Influencia de las condiciones de ejecución en la resistencia de anclajes en convencional y autocompactante

A través de los años las estructuras de hormigón armado han ido aumentando su cuota de mercado, sustituyendo a las estructuras de fábrica de piedra o ladrillo y restándole participación a las estructuras metálicas. Uno de los primeros problemas que surgieron al ejecutar las estructuras de hormigón armado, era cómo conectar una fase de una estructura de este tipo a una fase posterior o a una modificación posterior. Hasta los años 80-90 las conexiones de una fase de una estructura de hormigón armado, con otra posterior se hacían dejando en la primera fase placas de acero con garrotas embebidas en el hormigón fresco o barras grifadas recubiertas de poliestireno expandido. Una vez endurecido el hormigón se podían conectar nuevas barras, para la siguiente fase mediante soldadura a la placa de la superficie o enderezando las barras grifadas, para embeberlas en el hormigón fresco de la fase siguiente. Estos sistemas requerían conocer la existencia y alcance de la fase posterior antes de hormigonar la fase previa. Además requerían un replanteo muy exacto y complejo de los elementos de conexión. Otro problema existente en las estructuras de hormigón era la adherencia de un hormigón fresco a un hormigón endurecido previamente, ya que la superficie de contacto de ambos hormigones suponía un punto débil, con una adherencia baja. A partir de los años 80, la industria química de la construcción experimentó un gran avance en el desarrollo de productos capaces de generar una buena adherencia sobre el hormigón endurecido. Este avance tecnológico tenía aplicación tanto en la adherencia del hormigón fresco sobre el hormigón endurecido, como en la adherencia de barras post-instaladas en agujeros de hormigón endurecido. Este sistema se denominó “anclajes adherentes de barras de acero en hormigón endurecido”. La forma genérica de ejecutarlos es hacer una perforación cilíndrica en el soporte de hormigón, con una herramienta especifica como un taladro, limpiar la perforación, llenarla del material adherente y finalmente introducir la barra de acero. Los anclajes adherentes se dividen en anclajes cementosos y anclajes químicos, siendo estos últimos los más habituales, fiables, resistentes y fáciles de ejecutar. El uso del anclaje adherente de barras de acero en hormigón endurecido se ha extendido por todo el espectro productivo, siendo muy habitual tanto en construcción de obras de hormigón armado de obra civil y edificación, como en obras industriales, instalaciones o fijación de elementos. La ejecución de un anclaje de una barra de acero en hormigón endurecido depende de numerosas variables, que en su conjunto, o de forma aislada pueden afectar de forma notable a la resistencia del anclaje. Nos referimos a variables de los anclajes, que a menudo no se consideran tales como la dirección de la perforación, la máquina de perforación y el útil de perforación utilizado, la diferencia de diámetros entre el diámetro del taladro y la barra, el tipo de material de anclaje, la limpieza del taladro, la humedad del soporte, la altura del taladro, etc. La utilización en los últimos años de los hormigones Autocompactables, añade una variable adicional, que hasta ahora apenas ha sido estudiada. En línea con lo apuntado, la presente tesis doctoral tiene como objetivo principal el estudio de las condiciones de ejecución en la resistencia de los anclajes en hormigón convencional y autocompactable. Esta investigación se centra principalmente en la evaluación de la influencia de una serie de variables sobre la resistencia de los anclajes, tanto en hormigón convencional como en un hormigón autocompactable. Para este estudio ha sido necesaria la fabricación de dos soportes de hormigón sobre los cuales desarrollar los ensayos. Uno de los bloques se ha fabricado con hormigón convencional y el otro con hormigón autocompactable. En cada pieza de hormigón se han realizado 174 anclajes con barras de acero, variando los parámetros a estudiar, para obtener resultados de todas las variables consideradas. Los ensayos a realizar en ambos bloques son exactamente iguales, para poder comparar la diferencia entre un anclaje en un soporte de hormigón con vibrado convencional (HVC) y un hormigón autocompactante (HAC). De cada tipo de ensayo deseado se harán dos repeticiones en la misma pieza. El ensayo de arrancamiento de las barras se realizara con un gato hidráulico hueco, con un sistema de instrumentación de lectura y registro de datos en tiempo real. El análisis de los resultados, realizado con una potente herramienta estadística, ha permitido determinar y evaluar numéricamente la influencia de los variables consideradas en la resistencia de los anclajes realizados. Así mismo ha permitido diferenciar los resultados obtenidos en los hormigones convencionales y autocompactantes, tanto desde el punto de vista de la resistencia mecánica, como de las deformaciones sufridas en el arrancamiento. Se define la resistencia mecánica de un anclaje, como la fuerza desarrollada en la dirección de la barra, para hacer su arrancamiento del soporte. De la misma forma se considera desplazamiento, a la separación entre un punto fijo de la barra y otro del soporte, en la dirección de la barra. Dichos puntos se determinan cuando se ha terminado el anclaje, en la intersección de la superficie plana del soporte, con la barra. Las conclusiones obtenidas han permitido establecer qué variables afectan a la ejecución de los anclajes y en qué cuantía lo hacen, así como determinar la diferencia entre los anclajes en hormigón vibrado convencional y hormigón autocompactante, con resultados muy interesantes, que permiten valorar la influencia de dichas variables. Dentro de las conclusiones podemos destacar tres grupos, que denominaremos como de alta influencia, baja influencia y sin influencia. En todos los casos hay que hacer el estudio en términos de carga y de desplazamiento. Podemos considerar como de alta influencia, en términos de carga las variables de máquina de perforación y el material de anclaje. En términos de desplazamiento podemos considerar de alta influencia además de la máquina de perforación y el material de anclaje, el diámetro del taladro, así como la limpieza y humedad del soporte. Podemos considerar de baja influencia, en términos de carga las variables de tipo de hormigón, dirección de perforación, limpieza y humedad del soporte. En términos de desplazamiento podemos considerar de baja influencia el tipo de hormigón y la dirección de perforación. Podemos considerar en el apartado de “sin influencia”, en términos de carga las variables de diámetro de perforación y altura del taladro. En términos de desplazamiento podemos considerar como “sin influencia” la variable de altura del taladro. Podemos afirmar que las diferencias entre los valores de carga aumentan de forma muy importante en términos de desplazamiento. ABSTRACT Over the years the concrete structures have been increasing their market share, replacing the masonry structures of stone or brick and subtracting as well the participation of the metallic structures. One of the first problems encountered in the implementing of the reinforced concrete structures was connecting a phase structure of this type at a later stage or a subsequent amendment. Until the 80s and 90s the connections of one phase of a reinforced concrete structure with a subsequent first phase were done by leaving the steel plates embedded in the fresh concrete using hooks or bent bars coated with expanded polystyrene. Once the concrete had hardened new bars could be connected to the next stage by welding them to the surface plate or by straightening the bent bars to embed them in the fresh concrete of the next phase. These systems required a previous knowledge of the existence and scope of the subsequent phase before concreting the previous one. They also required a very precise and complex rethinking of the connecting elements. Another existing problem in the concrete structures was the adhesion of a fresh concrete to a previously hardened concrete, since the contact surface of both concretes leaded to a weak point with low adherence. Since the 80s, the chemicals construction industry experienced a breakthrough in the development of products that generate a good grip on the concrete. This technological advance had its application both in the grip on one hardened fresh concrete and in the adhesion of bar post-installed in holes of hardened concrete. This system was termed as adherent anchors of steel bars in hardened concrete. The generic way of executing this system is by firstly drilling a cylindrical hole in the concrete support using a specific tool such as a drill. Then, cleaning the bore and filling it with bonding material to lastly, introduce the steel bar. These adherent anchors are divided into cement and chemical anchors, the latter being the most common, reliable, durable and easy to run. The use of adhesive anchor of steel bars in hardened concrete has spread across the production spectrum turning itself into a very common solution in both construction of reinforced concrete civil engineering and construction, and industrial works, installations and fixing elements as well. The execution of an anchor of a steel bar in hardened concrete depends on numerous variables which together or as a single solution may significantly affect the strength of the anchor. We are referring to variables of anchors which are often not considered, such as the diameter difference between the rod and the bore, the drilling system, cleansing of the drill, type of anchor material, the moisture of the substrate, the direction of the drill, the drill’s height, etc. During recent years, the emergence of self-compacting concrete adds an additional variable which has hardly been studied so far. According to mentioned this thesis aims to study the main performance conditions in the resistance of conventional and self-compacting concrete anchors. This research is primarily focused on the evaluation of the influence of several variables on the strength of the anchoring, both in conventional concrete and self-compacting concrete. In order to complete this study it has been required the manufacture of two concrete supports on which to develop the tests. One of the blocks has been manufactured with conventional concrete and the other with self-compacting concrete. A total of 174 steel bar anchors have been made in each one of the concrete pieces varying the studied parameters in order to obtain results for all variables considered. The tests to be performed on both blocks are exactly the same in order to compare the difference between an anchor on a stand with vibrated concrete (HVC) and a self-compacting concrete (SCC). Each type of test required two repetitions in the same piece. The pulling test of the bars was made with a hollow jack and with an instrumentation system for reading and recording data in real time. The use of a powerful statistical tool in the analysis of the results allowed to numerically determine and evaluate the influence of the variables considered in the resistance of the anchors made. It has likewise enabled to differentiate the results obtained in the self-compacting and conventional concretes, from both the outlook of the mechanical strength and the deformations undergone by uprooting. The mechanical strength of an anchor is defined as the strength undergone in a direction of the bar to uproot it from the support. Likewise, the movement is defined as the separation between a fixed point of the bar and a fixed point from the support considering the direction of the bar. These points are only determined once the anchor is finished, with the bar, at the intersection in the flat surface of the support. The conclusions obtained have established which variables affect the execution of the anchors and in what quantity. They have also permitted to determine the difference between the anchors in vibrated concrete and selfcompacting concrete with very interesting results that also allow to assess the influence of these mentioned variables. Three groups are highlighted among the conclusions called high influence, low influence and no influence. In every case is necessary to perform the study in terms of loading and movement. In terms of loading, there are considered as high influence two variables: drilling machinery and anchorage material. In terms of movement, there are considered as high influence the drilling diameter and the cleaning and moisture of the support, besides the drilling machinery and the anchorage material. Variables such as type of concrete, drilling direction and cleaning and moisture of the support are considered of low influence in terms of load. In terms of movement, the type of concrete and the direction of the drilling are considered variables of low influence. Within the no influence section in terms of loading, there are included the diameter of the drilling and the height of the drill. In terms of loading, the height of the drill is considered as a no influence variable. We can affirm that the differences among the loading values increase significantly in terms of movement.

Refined facial disparity maps for automatic creation of 3D avatars

We propose a new method to automatically refine a facial disparity map obtained with standard cameras and under conventional illumination conditions by using a smart combination of traditional computer vision and 3D graphics techniques. Our system inputs two stereo images acquired with standard (calibrated) cameras and uses dense disparity estimation strategies to obtain a coarse initial disparity map, and SIFT to detect and match several feature points in the subjects face. We then use these points as anchors to modify the disparity in the facial area by building a Delaunay triangulation of their convex hull and interpolating their disparity values inside each triangle. We thus obtain a refined disparity map providing a much more accurate representation of the the subjects facial features. This refined facial disparity map may be easily transformed, through the camera calibration parameters, into a depth map to be used, also automatically, to improve the facial mesh of a 3D avatar to match the subjects real human features.

Sobre el comportamiento de micropilotes trabajando a flexión y/o cortante en estructuras de tierra

El empleo de los micropilotes en la ingeniería civil ha revolucionado las técnicas de estabilización de terraplenes a media ladera, ya que aunque los pilotes pueden ser la opción más económica, el uso de micropilotes permite llegar a sitios inaccesibles con menor coste de movimientos de tierras, realización de plataformas de trabajo de dimensiones reducidas, maquinaria necesaria es mucho más pequeña, liviana y versátil en su uso, incluyendo la posibilidad de situar la fabricación de morteros o lechadas a distancias de varias decenas de metros del elemento a ejecutar. Sin embargo, realizando una revisión de la documentación técnica que se tiene en el ámbito ingenieril, se comprobó que los sistemas de diseño de algunos casos (micropilotes en terraplenes a media ladera, micropilotes en pantallas verticales, micropilotes como “paraguas” en túneles, etc.) eran bastante deficientes o poco desarrollados. Premisa que permite concluir que el constructor ha ido por delante (como suele ocurrir en ingeniería geotécnica) del cálculo o de su análisis teórico. Del mismo modo se determinó que en su mayoría los micropilotes se utilizan en labores de recalce o como nueva solución de cimentación en condiciones de difícil acceso, casos en los que el diseño de los micropilotes viene definido por cargas axiales, de compresión o de tracción, consideraciones que se contemplan en reglamentaciones como la “Guía para el proyecto y la ejecución de micropilotes en obras de carretera” del Ministerio de Fomento. En los micropilotes utilizados para estabilizar terraplenes a media ladera y micropilotes actuando como muros pantalla, en los que éstos trabajan a esfuerzo cortante y flexión, no se dispone de sistemas de análisis fiables o no se introduce adecuadamente el problema de interacción terreno-micropilote. Además en muchos casos, los parámetros geotécnicos que se utilizan no tienen una base técnico-teórica adecuada por lo que los diseños pueden quedar excesivamente del lado de la seguridad, en la mayoría de los casos, o todo lo contrario. Uno de los objetivos principales de esta investigación es estudiar el comportamiento de los micropilotes que están sometidos a esfuerzos de flexión y cortante, además de otros objetivos de gran importancia que se describen en el apartado correspondiente de esta tesis. Cabe indicar que en este estudio no se ha incluido el caso de micropilotes quasi-horizontales trabajando a flexion (como los “paraguas” en túneles), por considerarse que estos tienen un comportamiento y un cálculo diferente, que está fuera del alcance de esta investigación. Se ha profundizado en el estudio del empleo de micropilotes en taludes, presentando casos reales de obras ejecutadas, datos estadísticos, problemas de diseño y ejecución, métodos de cálculo simplificados y modelación teórica en cada caso, efectuada mediante el empleo de elementos finitos con el Código Plaxis 2D. Para llevar a cabo los objetivos que se buscan con esta investigación, se ha iniciado con el desarrollo del “Estado del Arte” que ha permitido establecer tipología, aplicaciones, características y cálculo de los micropilotes que se emplean habitualmente. Seguidamente y a efectos de estudiar el problema dentro de un marco geotécnico real, se ha seleccionado una zona española de actuación, siendo ésta Andalucía, en la que se ha utilizado de manera muy importante la técnica de micropilotes sobre todo en problemas de estabilidad de terraplenes a media ladera. A partir de ahí, se ha realizado un estudio de las propiedades geotécnicas de los materiales (principalmente suelos y rocas muy blandas) que están presentes en esta zona geográfica, estudio que ha sido principalmente bibliográfico o a partir de la experiencia en la zona del Director de esta tesis. Del análisis realizado se han establecido ordenes de magnitud de los parámetros geotécnicos, principalmente la cohesión y el ángulo de rozamiento interno (además del módulo de deformación aparente o de un módulo de reacción lateral equivalente) para los diversos terrenos andaluces. Con el objeto de conocer el efecto de la ejecución de un micropilote en el terreno (volumen medio real del micropilote, una vez ejecutado; efecto de la presión aplicada en las propiedades del suelo circundante, etc.) se ha realizado una encuesta entre diversas empresas españolas especializadas en la técnica de los micropilotes, a efectos de controlar los volúmenes de inyección y las presiones aplicadas, en función de la deformabilidad del terreno circundante a dichos micropilotes, con lo que se ha logrado definir una rigidez a flexión equivalente de los mismos y la definición y características de una corona de terreno “mejorado” lograda mediante la introducción de la lechada y el efecto de la presión alrededor del micropilote. Con las premisas anteriores y a partir de los parámetros geotécnicos determinados para los terrenos andaluces, se ha procedido a estudiar la estabilidad de terraplenes apoyados sobre taludes a media ladera, mediante el uso de elementos finitos con el Código Plaxis 2D. En el capítulo 5. “Simulación del comportamiento de micropilotes estabilizando terraplenes”, se han desarrollado diversas simulaciones. Para empezar se simplificó el problema simulando casos similares a algunos reales en los que se conocía que los terraplenes habían llegado hasta su situación límite (de los que se disponía información de movimientos medidos con inclinómetros), a partir de ahí se inició la simulación de la inestabilidad para establecer el valor de los parámetros de resistencia al corte del terreno (mediante un análisis retrospectivo – back-análisis) comprobando a su vez que estos valores eran similares a los deducidos del estudio bibliográfico. Seguidamente se han introducido los micropilotes en el borde de la carretera y se ha analizado el comportamiento de éstos y del talud del terraplén (una vez construidos los micropilotes), con el objeto de establecer las bases para su diseño. De este modo y adoptando los distintos parámetros geotécnicos establecidos para los terrenos andaluces, se simularon tres casos reales (en Granada, Málaga y Ceuta), comparando los resultados de dichas simulaciones numéricas con los resultados de medidas reales de campo (desplazamientos del terreno, medidos con inclinómetros), obteniéndose una reproducción bastante acorde a los movimientos registrados. Con las primeras simulaciones se concluye que al instalar los micropilotes la zona más insegura de la ladera es la de aguas abajo. La superficie de rotura ya no afecta a la calzada que protegen los micropilotes. De ahí que se deduzca que esta solución sea válida y se haya aplicado masivamente en Andalucía. En esas condiciones, podría decirse que no se está simulando adecuadamente el trabajo de flexión de los micropilotes (en la superficie de rotura, ya que no les corta), aunque se utilicen elementos viga. Por esta razón se ha realizado otra simulación, basada en las siguientes hipótesis: − Se desprecia totalmente la masa potencialmente deslizante, es decir, la que está por delante de la fila exterior de micros. − La estratigrafía del terreno es similar a la considerada en las primeras simulaciones. − La barrera de micropilotes está constituida por dos elementos inclinados (uno hacia dentro del terraplén y otro hacia fuera), con inclinación 1(H):3(V). − Se puede introducir la rigidez del encepado. − Los micros están separados 0,556 m ó 1,00 m dentro de la misma alineación. − El empotramiento de los micropilotes en el sustrato resistente puede ser entre 1,5 y 7,0 m. Al “anular” el terreno que está por delante de los micropilotes, a lo largo del talud, estos elementos empiezan claramente a trabajar, pudiendo deducirse los esfuerzos de cortante y de flexión que puedan actuar sobre ellos (cota superior pero prácticamente muy cerca de la solución real). En esta nueva modelación se ha considerado tanto la rigidez equivalente (coeficiente ϴ) como la corona de terreno tratado concéntrico al micropilote. De acuerdo a esto último, y gracias a la comparación de estas modelaciones con valores reales de movimientos en laderas instrumentadas con problemas de estabilidad, se ha verificado que existe una similitud bastante importante entre los valores teóricos obtenidos y los medidos en campo, en relación al comportamiento de los micropilotes ejecutados en terraplenes a media ladera. Finalmente para completar el análisis de los micropilotes trabajando a flexión, se ha estudiado el caso de micropilotes dispuestos verticalmente, trabajando como pantallas discontinuas provistas de anclajes, aplicado a un caso real en la ciudad de Granada, en la obra “Hospital de Nuestra Señora de la Salud”. Para su análisis se utilizó el código numérico CYPE, basado en que la reacción del terreno se simula con muelles de rigidez Kh o “módulo de balasto” horizontal, introduciendo en la modelación como variables: a) Las diferentes medidas obtenidas en campo; b) El espesor de terreno cuaternario, que por lo que se pudo determinar, era variable, c) La rigidez y tensión inicial de los anclajes. d) La rigidez del terreno a través de valores relativos de Kh, recopilados en el estudio de los suelos de Andalucía, concretamente en la zona de Granada. Dicha pantalla se instrumentó con 4 inclinómetros (introducidos en los tubos de armadura de cuatro micropilotes), a efectos de controlar los desplazamientos horizontales del muro de contención durante las excavaciones pertinentes, a efectos de comprobar la seguridad del conjunto. A partir del modelo de cálculo desarrollado, se ha comprobado que el valor de Kh pierde importancia debido al gran número de niveles de anclajes, en lo concerniente a las deformaciones horizontales de la pantalla. Por otro lado, los momentos flectores son bastante sensibles a la distancia entre anclajes, al valor de la tensión inicial de los mismos y al valor de Kh. Dicho modelo también ha permitido reproducir de manera fiable los valores de desplazamientos medidos en campo y deducir los parámetros de deformabilidad del terreno, Kh, con valores del orden de la mitad de los medidos en el Metro Ligero de Granada, pero visiblemente superiores a los deducibles de ábacos que permiten obtener Kh para suelos granulares con poca cohesión (gravas y cuaternario superior de Sevilla) como es el caso del ábaco de Arozamena, debido, a nuestro juicio, a la cementación de los materiales presentes en Granada. En definitiva, de las anteriores deducciones se podría pensar en la optimización del diseño de los micropilotes en las obras que se prevean ejecutar en Granada, con similares características al caso de la pantalla vertical arriostrada mediante varios niveles de anclajes y en las que los materiales de emplazamiento tengan un comportamiento geotécnico similar a los estudiados, con el consiguiente ahorro económico. Con todo ello, se considera que se ha hecho una importante aportación para el diseño de futuras obras de micropilotes, trabajando a flexión y cortante, en obras de estabilización de laderas o de excavaciones. Using micropiles in civil engineering has transformed the techniques of stabilization of embankments on the natural or artificial slopes, because although the piles may be the cheapest option, the use of micropiles can reach inaccessible places with lower cost of earthworks, carrying out small work platforms. Machinery used is smaller, lightweight and versatile, including the possibility of manufacturing mortars or cement grouts over distances of several tens of meters of the element to build. However, making a review of the technical documentation available in the engineering field, it was found that systems designed in some cases (micropiles in embankments on the natural slopes, micropiles in vertical cut-off walls, micropiles like "umbrella" in tunnels, etc.) were quite poor or underdeveloped. Premise that concludes the builder has gone ahead (as usually happen in geotechnical engineering) of calculation or theoretical analysis. In the same way it was determined that most of the micropiles are used in underpinning works or as a new foundation solution in conditions of difficult access, in which case the design of micropiles is defined by axial, compressive or tensile loads, considered in regulations as the " Handbook for the design and execution of micropiles in road construction" of the Ministry of Development. The micropiles used to stabilize embankments on the slopes and micropiles act as retaining walls, where they work under shear stress and bending moment, there are not neither reliable systems analysis nor the problem of soil-micropile interaction are properly introduced. Moreover, in many cases, the geotechnical parameters used do not have a proper technical and theoretical basis for what designs may be excessively safe, or the opposite, in most cases. One of the main objectives of this research is to study the behavior of micro piles which are subjected to bending moment and shear stress, as well as other important objectives described in the pertinent section of this thesis. It should be noted that this study has not included the case of quasi-horizontal micropiles working bending moment (as the "umbrella" in tunnels), because it is considered they have a different behavior and calculation, which is outside the scope of this research. It has gone in depth in the study of using micropiles on slopes, presenting real cases of works made, statistics, problems of design and implementation, simplified calculation methods and theoretical modeling in each case, carried out by using FEM (Finite Element Method) Code Plaxis 2D. To accomplish the objectives of this research, It has been started with the development of the "state of the art" which stipulate types, applications, characteristics and calculation of micropiles that are commonly used. In order to study the problem in a real geotechnical field, it has been selected a Spanish zone of action, this being Andalusia, in which it has been used in a very important way, the technique of micropiles especially in embankments stability on natural slopes. From there, it has made a study of the geotechnical properties of the materials (mainly very soft soils and rocks) that are found in this geographical area, which has been mainly a bibliographic study or from the experience in the area of the Director of this thesis. It has been set orders of magnitude of the geotechnical parameters from analyzing made, especially the cohesion and angle of internal friction (also apparent deformation module or a side reaction module equivalent) for various typical Andalusian ground. In order to determine the effect of the implementation of a micropile on the ground (real average volume of micropile once carried out, effect of the pressure applied on the properties of the surrounding soil, etc.) it has conducted a survey among various skilled companies in the technique of micropiles, in order to control injection volumes and pressures applied, depending on the deformability of surrounding terrain such micropiles, whereby it has been possible to define a bending stiffness and the definition and characteristics of a crown land "improved" achieved by introducing the slurry and the effect of the pressure around the micropile. With the previous premises and from the geotechnical parameters determined for the Andalusian terrain, we proceeded to study the stability of embankments resting on batters on the slope, using FEM Code Plaxis 2D. In the fifth chapter "Simulation of the behavior of micropiles stabilizing embankments", there were several different numerical simulations. To begin the problem was simplified simulating similar to some real in which it was known that the embankments had reached their limit situation (for which information of movements measured with inclinometers were available), from there the simulation of instability is initiated to set the value of the shear strength parameters of the ground (by a retrospective analysis or back-analysis) checking these values were similar to those deduced from the bibliographical study Then micropiles have been introduced along the roadside and its behavior was analyzed as well as the slope of embankment (once micropiles were built ), in order to establish the basis for its design. In this way and taking the different geotechnical parameters for the Andalusian terrain, three real cases (in Granada, Malaga and Ceuta) were simulated by comparing the results of these numerical simulations with the results of real field measurements (ground displacements measured with inclinometers), getting quite consistent information according to registered movements. After the first simulations it has been concluded that after installing the micropiles the most insecure area of the natural slope is the downstream. The failure surface no longer affects the road that protects micropiles. Hence it is inferred that this solution is acceptable and it has been massively applied in Andalusia. Under these conditions, one could say that it is not working properly simulating the bending moment of micropiles (on the failure surface, and that does not cut them), although beam elements are used. Therefore another simulation was performed based on the following hypotheses: − The potentially sliding mass is totally neglected, that is, which is ahead of the outer row of micropiles. − Stratigraphy field is similar to the one considered in the first simulations. − Micropiles barrier is constituted by two inclined elements (one inward and one fill out) with inclination 1 (H): 3 (V). − You can enter the stiffness of the pile cap. − The microlies lines are separated 0.556 m or 1.00 m in the same alignment. − The embedding of the micropiles in the tough substrate can be between 1.5 and 7.0 m. To "annul" the ground that is in front of the micro piles, along the slope, these elements clearly start working, efforts can be inferred shear stress and bending moment which may affect them (upper bound but pretty close to the real) solution. In this new modeling it has been considered both equivalent stiffness coefficient (θ) as the treated soil crown concentric to the micropile. According to the latter, and by comparing these values with real modeling movements on field slopes instrumented with stability problems, it was verified that there is quite a significant similarity between the obtained theoretical values and the measured field in relation to the behavior of micropiles executed in embankments along the natural slope. Finally to complete the analysis of micropiles working in bending conditions, we have studied the case of micropiles arranged vertically, working as discontinued cut-off walls including anchors, applied to a real case in the city of Granada, in the play "Hospital of Our Lady of the Health ". CYPE numeric code, based on the reaction of the ground is simulated spring stiffness Kh or "subgrade" horizontal, introduced in modeling was used as variables for analysis: a) The different measurements obtained in field; b) The thickness of quaternary ground, so that could be determined, was variable, c) The stiffness and the prestress of the anchors. d) The stiffness of the ground through relative values of Kh, collected in the study of soils in Andalusia, particularly in the area of Granada. (previously study of the Andalusia soils) This cut-off wall was implemented with 4 inclinometers (introduced in armor tubes four micropiles) in order to control the horizontal displacements of the retaining wall during the relevant excavations, in order to ensure the safety of the whole. From the developed model calculation, it was found that the value of Kh becomes less important because a large number of anchors levels, with regard to the horizontal deformation of the cut-off wall. On the other hand, the bending moments are quite sensitive to the distance between anchors, the initial voltage value thereof and the value of Kh. This model has also been reproduced reliably displacement values measured in the field and deduce parameters terrain deformability, Kh, with values around half the measured Light Rail in Granada, but visibly higher than deductible of abacuses which can obtain Kh for granular soils with low cohesion (upper Quaternary gravels and Sevilla) such as Abacus Arozamena, because, in our view, to cementing materials in Granada. In short, previous deductions you might think on optimizing the design of micropiles in the works that are expected to perform in Granada, with similar characteristics to the case of the vertical cut-off wall braced through several levels of anchors and in which materials location have a geotechnical behavior similar to those studied, with the consequent economic savings. With all this, it is considered that a significant contribution have been made for the design of future works of micropiles, bending moment and shear stress working in slope stabilization works or excavations.

Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein

TVA, the cellular receptor for subgroup A avian leukosis viruses (ALV-A) can mediate viral entry when expressed as a transmembrane protein or as a glycosylphosphatidylinositol-linked protein on the surfaces of transfected mammalian cells. To determine whether mammalian cells can be rendered susceptible to ALV-A infection by attaching a soluble form of TVA to their plasma membranes, the TVA-epidermal growth factor (EGF) fusion protein was generated. TVA-EGF is comprised of the extracellular domain of TVA linked to the mature form of human EGF. Flow cytometric analysis confirmed that TVA-EGF is a bifunctional reagent capable of binding simultaneously to cell surface EGF receptors and to an ALV-A surface envelope-Ig fusion protein. TVA-EGF prebound to transfected mouse fibroblasts expressing either wild-type or kinase-deficient human EGF receptors, rendered these cells highly susceptible to infection by ALV-A vectors. Viral infection was blocked specifically in the presence of a recombinant human EGF protein, demonstrating that the binding of TVA-EGF to EGF receptors was essential for infectivity. These studies have demonstrated that a soluble TVA-ligand fusion protein can mediate viral infection when attached to specific cell surfaces, suggesting an approach for targeting retroviral infection to specific cell types.

PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.

Interaction of pigeon cytochrome c-(43–58) peptide analogs with either T cell antigen receptor or I-Ab molecule

We determined that a pigeon cytochrome c-derived peptide, p43–58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43–58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43–58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vβ usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR β chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR β chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.

Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development

Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.

Erythrocyte membrane vesiculation: Model for the molecular mechanism of protein sorting

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

The crystal structure of modified bovine fibrinogen

Here we report the crystal structure at ≈4-Å resolution of a selectively proteolyzed bovine fibrinogen. This key component in hemostasis is an elongated 340-kDa glycoprotein in the plasma that upon activation by thrombin self-assembles to form the fibrin clot. The crystals are unusual because they are made up of end-to-end bonded molecules that form flexible filaments. We have visualized the entire coiled-coil region of the molecule, which has a planar sigmoidal shape. The primary polymerization receptor pockets at the ends of the molecule face the same way throughout the end-to-end bonded filaments, and based on this conformation, we have developed an improved model of the two-stranded protofibril that is the basic building block in fibrin. Near the middle of the coiled-coil region, the plasmin-sensitive segment is a hinge about which the molecule adopts different conformations. This segment also includes the boundary between the three- and four-stranded portions of the coiled coil, indicating the location on the backbone that anchors the extended flexible Aα arm. We suggest that a flexible branch point in the molecule may help accommodate variability in the structure of the fibrin clot.

The role of phosphoglycans in Leishmania–sand fly interactions

Leishmania promastigotes synthesize an abundance of phosphoglycans, either attached to the cell surface through phosphatidylinositol anchors (lipophosphoglycan, LPG) or secreted as protein-containing glycoconjugates. These phosphoglycans are thought to promote the survival of the parasite within both its vertebrate and invertebrate hosts. The relative contributions of different phosphoglycan-containing molecules in Leishmania–sand fly interactions were tested by using mutants specifically deficient in either total phosphoglycans or LPG alone. Leishmania donovani promastigotes deficient in both LPG and protein-linked phosphoglycans because of loss of LPG2 (encoding the Golgi GDP-Man transporter) failed to survive the hydrolytic environment within the early blood-fed midgut. In contrast, L. donovani and Leishmania major mutants deficient solely in LPG expression because of loss of LPG1 (involved in biosynthesis of the core oligosaccharide LPG domain) had only a slight reduction in the survival and growth of promastigotes within the early blood-fed midgut. The ability of the LPG1-deficient promastigotes to persist in the midgut after blood meal excretion was completely lost, and this defect was correlated with their inability to bind to midgut epithelial cells in vitro. For both mutants, when phosphoglycan expression was restored to wild-type levels by reintroduction of LPG1 or LPG2 (as appropriate), then the wild-type phenotype was also restored. We conclude, first, that LPG is not essential for survival in the early blood-fed midgut but, along with other secreted phosphoglycan-containing glycoconjugates, can protect promastigotes from the digestive enzymes in the gut and, second, that LPG is required to mediate midgut attachment and to maintain infection in the fly during excretion of the digested blood meal.

VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

HCC-2, a human chemokine: Gene structure, expression pattern, and biological activity

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.