Multiscale analysis of the mechanical behavior of needle-punched nonwoven fabrics

Los fieltros son una familia de materiales textiles constituidos por una red desordenada de fibras conectadas por medio de enlaces térmicos, químicos o mecánicos. Presentan menor rigidez y resistencia (al igual que un menor coste de procesado) que sus homólogos tejidos, pero mayor deformabilidad y capacidad de absorción de energía. Los fieltros se emplean en diversas aplicaciones en ingeniería tales como aislamiento térmico, geotextiles, láminas ignífugas, filtración y absorción de agua, impacto balístico, etc. En particular, los fieltros punzonados fabricados con fibras de alta resistencia presentan una excelente resistencia frente a impacto balístico, ofreciendo las mismas prestaciones que los materiales tejidos con un tercio de la densidad areal. Sin embargo, se sabe muy poco acerca de los mecanismos de deformación y fallo a nivel microscópico, ni sobre como influyen en las propiedades mecánicas del material. Esta carencia de conocimiento dificulta la optimización del comportamiento mecánico de estos materiales y también limita el desarrollo de modelos constitutivos basados en mecanismos físicos, que puedan ser útiles en el diseño de componentes estructurales. En esta tesis doctoral se ha llevado a cabo un estudio minucioso con el fin de determinar los mecanismos de deformación y las propiedades mecánicas de fieltros punzonados fabricados con fibras de polietileno de ultra alto peso molecular. Los procesos de deformación y disipación de energía se han caracterizado en detalle por medio de una combinación de técnicas experimentales (ensayos mecánicos macroscópicos a velocidades de deformación cuasi-estáticas y dinámicas, impacto balístico, ensayos de extracción de una o múltiples fibras, microscopía óptica, tomografía computarizada de rayos X y difracción de rayos X de gran ángulo) que proporcionan información de los mecanismos dominantes a distintas escalas. Los ensayos mecánicos macroscópicos muestran que el fieltro presenta una resistencia y ductilidad excepcionales. El estado inicial de las fibras es curvado, y la carga se transmite por el fieltro a través de una red aleatoria e isótropa de nudos creada por el proceso de punzonamiento, resultando en la formación de una red activa de fibra. La rotación y el estirado de las fibras activas es seguido por el deslizamiento y extracción de la fibra de los puntos de anclaje mecánico. La mayor parte de la resistencia y la energía disipada es proporcionada por la extracción de las fibras activas de los nudos, y la fractura final tiene lugar como consecuencia del desenredo total de la red en una sección dada donde la deformación macroscópica se localiza. No obstante, aunque la distribución inicial de la orientación de las fibras es isótropa, las propiedades mecánicas resultantes (en términos de rigidez, resistencia y energía absorbida) son muy anisótropas. Los ensayos de extracción de múltiples fibras en diferentes orientaciones muestran que la estructura de los nudos conecta más fibras en la dirección transversal en comparación con la dirección de la máquina. La mejor interconectividad de las fibras a lo largo de la dirección transversal da lugar a una esqueleto activo de fibras más denso, mejorando las propiedades mecánicas. En términos de afinidad, los fieltros deformados a lo largo de la dirección transversal exhiben deformación afín (la deformación macroscópica transfiere directamente a las fibras por el material circundante), mientras que el fieltro deformado a lo largo de la dirección de la máquina presenta deformación no afín, y la mayor parte de la deformación macroscópica no es transmitida a las fibras. A partir de estas observaciones experimentales, se ha desarrollado un modelo constitutivo para fieltros punzonados confinados por enlaces mecánicos. El modelo considera los efectos de la deformación no afín, la conectividad anisótropa inducida durante el punzonamiento, la curvatura y re-orientación de la fibra, así como el desenredo y extracción de la fibra de los nudos. El modelo proporciona la respuesta de un mesodominio del material correspondiente al volumen asociado a un elemento finito, y se divide en dos bloques. El primer bloque representa el comportamiento de la red y establece la relación entre el gradiente de deformación macroscópico y la respuesta microscópica, obtenido a partir de la integración de la respuesta de las fibras en el mesodominio. El segundo bloque describe el comportamiento de la fibra, teniendo en cuenta las características de la deformación de cada familia de fibras en el mesodominio, incluyendo deformación no afín, estiramiento, deslizamiento y extracción. En la medida de lo posible, se ha asignado un significado físico claro a los parámetros del modelo, por lo que se pueden identificar por medio de ensayos independientes. Las simulaciones numéricas basadas en el modelo se adecúan a los resultados experimentales de ensayos cuasi-estáticos y balísticos desde el punto de vista de la respuesta mecánica macroscópica y de los micromecanismos de deformación. Además, suministran información adicional sobre la influencia de las características microstructurales (orientación de la fibra, conectividad de la fibra anisótropa, afinidad, etc) en el comportamiento mecánico de los fieltros punzonados. Nonwoven fabrics are a class of textile material made up of a disordered fiber network linked by either thermal, chemical or mechanical bonds. They present lower stiffness and strength (as well as processing cost) than the woven counterparts but much higher deformability and energy absorption capability and are used in many different engineering applications (including thermal insulation, geotextiles, fireproof layers, filtration and water absorption, ballistic impact, etc). In particular, needle-punched nonwoven fabrics manufactured with high strength fibers present an excellent performance for ballistic protection, providing the same ballistic protection with one third of the areal weight as compared to dry woven fabrics. Nevertheless, very little is known about their deformation and fracture micromechanisms at the microscopic level and how they contribute to the macroscopic mechanical properties. This lack of knowledge hinders the optimization of their mechanical performance and also limits the development of physically-based models of the mechanical behavior that can be used in the design of structural components with these materials. In this thesis, a thorough study was carried out to ascertain the micromechanisms of deformation and the mechanical properties of a needle-punched nonwoven fabric made up by ultra high molecular weight polyethylene fibers. The deformation and energy dissipation processes were characterized in detail by a combination of experimental techniques (macroscopic mechanical tests at quasi-static and high strain rates, ballistic impact, single fiber and multi fiber pull-out tests, optical microscopy, X-ray computed tomography and wide angle X-ray diffraction) that provided information of the dominant mechanisms at different length scales. The macroscopic mechanical tests showed that the nonwoven fabric presented an outstanding strength and energy absorption capacity. It was found that fibers were initially curved and the load was transferred within the fabric through the random and isotropic network of knots created by needlepunching, leading to the formation of an active fiber network. Uncurling and stretching of the active fibers was followed by fiber sliding and pull-out from the entanglement points. Most of the strength and energy dissipation was provided by the extraction of the active fibers from the knots and final fracture occurred by the total disentanglement of the fiber network in a given section at which the macroscopic deformation was localized. However, although the initial fiber orientation distribution was isotropic, the mechanical properties (in terms of stiffness, strength and energy absorption) were highly anisotropic. Pull-out tests of multiple fibers at different orientations showed that structure of the knots connected more fibers in the transverse direction as compared with the machine direction. The better fiber interconnection along the transverse direction led to a denser active fiber skeleton, enhancing the mechanical response. In terms of affinity, fabrics deformed along the transverse direction essentially displayed affine deformation {i.e. the macroscopic strain was directly transferred to the fibers by the surrounding fabric, while fabrics deformed along the machine direction underwent non-affine deformation, and most of the macroscopic strain was not transferred to the fibers. Based on these experimental observations, a constitutive model for the mechanical behavior of the mechanically-entangled nonwoven fiber network was developed. The model accounted for the effects of non-affine deformation, anisotropic connectivity induced by the entanglement points, fiber uncurling and re-orientation as well as fiber disentanglement and pull-out from the knots. The model provided the constitutive response for a mesodomain of the fabric corresponding to the volume associated to a finite element and is divided in two blocks. The first one was the network model which established the relationship between the macroscopic deformation gradient and the microscopic response obtained by integrating the response of the fibers in the mesodomain. The second one was the fiber model, which took into account the deformation features of each set of fibers in the mesodomain, including non-affinity, uncurling, pull-out and disentanglement. As far as possible, a clear physical meaning is given to the model parameters, so they can be identified by means of independent tests. The numerical simulations based on the model were in very good agreement with the experimental results of in-plane and ballistic mechanical response of the fabrics in terms of the macroscopic mechanical response and of the micromechanisms of deformation. In addition, it provided additional information about the influence of the microstructural features (fiber orientation, anisotropic fiber connectivity, affinity) on the mechanical performance of mechanically-entangled nonwoven fabrics.

Depth Averaged Models for Fast Landslide Propagation: Mathematical, Rheological and Numerical Aspects

This paper presents an overview of depth averaged modelling of fast catastrophic landslides where coupling of solid skeleton and pore fluid (air and water) is important. The first goal is to show how Biot-Zienkiewicz models can be applied to develop depth integrated, coupled models. The second objective of the paper is to consider a link which can be established between rheological and constitutive models. Perzyna´s viscoplasticity can be considered a general framework within which rheological models such as Bingham and cohesive frictional fluids can be derived. Among the several alternative numerical models, we will focus here on SPH which has not been widely applied by engineers to model landslide propagation. We propose an improvement, based on combining Finite Difference meshes associated to SPH nodes to describe pore pressure evolution inside the landslide mass. We devote a Section to analyze the performance of the models, considering three sets of tests and examples which allows to assess the model performance and limitations: (i) Problems having an analytical solution, (ii) Small scale laboratory tests, and (iii) Real cases for which we have had access to reliable information

Deciduous tree reconstruction algorithm based on cylinder fitting from mobile terrestrial laser scanned point clouds

Vector reconstruction of objects from an unstructured point cloud obtained with a LiDAR-based system (light detection and ranging) is one of the most promising methods to build three dimensional models of orchards. The cylinder fitting method for woody structure reconstruction of leafless trees from point clouds obtained with a mobile terrestrial laser scanner (MTLS) has been analysed. The advantage of this method is that it performs reconstruction in a single step. The most time consuming part of the algorithm is generation of the cylinder direction, which must be recalculated at the inclusion of each point in the cylinder. The tree skeleton is obtained at the same time as the cluster of cylinders is formed. The method does not guarantee a unique convergence and the reconstruction parameter values must be carefully chosen. A balanced processing of clusters has also been defined which has proven to be very efficient in terms of processing time by following the hierarchy of branches, predecessors and successors. The algorithm was applied to simulated MTLS of virtual orchard models and to MTLS data of real orchards. The constraints applied in the method have been reviewed to ensure better convergence and simpler use of parameters. The results obtained show a correct reconstruction of the woody structure of the trees and the algorithm runs in linear logarithmic time

Prototipos experimentales modernos: la colonia de Schorlemerallee y las villas Am Rupenhorn de los Luckhardt y Anker. Berlín 1924-1930

La colonia experimental de Schorlemerallee y las villas Am Rupenhorn son dos proyectos concluidos en 1930 por los hermanos Wassili y Hans Luckhardt con Alfons Anker en Berlín. Ambos proyectos forman parte del mismo proceso, que comienza en la Colonia -una exploración sobre el lenguaje moderno en una serie de fases sucesivas- y culmina con las Villas. Éstas últimas, realizadas inmediatamente después de la Colonia, son la síntesis de esa experiencia, aunque finalmente acabaron trascendiéndola, ya que se convirtieron en un modelo sobre la casa en la naturaleza, sobre la idea de la villa clásica y sobre los nuevos modos de habitar, alcanzando con el tiempo la condición de canon moderno. A pesar de ello, no es esta condición lo más importante. Lo singular en este caso, es el propio proceso de proyecto –Colonia versus Villas- un verdadero experimento en su concepción, método y resultados, a través del cual sus autores investigan nuevas tecnologías aplicadas a nuevas formas de habitar y desarrollan un nuevo lenguaje, cuyo resultado son unos prototipos tecnológicos, con los que pretenden, como diría Mies van der Rohe: “Me he esforzado por construir una arquitectura para una sociedad tecnológica. He intentado que todo resultara razonable y claro.....para que cualquiera pueda hacer arquitectura.” El momento y lugar no pueden ser más propicios: Berlín entre 1924 y 1930, en el mismo origen del Movimiento Moderno. El experimento se plantea con auténtico rigor científico. Los arquitectos diseñan, construyen y financian su proyecto, controlando todas sus variables. Especialmente, por lo insólito, es el control de la variable económica. Porque este factor, la economía, es para ellos una clave fundamental del proceso. Se trataba de demostrar que la Nueva Arquitectura (o Neues Bauen, como les gustaba denominarla) era capaz de construir mejor y más rápido la vivienda para una nueva sociedad. La revolución y la vanguardia van de la mano: son el Zeitgeist o espíritu de la época, un contexto que es parte sustancial del proceso, y como lo calificarían los Smithson, un contexto heroico. El concepto se centra en la tríada Bauhaus: diseño + tecnología x economía. En cuanto al método, se fijan una serie de parámetros –las variables del experimento- que se agrupan en tres categorías distintas: topología, tipología y tecnología. La combinación de las variables de cada categoría dará lugar a un sistema con unas características determinadas: una definición del espacio, una forma, un lenguaje y una tecnología, características que permiten establecer las reglas para su desarrollo. Los sistemas resultantes son tres, denominados según su doble condición tipológica/ tecnológica: 1. Sistema de muro de carga: Viviendas adosadas en zig-zag o Mauerwerksbauten. 2. Sistema de esqueleto de acero: Viviendas aisladas o Stahlskelettbauten 3. Sistema de hormigón armado: Viviendas en hilera recta o Betonbauten Las villas Am Rupenhorn se plantean a continuación como verificación de este proceso: la síntesis de las categorías desarrolladas en la Colonia. Pero llegan en un momento de gracia, justo cuando los Luckhardt y Anker se encuentran profundamente implicados en el proceso de desarrollo de un nuevo lenguaje y con la reciente experiencia de la Colonia, que ha sido un éxito en casi todos los aspectos posibles. “En 1930, están en la cumbre”, como diría su mejor crítico y antiguo colaborador: Achim Wendschuh. En las Villas, los arquitectos integran su lenguaje, ya plenamente moderno, con sus experiencias previas: las que los relacionan con su reciente expresionismo (que se podría calificar como Kunstwollen) y con la tradición clásica de la cultura arquitectónica alemana: el sentido del material que deben a Semper y la sensibilidad hacia el paisaje, que toman de Schinkel. El extraordinario interés de las Villas se debe a factores como el tratamiento de la relación dual, poco habitual en la arquitectura moderna, la síntesis de lenguajes y las circunstancias de su momento histórico, factores que las han convertido en una propuesta única e irrepetible de una de las vías experimentales más interesantes y desconocidas de la Modernidad. ABSTRACT The experimental Housing Estate of Schorlemerallee and the Am Rupenhorn Villas are two projects completed by the brothers Wassili and Hans Luckhardt with Alfons Anker in Berlin in 1930. Both projects are part of the same process, starting with the Housing Estate --an exploration of the modern language in a series of phases- which culminates with the Villas project. The Villas Am Ruperhorn, designed immediately after the Housing development, are the synthesis and crowning point of this experience, even finally over passing it, since they have become a model of the house in nature, related with both the ideal of the classical villa and the new ways of life, reaching the condition of a modern canon. However, this is not its most important issue. The most remarkable condition is the project process itself -Housing versus Villas- a true experiment in concept, method and results, in which the authors research new technologies for new ways of living, developing an innovative language, with results in new prototypes, in the way Mies van der Rohe was looking for: “I have tried to make an architecture for a technological society. I have wanted to keep everything reasonable and clear… to have an architecture that anybody can do." The time and place could not be more favourable: Berlin from 1924 to 1930, in the very origin of Modern Movement. The experiment takes place with genuine scientific accuracy. Architects design, build and finance their own project, controlling all variables. Especially, and quite unusual, the control of the economic variable. Precisely the economic factor is for them a fundamental key to the process. It was shown to prove that the new architecture (or Neues Bauen, as they liked to call it) was able to build not only faster, better and more efficient dwellings for a new society, but also at lower cost. Revolution and Avant-garde use to move forward together, because they share the Zeitgeist --or time's spirit--, a context which is a substantial part of the process, and as the Alison & Peter Smithsons would describe, an heroic context. The concept focuses on the Bauhaus triad: Design + Technology x Economy. For the method, a number of variables are fixed --the experimental parameters-- that are later grouped into three distinct categories: Topology, Typology and Technology. The combination of these variables within each category gives way to several systems, with specific characteristics: a definition of space, a form, a language and a technology, thus allowing to establish the rules for its development: The resulting systems are three, called by double typological / technological issue: 1. Terraced Housing in zig-zag or Mauerwerksbauten (bearing wall system) 2. Detached Housing or Stahlskelettbauten (steel skeleton system) 3. Terraced Housing in one row or Betonbauten (reinforced concrete system) The Am Rupenhorn Villas are planned as the check of this process: the synthesis of the categories developed all through the Housing Estate research. The Am Ruperhorn project is developed in a crucial moment, just as the Luckhardts and Anker are deeply involved in the definition process of a new language after the recent experience of Schorlemerallee, which has been a success in almost all possible aspects. "In 1930, they are on the top” has said his best critic and long-time collaborator, Achim Wendschuh. In the Villas, the authors make up their fully modern language with their own background, related with their recent Expressionist trend (Kunstwollen) and with the classical tradition of the German architectural culture: the notion of material related with Semper and the sensible approach to the landscape, linked with Schinkel. Its extraordinary interest lay on diverse factors, such as dual relationships, unusual in modern architecture, synthesis of languages and circumstances of their historical moment, all factors that have become a unique and unrepeatable proposal in one of the most extraordinary experimental ways of Modernity.

Multi-textured 3D humanoid reconstruction through passive and active automatic techniques = Reconstrucción de humanoides 3D multi-texturados mediante técnicas pasivas y activas automáticas

The proliferation of video games and other applications of computer graphics in everyday life demands a much easier way to create animatable virtual human characters. Traditionally, this has been the job of highly skilled artists and animators that painstakingly model, rig and animate their avatars, and usually have to tune them for each application and transmission/rendering platform. The emergence of virtual/mixed reality environments also calls for practical and costeffective ways to produce custom models of actual people. The purpose of the present dissertation is bringing 3D human scanning closer to the average user. For this, two different techniques are presented, one passive and one active. The first one is a fully automatic system for generating statically multi-textured avatars of real people captured with several standard cameras. Our system uses a state-of-the-art shape from silhouette technique to retrieve the shape of subject. However, to deal with the lack of detail that is common in the facial region for these kind of techniques, which do not handle concavities correctly, our system proposes an approach to improve the quality of this region. This face enhancement technique uses a generic facial model which is transformed according to the specific facial features of the subject. Moreover, this system features a novel technique for generating view-independent texture atlases computed from the original images. This static multi-texturing system yields a seamless texture atlas calculated by combining the color information from several photos. We suppress the color seams due to image misalignments and irregular lighting conditions that multi-texturing approaches typically suffer from, while minimizing the blurring effect introduced by color blending techniques. The second technique features a system to retrieve a fully animatable 3D model of a human using a commercial depth sensor. Differently to other approaches in the current state of the art, our system does not require the user to be completely still through the scanning process, and neither the depth sensor is moved around the subject to cover all its surface. Instead, the depth sensor remains static and the skeleton tracking information is used to compensate the user’s movements during the scanning stage. RESUMEN La popularización de videojuegos y otras aplicaciones de los gráficos por ordenador en el día a día requiere una manera más sencilla de crear modelos virtuales humanos animables. Tradicionalmente, estos modelos han sido creados por artistas profesionales que cuidadosamente los modelan y animan, y que tienen que adaptar específicamente para cada aplicación y plataforma de transmisión o visualización. La aparición de los entornos de realidad virtual/mixta aumenta incluso más la demanda de técnicas prácticas y baratas para producir modelos 3D representando personas reales. El objetivo de esta tesis es acercar el escaneo de humanos en 3D al usuario medio. Para ello, se presentan dos técnicas diferentes, una pasiva y una activa. La primera es un sistema automático para generar avatares multi-texturizados de personas reales mediante una serie de cámaras comunes. Nuestro sistema usa técnicas del estado del arte basadas en shape from silhouette para extraer la forma del sujeto a escanear. Sin embargo, este tipo de técnicas no gestiona las concavidades correctamente, por lo que nuestro sistema propone una manera de incrementar la calidad en una región del modelo que se ve especialmente afectada: la cara. Esta técnica de mejora facial usa un modelo 3D genérico de una cara y lo modifica según los rasgos faciales específicos del sujeto. Además, el sistema incluye una novedosa técnica para generar un atlas de textura a partir de las imágenes capturadas. Este sistema de multi-texturización consigue un atlas de textura sin transiciones abruptas de color gracias a su manera de mezclar la información de color de varias imágenes sobre cada triángulo. Todas las costuras y discontinuidades de color debidas a las condiciones de iluminación irregulares son eliminadas, minimizando el efecto de desenfoque de la interpolación que normalmente introducen este tipo de métodos. La segunda técnica presenta un sistema para conseguir un modelo humano 3D completamente animable utilizando un sensor de profundidad. A diferencia de otros métodos del estado de arte, nuestro sistema no requiere que el usuario esté completamente quieto durante el proceso de escaneado, ni mover el sensor alrededor del sujeto para cubrir toda su superficie. Por el contrario, el sensor se mantiene estático y el esqueleto virtual de la persona, que se va siguiendo durante el proceso, se utiliza para compensar sus movimientos durante el escaneado.

Health monitoring of a new hysteretic damper subjected to earthquakes on a shaking table

This paper presents the experimental results obtained by applying frequency-domain structural health monitoring techniques to assess the damage suffered on a special type of damper called Web Plastifying Damper (WPD). The WPD is a hysteretic type energy dissipator recently developed for the passive control of structures subjected to earthquakes. It consists of several I-section steel segments connected in parallel. The energy is dissipated through plastic deformations of the web of the I-sections, which constitute the dissipative parts of the damper. WPDs were subjected to successive histories of dynamically-imposed cyclic deformations of increasing magnitude with the shaking table of the University of Granada. To assess the damage to the web of the I-section steel segments after each history of loading, a new damage index called Area Index of Damage (AID) was obtained from simple vibration tests. The vibration signals were acquired by means of piezoelectric sensors attached on the I-sections, and non-parametric statistical methods were applied to calculate AID in terms of changes in frequency response functions. The damage index AID was correlated with another energy-based damage index-ID- which past research has proven to accurately characterize the level of mechanical damage. The ID is rooted in the decomposition of the load-displacement curve experienced by the damper into the so-called skeleton and Bauschinger parts. ID predicts the level of damage and the proximity to failure of the damper accurately, but it requires costly instrumentation. The experiments reported in this paper demonstrate a good correlation between AID and ID in a realistic seismic loading scenario consisting of dynamically applied arbitrary cyclic loads. Based on this correlation, it is possible to estimate ID indirectly from the AID, which calls for much simpler and less expensive instrumentation.

Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.

Structural protein 4.1 is located in mammalian centrosomes

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

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.

A widely expressed βIII spectrin associated with Golgi and cytoplasmic vesicles

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the β-spectrin gene family by homology searches of the GenBank databases and by 5′ rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called βIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. βIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human βIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human βIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that βIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and β-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with βIII spectrin. βIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice

The homozygous disruption of the mouse AP-2 gene yields a complex and lethal phenotype that results from defective development of the neural tube, head, and body wall. The severe and pleiotropic developmental abnormalities observed in the knockout mouse suggested that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, we have now analyzed chimeric mice composed of both wild-type and AP-2-null cells. The phenotypes obtained from these chimeras indicate that there is an independent requirement for AP-2 in the formation of the neural tube, body wall, and craniofacial skeleton. In addition, these studies reveal that AP-2 exerts a major influence on eye formation, which is a critical new role for AP-2 that was masked previously in the knockout mice. Furthermore, we also have uncovered an unexpected influence of AP-2 on limb pattern formation; this influence is typified by major limb duplications. The range of phenotypes observed in the chimeras displays a significant overlap with those caused by teratogenic levels of retinoic acid, strongly suggesting that AP-2 is an important component of the mechanism of action of this morphogen.

Developmental Expression of Spectrins in Rat Skeletal Muscle

Skeletal muscle contains spectrin (or spectrin I) and fodrin (or spectrin II), members of the spectrin supergene family. We used isoform-specific antibodies and cDNA probes to investigate the molecular forms, developmental expression, and subcellular localization of the spectrins in skeletal muscle of the rat. We report that β-spectrin (βI) replaces β-fodrin (βII) at the sarcolemma as skeletal muscle fibers develop. As a result, adult muscle fibers contain only α-fodrin (αII) and the muscle isoform of β-spectrin (βIΣ2). By contrast, other types of cells present in skeletal muscle tissue, including blood vessels and nerves, contain only α- and β-fodrin. During late embryogenesis and early postnatal development, skeletal muscle fibers contain a previously unknown form of spectrin complex, consisting of α-fodrin, β-fodrin, and the muscle isoform of β-spectrin. These complexes associate with the sarcolemma to form linear membrane skeletal structures that otherwise resemble the structures found in the adult. Our results suggest that the spectrin-based membrane skeleton of muscle fibers can exist in three distinct states during development.

Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice

Androgens may regulate the male skeleton directly through a stimulation of androgen receptors or indirectly through aromatization of androgens into estrogen and, thereafter, through stimulation of estrogen receptors (ERs). The relative importance of ER subtypes in the regulation of the male skeleton was studied in ERα-knockout (ERKO), ERβ-knockout (BERKO), and double ERα/β-knockout (DERKO) mice. ERKO and DERKO, but not BERKO, demonstrated decreased longitudinal as well as radial skeletal growth associated with decreased serum levels of insulin-like growth factor I. Therefore, ERα, but not ERβ, mediates important effects of estrogen in the skeleton of male mice during growth and maturation.

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.