, no consiguiendo llegar a un consenso en la designación del método de ensayo más adecuado al tipo de material y uso. La tesis concluye que se deben tener en cuenta las necesidades del ciudadano. Seleccionar el material adecuado al lugar y buscar las características adecuadas del material en las condiciones de uso previstas. Conocer el lugar y el uso al que se destina de forma que definamos las características fundamentales que se mantienen inalterables durante un periodo de tiempo razonable ABSTRACT The increasing need for citizens to recover their relationship with their environment as they seek to improve quality of life has made us reconsider cities and the elements that constitute them. This piece of research is focused on urban pavements as an essential element of connection between the citizen and their environment in the urban area. The urban area offers pavements as an external and visible part of its skin, its epidermis, upon which all activity is supported and carried out, giving shape and influencing in the quality of space. Citizens should be allowed to use urban pavements on public roads with total safety and comfort. The main objective of this research is to be able to establish criteria for the design and use of urban pavements, according to both functional parameters (study of human walking – normal and pathological, ergonometric, anthropometric, and biomechanically) as well as formal or design parameters (related to size, shape, colour, texture, joinings, usability, durability and resilience). Further external considerations are taken into account such as pollution, location, use and climate. Current regulations, both national and internationally, do not establish a standard degree of safety for exterior pavements. The situation is complicated further by the existence of a number of test methods and European directives, that do not reach a consensus on the most appropriate test methods on usage and materials. The thesis concludes that the focus should be citizen-centric. Materials should be chosen according to how appropriate they are for the location and the designated kind of usage. Understanding and knowing the site and the proposed kind of use are of fundamental necessity when defining the characteristics that are unchanging over a reasonable period of time.

Fernando García Mercadal y el Movimiento Moderno

La tesis plantea el estudio de la figura de Fernando García Mercadal (1896- 1985) y su obra, en el contexto del proyecto moderno perteneciente a la racionalidad teórica colectiva de la segunda generación del Movimiento Moderno en Europa, y explora la vida y circunstancias de un arquitecto que aunque nacido en el XIX, trajo la modernidad a España. El Movimiento Moderno en España, se articula en las décadas de los años 20 y 30 a través del GATEPAC y un grupo de arquitectos racionalistas en Madrid que Carlos Flores denomina Generación del 1925. Fernando García Mercadal, primero de su promoción en 1921 y pensionado en Roma, pertenece a ambos y pronto se convierte junto con Josep Lluis Sert, en una de las figuras más relevantes del panorama moderno español. Único miembro fundador del GATEPAC que había nacido en el siglo XIX, publica habitualmente en la revista AC (1931-1937), y en la revista Arquitectura desde 1920. Organiza en la Residencia de Estudiantes, entre 1928 y 1932, un famoso ciclo de Conferencias que imparten Le Corbusier, Mendelsohn, Van Doesburg, Gropius y Giedion. También asiste a la reunión constituyente de los CIAM en La Sarraz en 1928, al CIAM II en Frankfurt en 1929 y al CIAM III en Bruselas en 1930. Fue profesor en la Escuela de Arquitectura de Madrid y Arquitecto Jefe de la Oficina de Urbanismo y de Parques y Jardines del Ayuntamiento de Madrid, cargo al que accede por oposición en 1932. Tras la guerra fue depurado e inhabilitado profesionalmente, hasta que en 1946 comienza a trabajar en el Departamento de Arquitectura del I.N.P. donde continúa hasta los años 70. En 1979 es nombrado Académico de número de la Real Academia de Bellas Artes de San Fernando y muere en Madrid en 1985. Estos son los datos conocidos y aunque en la tesis se aportan nuevos datos inéditos, es en las premisas para su interpretación donde este trabajo de investigación pone el acento. En la historiografía de nuestra arquitectura moderna, pervive una tendencia a suponer una desconexión entre la vanguardia arquitectónica en los años 20 y 30 y el movimiento moderno internacional. El manto de silencio que cayó sobre la generación anterior a la guerra, una vez terminada esta, contribuye a reforzar la idea, cuyo origen y causas exceden el ámbito de trabajo, aunque se tratarán tangencialmente por ser ineludible hacerlo. La tesis pone en cuestión ese planteamiento desde el convencimiento fundamentado en los datos y en la consulta de fuentes originales, que la arquitectura española anterior a la guerra, aun con las circunstancias particulares del país, estuvo en sintonía con la europea, siendo esta la hipótesis de partida de la investigación. Las aportaciones más significativas que, a mi entender, presenta la tesis doctoral, y tienen mayor originalidad, son las siguientes; 1.Puesta en valor de la arquitectura de FGM, antes y después de la guerra, abandonando la idea de su supuesta renuncia a las ideas modernas a partir de los 30; 2. Puesta en valor, con aportación de datos concretos, de la Intensa relación mantenida por FGM y otros arquitectos españoles del Movimiento Moderno con los arquitectos de igual tendencia en el resto de Europa, a través de contactos recíprocos y continuos; 3. Estudio de la obra de FGM en el marco del Movimiento Moderno como una nueva arquitectura basada no tanto en la epidermis, como en una forma de hacer y encarar los problemas donde el proceso es tan importante como el resultado; con el Urbanismo como una nueva ciencia, y con el desarrollo de nuevos programas funcionales acordes a las necesidades de la sociedad contemporánea como el Rincón de Goya y el Hospital de Zaragoza. Se manejan tres métodos de trabajo. Los dos primeros aportan un nuevo enfoque al análisis crítico de FGM y su obra, situándole en el contexto internacional, además del español. El tercero, de carácter instrumental, permite el manejo y sistematización de la documentación. El primer método, consiste en aplicar el criterio generacional de Pevsner o Giedion entre otros, al Movimiento Moderno en España, situando a Fernando García Mercadal como arquitecto de la segunda generación, para hacer un análisis crítico comparativo de su trayectoria con otros arquitectos europeos de la misma, que permita establecer semejanzas y diferencias. El segundo método, complementario al anterior, consiste en estudiar las relaciones internacionales de FGM con las figuras más próximas a él y sus posibles influencias. Por último, y en relación con la documentación de obras y proyectos, se manejan a su vez dos criterios. Para la obra completa, y debido a la inexistencia de originales del Legado García Mercadal, se utiliza con carácter general el material ya publicado, disperso en libros y revistas. En el caso de las cinco obras seleccionadas, se completa con búsqueda en archivos, toma de datos in situ actualizados cuando es posible, y una recopilación de su repercusión en prensa escrita de su momento. ABSTRACT This research study focuses on Fernando Garcia Mercadal (1896-1985) and his work in the context of the Modern project as part of the collective theoretical rationality of the second generation of European Modern architecture. It explores the life and circumstances of the architect, who even though born in 19th century introduced Modern architecture in Spain. Modern architecture (Modern Movement) in Spain covered two decades between 20's and 30's through GATEPAC and a group of rationalists in Madrid that Carlos Flores named “generation of '25”. Fernando Garcia Mercadal, top of his class in 1921 and granted with the scholarship in Rome, belonged to both groups and early in his career he became, along with Josep Lluis Sert, one of the most relevant figures of Modern Architecture in Spain. He was the only member of GATEPAC who was born in 19th century. He frequently published on the magazine “AC” (1931-1937) and on “Arquitecture” magazine since 1920. He organized a series of famous lectures at “Residencia de Estudiantes” (Madrid) between 1928 and 1932 in which Le Corbusier, Mendelsohn, Van Doesburg, Gropius or Giedion took part. He was a member of the constituent meeting of CIAM in La Sarraz in 1928, CIAM II in Frankfurt in 1929 and CIAM III in Brussels in 1930. Mercadal was a teacher at Escuela de Arquitectura de Madrid and the Chief of the Urban Planning, Parks and Gardens Office of the Council of Madrid. He earned his position by public examination in 1932. After the civil war he was disqualified professionally until 1946, when he started working at the Architecture Department at INP until 70's. In 1979 he was elected as an academic member of “Real Academia de Bellas Artes de San Fernando” and later died in 1985. These are the well-known facts and even though this research study provides unpublished facts, it focuses on the premises to interpret them. Throughout the historiographical discourse of Spanish Modern architecture there is a tendency to assume a disconnection between the Avant-garde architecture movements in 20's and 30's and International Modern architecture (Modern Movement). The generation preceding the war remained silent in regard to this issue and once the war was over, they contributed to support the disconnection. The origin and cause exceed the frame of this work, even though they are incidentally commented due to its importance. This research study questions the approach explained above, based on the facts and the original sources. Its first hypothesis states that Spanish architecture before the civil war was, under its own circumstances, in tune with European architecture. The most important and original contributions of this research study are the following under my point of view: 1. To highlight the architecture of FGM, before and after the war, leaving behind his reputed withdrawal of Modernity after 30's; 2. To highlight the intense relationship between FGM and other Modern Spanish architects and other European architects sharing the same ideas, providing detailed facts; 3. Study of FGM's work in the context of Modern architecture as a new architecture based on its know-how and the way problems are faced. The process is as important as the result, not so much based on the dermis; with urban planning as the new science and with the development of the new functional programs based on the needs of contemporary society as in Rincón de Goya or Hospital de Zaragoza. Three work methods are used. The first two add a new vision of the critical analysis related to FGM and his work, positioning him in the international context in addition to Spain. The third is used as an instrument to manage and systematize the documentation. The first method applies the generational criteria of Pevsner or Giedion (among others) to Modern architecture in Spain, positioning Fernando Garcia Mercadal as a second generation architect. A critical-comparative analysis of his career and contemporary European architects is made to establish similarities and differences. The second method is complementary to the previous one and studies the international relationships of FGM with other recognised architects that were close to him and their possible influences. At last, in relation to his works and projects, two methods are used. For the complete works, due to the lack of originals, published material found on magazines and books is used as the source. In the case of the five selected buildings, it is complemented with archive search, onsite data collection when possible and the impact on the press at that moment.

Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3–33.3% of the colonies thus formed were β-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were β-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Regulation of sulfur assimilation in higher plants: A sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana

Proton/sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants. Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation. In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and serine acetyltransferase (SAT1) were inducible by sulfate starvation in A. thaliana. The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.

The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains

Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3′ end of the EDA gene. All transcripts recovered have the same 5′ exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.

CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA+ memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA+ T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.

Spatio-temporally controlled site-specific somatic mutagenesis in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination–excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (≈40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli β-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.

Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.

A transgenic mouse model with an inducible skin blistering disease phenotype

One of the current limitations of gene transfer protocols involving mammalian genomes is the lack of spatial and temporal control over the desired gene manipulation. Starting from a human keratin gene showing a complex regulation as a template, we identified regulatory sequences that confer inducible gene expression in a subpopulation of keratinocytes in stratified epithelia of adult transgenic mice. We used this cassette to produce transgenic mice with an inducible skin blistering phenotype mimicking a form of epidermolytic hyperkeratosis, a keratin gene disorder. Upon induction by topical application of a phorbol ester, the mutant keratin transgene product accumulates in the differentiating layers of epidermis, leading to keratinocyte lysis after application of mechanical trauma. This mouse model will allow for a better understanding of the complex relationship between keratin mutation, keratinocyte cytoarchitecture, and hypersensitivity to trauma. The development of an inducible expression vector showing an exquisite cellular specificity has important implications for manipulating genes in a spatially and temporally controlled fashion in transgenic mice, and for the design of gene therapy strategies using skin as a tissue source for the controlled delivery of foreign substances.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis

H-2Kb-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181–188 (TRP-2) and connexin 37 residues 52–59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Iab+ cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.

Mouse Deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix–loop–helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.