Distribution of phytolithes in CRP sediment cores from the Ross Sea, Antarctica

Phytoliths (siliceous plant microfossils) have been recovered from Cenozoic sediments (c. 34 to 17 Ma) in the CRP-2/2A and CRP-3 drillholes cored off Cape Roberts, Victoria Land Basin, Antarctica. The phytolith assemblages are sparse, but well-preserved and dominated by spherical forms similar to those of modern trees or shrubs. Rare phytoliths comparable to modern grass forms are also present. However, due to the paucity of phytolith data, any interpretations made are necessarily tentative. The assemblages of CRP-2/2A and the upper c. 250 m of CRP-3 are interpreted as representing a predominantly woody vegetation, including Nothofagus and Libocedrus with local areas of grass in the more exposed locations. A cool climate is interpreted to have prevailed throughout both cores. However, beneath c. 250 metres below sea floor in CRP-3, the dominant woody vegetation is supplemented by pockets of Palmae, ?Proteaceae and 'warm' climate grasses. This association represents vegetation growth in sheltered, moist sites - possibly north-facing mid-slopes or the coastal fringe. It may also represent remnant vegetation that grew in moist, temperate conditions during the Middle to Late Eocene, previously interpreted from the Southern McMurdo Sound erratics and lower part of the CIROS-1 drillhole. The phytolith analysis compares well to the terrestrial palynomorph record from both cores and provides additional independent taxonomic and climatic interpretations.

Geochemistry at DSDP Site 76-533

Bulk X-ray mineralogy of 47 hemipelagic mud and clay samples from the Blake Outer Ridge has revealed that the sediments contain low magnesian calcite, calcian dolomite, ferroan dolomite, and magnesian siderite. Dolomite and siderite are authigenic and occur as rhombohedrons scattered through the sediments, whereas calcite is mostly biogenic. Pliocene dolomitic lenses are made up of interlocking polyhedral grains of ferroan dolomite. The contents of authigenic dolomite and siderite are 3 to 8% in carbonate sediments and 70 to 89% in dolomitic lenses. Dolomite occurs largely in the cores above 192 m sub-bottom depth, whereas siderite occurs in the cores below 87 m. The distribution and occurrence of dolomite and siderite have determined the diagenetic zonation of carbonates as Zone I (dolomitic zone, top-90 m), Zone II (transition zone, 90-180 m), and Zone III (sideritic zone, 180 m-bottom). Measurements of major and minor elements in the untreated total sediment samples and the insoluble residues after digestion in acid-reducing solution have revealed that the soluble fraction concentrates carbonates and ferromanganese associations (Ca, Mg, Sr, Fe, and Mn). Typical "hydrogenous elements" (Co, Cu, Ni, and V) are more concentrated in the insoluble residues rather than in the soluble fraction; the concentrations of these four elements are low and comparable to modern offshore mud, probably because the Site 533 sediments were deposited at a high rate of sedimentation. The contents of Fe2O3 and MnO are somewhat high for rapidly accumulated mud, particularly in the Pliocene sediments (8.09 and 0.26%, respectively, on a Carbonate-free basis). The high Fe and Mn contents are mainly due to the high contribution of the leacheable nonlithogenous fraction; leacheable Fe and Mn originate in the ferromanganese oxide accumulated on the seafloor. Only a small amount of ferric oxide was converted to iron sulfide in the surficial part of Zone I. Most ferromanganese oxide was reduced and precipitated as ferroan dolomite and magnesian siderite in Zones II and III under high alkalinity and high pH conditions in the organic-matter-rich sediments. Fe2+ and Mn2+ in the deeper sediments beneath Zone III possibly migrated upward and concentrated as siderite in Zone III, hence resulting in high contents of Fe and Mn in the Pliocene sediments. Analysis of carbonate zonation on the Blake Outer Ridge has revealed that the zonation is subparallel to the bedding plane rather than to the present seafloor. The sediments at Site 103 on the flank region of the Ridge are lacking Zone I and most of Zone II, probably the result of erosion of the most of the Pleistocene and Pliocene sediments by the enhanced bottom currents during the Pleistocene.

Programmed design of ship forms

This paper describes a new category of CAD applications devoted to the definition and parameterization of hull forms, called programmed design. Programmed design relies on two prerequisites. The first one is a product model with a variety of types large enough to face the modeling of any type of ship. The second one is a design language dedicated to create the product model. The main purpose of the language is to publish the modeling algorithms of the application in the designer knowledge domain to let the designer create parametric model scripts. The programmed design is an evolution of the parametric design but it is not just parametric design. It is a tool to create parametric design tools. It provides a methodology to extract the design knowledge by abstracting a design experience in order to store and reuse it. Programmed design is related with the organizational and architectural aspects of the CAD applications but not with the development of modeling algorithms. It is built on top and relies on existing algorithms provided by a comprehensive product model. Programmed design can be useful to develop new applications, to support the evolution of existing applications or even to integrate different types of application in a single one. A three-level software architecture is proposed to make the implementation of the programmed design easier. These levels are the conceptual level based on the design language, the mathematical level based on the geometric formulation of the product model and the visual level based on the polyhedral representation of the model as required by the graphic card. Finally, some scenarios of the use of programmed design are discussed. For instance, the development of specialized parametric hull form generators for a ship type or a family of ships or the creation of palettes of hull form components to be used as parametric design patterns. Also two new processes of reverse engineering which can considerably improve the application have been detected: the creation of the mathematical level from the visual level and the creation of the conceptual level from the mathematical level. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction

Generación de modelos físicos personalizados y segmentación de la pared arterial y el trombo intraluminal en imágenes de aneurismas aórticos abdominales

Esta tesis doctoral está encuadrada dentro del marco general de la ingeniería biomédica aplicada al tratamiento de las enfermedades cardiovasculares, enfermedades que provocan alrededor de 1.9 millones (40%) de muertes al año en la Unión Europea. En este contexto surge el proyecto europeo SCATh-Smart Catheterization, cuyo objetivo principal es mejorar los procedimientos de cateterismo aórtico introduciendo nuevas tecnologías de planificación y navegación quirúrgica y minimizando el uso de fluoroscopía. En particular, esta tesis aborda el modelado y diagnóstico de aneurismas aórticos abdominales (AAA) y del trombo intraluminal (TIL), allí donde esté presente, así como la segmentación de estas estructuras en imágenes preoperatorias de RM. Los modelos físicos específicos del paciente, construidos a partir de imágenes médicas preoperatorias, tienen múltiples usos, que van desde la evaluación preoperatoria de estructuras anatómicas a la planificación quirúrgica para el guiado de catéteres. En el diagnóstico y tratamiento de AAA, los modelos físicos son útiles a la hora de evaluar diversas variables biomecánicas y fisiológicas de las estructuras vasculares. Existen múltiples técnicas que requieren de la generación de modelos físicos que representen la anatomía vascular. Una de las principales aplicaciones de los modelos físicos es el análisis de elementos finitos (FE). Las simulaciones de FE para AAA pueden ser específicas para el paciente y permiten modelar estados de estrés complejos, incluyendo los efectos provocados por el TIL. La aplicación de métodos numéricos de análisis tiene como requisito previo la generación de una malla computacional que representa la geometría de interés mediante un conjunto de elementos poliédricos, siendo los hexaédricos los que presentan mejores resultados. En las estructuras vasculares, generar mallas hexaédricas es un proceso especialmente exigente debido a la compleja anatomía 3D ramificada. La mayoría de los AAA se encuentran situados en la bifurcación de la arteria aorta en las arterias iliacas y es necesario modelar de manera fiel dicha bifurcación. En el caso de que la sangre se estanque en el aneurisma provocando un TIL, éste forma una estructura adyacente a la pared aórtica. De este modo, el contorno externo del TIL es el mismo que el contorno interno de la pared, por lo que las mallas resultantes deben reflejar esta particularidad, lo que se denomina como "mallas conformadas". El fin último de este trabajo es modelar las estructuras vasculares de modo que proporcionen nuevas herramientas para un mejor diagnóstico clínico, facilitando medidas de riesgo de rotura de la arteria, presión sistólica o diastólica, etc. Por tanto, el primer objetivo de esta tesis es diseñar un método novedoso y robusto para generar mallas hexaédricas tanto de la pared aórtica como del trombo. Para la identificación de estas estructuras se utilizan imágenes de resonancia magnética (RM). Deben mantenerse sus propiedades de adyacencia utilizando elementos de alta calidad, prestando especial atención al modelado de la bifurcación y a que sean adecuadas para el análisis de FE. El método tiene en cuenta la evolución de la línea central del vaso en el espacio tridimensional y genera la malla directamente a partir de las imágenes segmentadas, sin necesidad de reconstruir superficies triangulares. Con el fin de reducir la intervención del usuario en el proceso de generación de las mallas, es también objetivo de esta tesis desarrollar un método de segmentación semiautomática de las distintas estructuras de interés. Las principales contribuciones de esta tesis doctoral son: 1. El diseño, implementación y evaluación de un algoritmo de generación de mallas hexaédricas conformadas de la pared y el TIL a partir de los contornos segmentados en imágenes de RM. Se ha llevado a cabo una evaluación de calidad que determine su aplicabilidad a métodos de FE. Los resultados demuestran que el algoritmo desarrollado genera mallas conformadas de alta calidad incluso en la región de la bifurcación, que son adecuadas para su uso en métodos de análisis de FE. 2. El diseño, implementación y evaluación de un método de segmentación automático de las estructuras de interés. La luz arterial se segmenta de manera semiautomática utilizando un software disponible a partir de imágenes de RM con contraste. Los resultados de este proceso sirven de inicialización para la segmentación automática de las caras interna y externa de la pared aórtica utilizando métodos basado en modelos de textura y forma a partir de imágenes de RM sin contraste. Los resultados demuestran que el algoritmo desarrollado proporciona segmentaciones fieles de las distintas estructuras de interés. En conclusión, el trabajo realizado en esta tesis doctoral corrobora las hipótesis de investigación postuladas, y pretende servir como aportación para futuros avances en la generación de modelos físicos de geometrías biológicas. ABSTRACT The frame of this PhD Thesis is the biomedical engineering applied to the treatment of cardiovascular diseases, which cause around 1.9 million deaths per year in the European Union and suppose about 40% of deaths per year. In this context appears the European project SCATh-Smart Catheterization. The main objective of this project is creating a platform which improves the navigation of catheters in aortic catheterization minimizing the use of fluoroscopy. In the framework of this project, the specific field of this PhD Thesis is the diagnosis and modeling of abdominal aortic aneurysm (AAAs) and the intraluminal thrombus (ILT) whenever it is present. Patient-specific physical models built from preoperative imaging are becoming increasingly important in the area of minimally invasive surgery. These models can be employed for different purposes, such as the preoperatory evaluation of anatomic structures or the surgical planning for catheter guidance. In the specific case of AAA diagnosis and treatment, physical models are especially useful for evaluating pressures over vascular structures. There are multiple techniques that require the generation of physical models which represent the target anatomy. Finite element (FE) analysis is one the principal applications for physical models. FE simulations for AAA may be patient-specific and allow modeling biomechanical and physiological variables including those produced by ILT, and also the segmentation of those anatomical structures in preoperative MR images. Applying numeric methods requires the generation of a proper computational mesh. These meshes represent the patient anatomy using a set of polyhedral elements, with hexahedral elements providing better results. In the specific case of vascular structures, generating hexahedral meshes is a challenging task due to the complex 3D branching anatomy. Each patient’s aneurysm is unique, characterized by its location and shape, and must be accurately represented for subsequent analyses to be meaningful. Most AAAs are located in the region where the aorta bifurcates into the iliac arteries and it is necessary to model this bifurcation precisely and reliably. If blood stagnates in the aneurysm and forms an ILT, it exists as a conforming structure with the aortic wall, i.e. the ILT’s outer contour is the same as the wall’s inner contour. Therefore, resulting meshes must also be conforming. The main objective of this PhD Thesis is designing a novel and robust method for generating conforming hexahedral meshes for the aortic wall and the thrombus. These meshes are built using largely high-quality elements, especially at the bifurcation, that are suitable for FE analysis of tissue stresses. The method accounts for the evolution of the vessel’s centerline which may develop outside a single plane, and generates the mesh directly from segmented images without the requirement to reconstruct triangular surfaces. In order to reduce the user intervention in the mesh generation process is also a goal of this PhD. Thesis to develop a semiautomatic segmentation method for the structures of interest. The segmentation is performed from magnetic resonance image (MRI) sequences that have tuned to provide high contrast for the arterial tissue against the surrounding soft tissue, so that we determine the required information reliably. The main contributions of this PhD Thesis are: 1. The design, implementation and evaluation of an algorithm for generating hexahedral conforming meshes of the arterial wall and the ILT from the segmented contours. A quality inspection has been applied to the meshes in order to determine their suitability for FE methods. Results show that the developed algorithm generates high quality conforming hexahedral meshes even at the bifurcation region. Thus, these meshes are suitable for FE analysis. 2. The design, implementation and evaluation of a semiautomatic segmentation method for the structures of interest. The lumen is segmented in a semiautomatic way from contrast filled MRI using an available software. The results obtained from this process are used to initialize the automatic segmentation of the internal and external faces of the aortic wall. These segmentations are performed by methods based on texture and shape models from MRI with no contrast. The results show that the algorithm provides faithful segmentations of the structures of interest requiring minimal user intervention. In conclusion, the work undertaken in this PhD. Thesis verifies the investigation hypotheses. It intends to serve as basis for future physical model generation of proper biological anatomies used by numerical methods.

A CAD development strategy for the next years

This paper suggests a new strategy to develop CAD applications taking into account some of the most interesting proposals which have recently appeared in the technology development arena. Programming languages, operating systems, user devices, software architecture, user interfaces and user experience are among the elements which are considered for a new development framework. This strategy considers the organizational and architectural aspects of the CAD application together with the development framework. The architectural and organizational aspects are based on the programmed design concept, which can be implemented by means of a three-level software architecture. These levels are the conceptual level based on a declarative language, the mathematical level based on the geometric formulation of the product model and the visual level based on the polyhedral representation of the model as required by the graphic card. The development framework which has been considered is Windows 8. This operating system offers three development environments, one for web pplications (HTML5 + CSS3 + JavaScript), and other for native applications C/C++) and of course yet another for .NET applications (C#, VB, F#, etc.). The use rinterface and user experience for non-web application is described ith XAML (a well known declarative XML language) and the 3D API for games and design applications is DirectX. Additionally, Windows 8 facilitates the use of hybrid solutions, in which native and managed code can interoperate easily. Some of the most remarkable advantages of this strategy are the possibility of targeting both desktop and touch screen devices with the same development framework, the usage of several programming paradigms to apply the most appropriate language to each domain and the multilevel segmentation of developers and designers to facilitate the implementation of an open network of collaborators.

Desarrollo de un proceso de razonamiento visual para la valoración del patrimonio

El tema de la presente tesis es la valoración del patrimonio y en ella se considera que el patrimonio es un proceso cultural interesado en negociar, crear y recrear recuerdos, valores y significados culturales. Actualmente el patrimonio como proceso se está consolidando en la literatura científica, aunque la idea de que es una ‘cosa’ es dominante en el debate internacional y está respaldada tanto por políticas como prácticas de la UNESCO. El considerar el patrimonio como un proceso permite una mirada crítica, que subraya la significación. Es decir, supone el correlato que conlleva definir algo como ‘patrimonio’, o hacer que lo vaya siendo. Esta visión del concepto permite la posibilidad de comprender no sólo lo que se ha valorado, sino también lo que se ha olvidado y el porqué. El principal objetivo de esta investigación es explorar las características de un proceso de razonamiento visual para aplicarlo en el de valoración del patrimonio. Éste que se presenta, implica la creación de representaciones visuales y sus relaciones, además su meta no está centrada en producir un ambiente que sea indiferenciado de la realidad física. Con él se pretende ofrecer la posibilidad de comunicar la dimensión ‘poliédrica’ del patrimonio. Para que este nuevo proceso que propongo sea viable y sostenible, existe la necesidad de tener en cuenta el fin que se quiere lograr: la valoración. Es importante considerar que es un proceso en el cual las dinámicas de aprendizaje, comportamientos y exploración del patrimonio están directamente relacionadas con su valoración. Por lo tanto, hay que saber cómo se genera la valoración del patrimonio, con el fin de ser capaces de desarrollar el proceso adaptado a estas dinámicas. La hipótesis de esta tesis defiende que un proceso de razonamiento visual para la valoración del patrimonio permite que las personas involucradas en el proceso inicien un proceso de interacción con un elemento patrimonial y su imagen mental para llegar a ciertas conclusiones con respecto a su valor y significado. El trabajo describe la metodología que da lugar al proceso de razonamiento visual para el patrimonio, que ha sido concebido sobre un modelado descriptivo de procesos, donde se han caracterizado tres niveles: meta-nivel, de análisis y operacional. En el modelado del proceso los agentes, junto con el patrimonio, son los protagonistas. El enfoque propuesto no es sólo sobre el patrimonio, sino sobre la compleja relación entre las personas y el patrimonio. Los agentes humanos dan valor a los testimonios de la vida pasada y les imbuyen de significado. Por lo tanto, este enfoque de un proceso de razonamiento visual sirve para detectar los cambios en el valor del patrimonio, además de su dimensión poliédrica en términos espaciales y temporales. Además se ha propuesto una nueva tipología de patrimonio necesaria para sustentar un proceso de razonamiento visual para su valoración. Esta tipología está apoyada en la usabilidad del patrimonio y dentro de ella se encuentran los siguientes tipos de patrimonio: accesible, cautivo, contextualizado, descontextualizado, original y vicarial. El desarrollo de un proceso de razonamiento visual para el patrimonio es una propuesta innovadora porque integra el proceso para su valoración, contemplando la dimensión poliédrica del patrimonio y explotando la potencialidad del razonamiento visual. Además, los posibles usuarios del proceso propuesto van a tener interacción de manera directa con el patrimonio e indirecta con la información relativa a él, como por ejemplo, con los metadatos. Por tanto, el proceso propuesto posibilita que los posibles usuarios se impliquen activamente en la propia valoración del patrimonio. ABSTRACT The subject of this thesis is heritage valuation and it argues that heritage is a cultural process that is inherited, transmitted, and transformed by individuals who are interested in negotiating, creating and recreating memories and cultural meanings. Recently heritage as a process has seen a consolidation in the research, although the idea that heritage is a ‘thing’ is dominant in the international debate and is supported by policies and practice of UNESCO. Seeing heritage as a process enables a critical view, underscoring the significance. That is, it is the correlate involved in defining something as ‘heritage’, or converting it into heritage. This view of the concept allows the possibility to understand not only what has been valued, but also what has been forgotten and why. The main objective of this research is to explore the characteristics of a visual reasoning process in order to apply it to a heritage valuation. The goal of the process is not centered on producing an environment that is undifferentiated from physical reality. Thus, the objective of the process is to provide the ability to communicate the ‘polyhedral’ dimension of heritage. For this new process to be viable and sustainable, it is necessary to consider what is to be achieved: heritage valuation. It is important to note that it is a process in which the dynamics of learning, behavior and exploration heritage are directly related to its valuation. Therefore, we need to know how this valuation takes place in order to be able to develop a process that is adapted to these dynamic. The hypothesis of this thesis argues that a visual reasoning process for heritage valuation allows people involved in the process to initiate an interaction with a heritage and to build its mental image to reach certain conclusions regarding its value and meaning. The thesis describes the methodology that results in a visual reasoning process for heritage valuation, which has been based on a descriptive modeling process and have characterized three levels: meta, analysis and operational -level. The agents are the protagonists in the process, along with heritage. The proposed approach is not only about heritage but the complex relationship between people and heritage. Human operators give value to the testimonies of past life and imbue them with meaning. Therefore, this approach of a visual reasoning process is used to detect changes in the value of heritage and its multifaceted dimension in spatial and temporal terms. A new type of heritage required to support a visual reasoning process for heritage valuation has also been proposed. This type is supported by its usability and it covers the following types of heritage: available, captive, contextualized, decontextualized, original and vicarious. The development of a visual reasoning process for heritage valuation is innovative because it integrates the process for valuation of heritage, considering the multifaceted dimension of heritage and exploiting the potential of visual reasoning. In addition, potential users of the proposed process will have direct interaction with heritage and indirectly with the information about it, such as the metadata. Therefore, the proposed process enables potential users to be actively involved in their own heritage valuation.

Haus des Rundfunks en Berlin, de Hans Poelzig, 1930

Poelzig affirmed that the technical ideal consisted of using the least amount of shapes and materials possible. He understood architecture as an art, as the expression of form; In contrast to the indifference of the most widely recognized values of modernism: technical innovation in construction along with the functionality of buildings. The Haus des Rundfunks is a powerful hermetic factory in the shape of a triangle and of uniform height. Mass, geometric rigidity and the absence of textures make up a building which relinquishes expressing the new media, with a perimeter ring consisting of offices and larger spaces that are more characteristic towards their inside. Geometry is used as a means to organize and limit the shapes of the buildings. The arrangement and division of different intercommunicating spaces are subjected to the crudeness of their geometric limits. Interior spaces ranging from the most static to the most dynamic are defined through the qualities of materials and geometries. This turns the spaces into geometric grids which decompose the wall, floor and ceiling planes into fragments, regardless of the constructive reality provided by a metallic frame structure. Its polyhedral lamps enclose artificial and emphasize the value of the grid. Poelzig limits himself to dealing with planes within a closed volume of uniform height, and maintains geometric rigidity compatible with a certain degree of figurativeness. This article tries to bring to life this exemplary building, marked by its passivity and hermetism.

Arquitecturas en árboles: atenciones contemporáneas, 1990-2010

La tesis abre su mirada hacia uno de los discursos más interesantes, a mi juicio, de la cultura arquitectónica contemporánea: el de la relación establecida entre el ser humano y la naturaleza. Algunos autores creen que esta relación se establece como simbiosis, como un organismo arquitectónico en correlación y analogía con su entorno y emplazamiento. Otros ven una correspodencia en la que la arquitectura se ubica como un artefacto que ”dialoga” y que no imita a la naturaleza. Las arquitecturas en árboles se desarrollan en estos dos sentidos. El panorama de estas arquitecturas está abierto, es incipiente, laberíntico, poliédrico y caleidoscópico. Metodológicamente ha sido necesario acotarlo y el período de estudio elegido ha sido el más fructífero de la historia y cultura de las estructuras en los árboles. En los últimos veinticinco años, éstas han vuelto a generar atracción por el auge de la sensibilización medioambiental. También se ha producido un incremento de las culturas del ocio (con acento en el deseo por encima de la utilidad o la necesidad) y de la introspección (en busca de un retiro espiritual alejado de la cultura de la globalización). Hacia 1990 se producen acontecimientos relevantes que son los precursores de este desarrollo: la aparición en 1988 del libro “Treehouses” de Aikman abre de manera desenfadada esta cultura arquitectónica. Para acotar el final del período de investigación se ha dejado un margen desde 2010 a 2015 por prudencia investigadora. Los objetivos de la tesis se han centrado: Primero, en encuadrar el concepto de “arquitecturas en árboles”; Segundo, en descubrir los motivos, ideas que inspiraron las arquitecturas en árboles anteriores al período de estudio y su posible repercusión en sus descendientes contemporáneas. Principalmente son atenciones ideológicas, culturales, sociales, filosóficas, simbólicas, míticos y funcionales. Tercero, en confeccionar una taxonomía de las intenciones, consideraciones y desencadenantes básicos que pudieran establecerse como claves del proceso imaginario, conceptivo y constructivo de la actuales arquitecturas en árboles (1990-2010). Para ello nos hemos puesto bajo el paraguas metodológico del concepto de atención, defendido por Quaroni, Ferrater, Broadbent y Seguí. Hemos planteado una reflexión sobre el tipo de atenciones y su idoneaidad llegando a distinguir las siguientes atenciones básicas: 1) Las atenciones históricas (del pasado): englobando bajo esta denominación el entramado rizomático de atenciones sociales, culturales, antropológicas, filosóficas, míticas y simbólicas. Estas atenciones permiten al lector posicionarse de manera efectiva en este amplio campo, para entrar con posterioridad en el cuerpo de la investigación. Posteriormente descubriremos que éstas se convierten en atenciones crítico-modales para las arquitecturas contemporáneas. 2) Las atenciones funcionales contemporáneas: en las que estableceremos una conexión multiple con otras atenciones ergonómicas, organizativas, tipológicas y medioambientales básicas que están claramente influenciadas por aquellas. Quedan establecidas en una taxonomía inédita de treinta ocho subcategorías que nos alejan del estereotipo de la casa en el árbol como lugar de defensa y protección, o como espacio lúdico-infantil 3) Las atenciones constructivas contemporáneas: que las relacionaremos con las biológicas, estructurales-portantes, tipológico-constructivas, medioambientales básicas (de los materiales), y que son tenidas en cuenta por los contructores. Asimismo, se revelan los sistemas de construcción de arquitecturas portantes y autoportantes (artificiales-naturales) y las 100% naturales. Además, se decubren los seis mecanismos estructurales de sujeción de habitáculos en árboles más utilizados, así como cuáles son los árboles idóneos para soportar estas estructuras. 4) Las atenciones formales contemporáneas: con sus conexiones hacia las atenciones presenciales, espaciales organizativas y medioambientales básicas. Se establecen taxonomías sistemáticas formales simples y complejas (pragmática, icónica, analógica y canónica). Se estudian con profundidad la analogía con otras realidades y la estructuración geométrica formal (canónica) por considerarlas las más relevantes. Se culmina la investigación con una reflexión sobre el conjunto rizomático de atenciones, disciplinas, nociones y conceptos inherentes al campo de las arquitecturas en árboles, ejemplificándolo con un ideograma. La última vista se fija sobre el arco temporal de estas arquitecturas reflejando las cincuenta más significativas por su mayor coherencia de competencia atencional, total o parcial. ABSTRACT This dissertation looks into one the most interesting discourses in contemporary architectural culture: the relationship between human beings and nature. Some scholars think of this relationship as a symbiosis, as an architectural organism armonically correlated with its environment and location. Others believe that a correspondence exists where architecture stands as an artifact ‘in dialogue’ with, and not as an imitation of, nature. Treehouses develop in both senses. The realm of these architectures is open, embryonic, labyrinthine, polyhedral and kaleidoscopic. It was necessary to methodologically delimit it. We chose for the study the most fruitful period in the history and culture of treehouses. During the last twenty-five years, the latter have exerted great appeal once more due to the rise in environmental awareness. Likewise, leisure cultures (highlighting desire over utility or necessity) and introspection (seeking spiritual retreat far from the culture of globalization) have significantly increased. Around 1990, some relevant events announce this development: the publication of the book Treehouses by Aikman in 1988 inaugurates in an informal fashion such architectural culture. As for the final limit of the period addressed, an open margin between 2010 and 2015 has been established, out of scholarly prudence. The dissertation objectives have focused on: First, framing the concept of “treehouses”. Second, discovering the motifs and ideas that inspired treehouses in previous periods, and their possible influence on their contemporary successors. These consist basically of idelogical, cultural, social, philosophical, symbolic, mythical and functional attentions. Third, elaborate a taxonomy of the different basic intentions, considerations and triggers that might appear as crucial for the imaginary, conceptual and constructive process of current treehouses (1990-2010). To this end, we stood under the methodological umbrella of the notion of attention as defined by Quaroni, Ferrater, Broadbent and Seguí. We have proposed a reflection on the different kind of attentions and their suitability, distinguishing the following basic attentions: 1) Historical attentions (from the past): this denomination comprises the rhizomatic fabric of social, cultural, anthropological, philosophical, mythical and symbolic attentions. These attentions allow the reader to get a foothold on this broad field before being able to go into the main body of research. We will later discover that these become critical modal attentions for contemporary architectures. 2) Contemporary functional attentions. A multiple connection will be established with other basic ergonomic, organisational, typological and environmental attentions clearly influenced by them. They are classified in an unprecedented taxonomy of thirty-eight subcategories that move us away from the stereotype of the tree house as a site of defense and protection, or as a children’s playground. 3) Contemporary constructive attentions. They will be linked with basic biological, structural-carrying, typological-constructive, environmental attentions (of materials) taken into account by constructors. Likewise, the construction systems for carrying and self-carrying (artificial-natural) architectures, as well as for 100% natural ones, are revealed. Besides, we disclose the six structural mechanisms employed to fasten cabins to trees, and which the most suitable trees to support such structures are. 4) Contemporary formal attentions, including their connections with spatial, organizational and basic environmental attentions. Simple and complex formal systematic taxonomies (pragmatic, iconic, analogical and canonical) have been established. Their analogy to other realities and to formal geometrical (canonical) structuring is studied in depth as utterly significant. The research concludes by reflecting on the rhizomatic set of attentions, disciplines, notions and concepts inherent in the field of treehouses through an ideogram. The final view engages with the time span of these architectures by highlighting the fifty most noteworthy on account of the higher consistency of their attentional competence, either total or partial.

Rem : a los dos lados del espejo = (Rem at both sides of the mirror)

Cuando se trata de Rem Koolhaas, su espejo no refleja una sola imagen sino múltiples, es un prisma poliédrico. Su espejo nos devuelve el Rem mediático, el intelectual, el conceptualizador, el constructor, el analista, el periodista, el actor... En el caso de esta investigación, fijamos el punto de mira en el Rem COMUNICADOR. “Rem a los dos lados del espejo” se enmarca en una investigación sobre los medios de comunicación de arquitectura, su reflejo en la producción arquitectónica y viceversa. Se trata de llegar a discernir si comunicación y producción arquitectónica colisionan y confluyen en el caso de grandes comunicadores como Rem Koolhaas, si el mensaje y el medio transmisor adquieren las mismas cualidades. Centrándose en la figura de Rem Koolhaas, la tesis aborda la evolución de su faceta comunicativa y las transformaciones sucesivas en el campo de la comunicación arquitectónica, en paralelo a su evolución conceptual a lo largo de su trayectoria. La investigación, por tanto, no se centra tanto en su componente teórica o en la práctica arquitectónica de OMA, sino en la exposición de su producción al mundo, especialmente a través de sus ensayos y libros. “Delirious New York” y “SMLXL” son un reflejo del momento conceptual en que se inscriben, y contienen mucha información sobre los referentes gráficos que irremediablemente han influido en su composición. Especialmente, la aparición de “SMLXL” supuso un revulsivo para el mundo de la comunicación arquitectónica, porque puso el foco sobre la importancia de dejar atrás un discurso narrativo linea y unifocal, para afrontar la comunicación barajando múltiples variables, y aproximaciones, en un proceso similar al desarrollo de un proyecto de arquitectura. Presenta un diseño muy novedoso y una edición extremadamente cuidada, que atiende a parámetros mucho más ambiciosos que los meramente narrativos. Profundiza en la necesidad de una temática global, planteando cuál es la aproximación más apropiada para cada uno de los proyectos que describe, transmitiendo al lector una percepción más allá de lo estrictamente visual, más próximo a lo sensorial. Además, su enorme repercusión a nivel internacional y el gran interés que despertó (no solamente entre los arquitectos, sino también entre diseñadores gráficos, publicistas, personas provenientes de todo tipo de tendencias artísticas y público en general), provocó la globalización del fenómeno de las publicaciones arquitectónicas y puso de manifiesto la importancia de la comunicación como una disciplina en sí misma, dentro de la producción arquitectónica en la era actual. A pesar de la importancia de “SMLXL” a todos los niveles, la presente tesis plantea que, donde realmente se culmina esa experiencia comunicativa, es en “Content”, al incluir nuevos parámetros relacionados con la fusión conceptual de continente y contenido. Es en esta publicación donde el objeto de la comunicación y la expresión de la misma se convierten en un único elemento, que se rige por leyes similares. En este caso, la ley fundamental es la aplicación hasta sus máximas consecuencias de la “cultura de la congestión”, tanto en el mensaje como en el medio, generando lo que hemos convenido en denominar “comunicación congestiva”. Esta concepción deviene en que necesariamente se materialice como un producto efímero, desechable, casi virtual, porque responde a las condiciones de un momento muy concreto y específico y fuera de ese contexto pierde su significación.. La “cultura de la congestión” empieza a surgir en los planteamientos de Koolhaas en la Architectural Association School of Architecture de Londres, bajo la tutela de Elia Zenghelis. Posteriormente se desarrolla en su manifiesto retroactivo sobre Manhattan, “Delirious New York”, donde declara la guerra abierta al urbanismo del movimiento moderno y afirma que la ciudad realmente contemporánea es aquella que es fruto de un desarrollo no planificado, hiperdensa y posible gracias a los avances tecnológicos de su era. Finalmente comienza a materializarse en la Diploma Unit 9 de la AA, donde entra como profesor en 1975, dejando una huella indeleble en las generaciones posteriores de arquitectos que pasaron dicha unidad. Rem Koolhaas es ante todo un intelectual y por ello, todo el constructo teórico sobre la metrópolis comienza a reflejarse en su obra a través de OMA desde el comienzo de su producción. Podemos decir a grandes rasgos que su carrera está marcada por dos hitos históricos fundamentales que determinan tres etapas diferenciadas en su producción. En sus primeros años de profesión, Koolhaas sigue fascinado por la metrópolis urbana y la aplicación del método paranoico crítico a su producción arquitectónica. Es un arquitecto profundamente surrealista. Entiende este método como una estrategia de conocimiento y aproximación al mundo que le rodea: “dejar salir el inconsciente pero sostenerlo con las muletas de la racionalidad”. Pero lo que en realidad le interesa es su aplicación a la gran escala, el “Bigness”, y por ello, participa en proyectos muy ambiciosos de los que surgen conceptos que, más allá de resultar premiados o no, han dejado una huella ideológica en el devenir de la arquitectura. Entre estos proyectos, cabe destacar su propuesta para el Parque de la Villette o la Très Grande Bibliotèque de París. Sus proyectos de esta época destilan una gran carga conceptual, que devienen en unos interiores sorprendentes pero una apariencia exterior sobria o incluso podríamos decir "povera", por el uso de materiales efímeros, poco habituales en la macro-arquitectura hasta ese momento. Súbitamente, en 1997, explotó el denominado “Efecto Bilbao”, de la mano de Frank Gehry (1). El Museo Guggenheim de Bilbao, con su espectacularidad, sus formas pregnantes e imposibles, impacta al mundo. Nace la era de la “Arquitectura del Espectáculo”; la transformación de la ciudad a través de ICONOS que actúen como nodos de atracción y concentración en torno a los cuales supuestamente se revitaliza la actividad económica, cultural y sociopolítica de la ciudad, como si a través de un único gesto se pudieran regenerar todos los tejidos internos de la urbe. Rem Koolhaas comprende rápidamente que la aproximación a la ciudad ha cambiado y, sobre todo, el mercado. En el mundo de la globalización, la única manera de llegar a materializar el “Bigness”, es encerrando sus ejercicios intelectuales en formas pregnantes, bellas, icónicas, espectaculares. Koolhaas encuentra su marca personal en la estética “Stealth”, proveniente de los aviones de combate facetados para evitar los radares, elaborados en los años 80. De esta época surgen proyectos como la Casa da Música de Oporto o la Biblioteca de Seattle; ambos edificios son iconos facetados, de belleza pregnante, que dejan una huella indeleble en la ciudad y provocan, al igual que el Guggenheim, un cierto efecto de recuperación y revitalización en el entorno en que se asientan, al menos de manera temporal. En cualquier caso, Koolhaas nunca abandona los ejercicios meramente teóricos, pero segrega su actividad en dos: OMA produce aquello que tiene vocación de ser construido y se rige por los parámetros del mercado global y AMO, la otra cara del espejo de Rem, aplica el pensamiento arquitectónico a campos no explorados, sin la dependencia de agentes externos, pudiendo permitirse ser un laboratorio puramente experimental. En este escenario, llega el 11 de septiembre de 2001 y el ataque a las Torres Gemelas de Nueva York tiene efectos devastadores a todos los niveles, significando, en un período de tiempo sorprendentemente corto, un cambio en el orden mundial. Rem Koolhaas da entonces un giro de 180 grados, dirige su mirada hacia China, donde entiende que sus aportaciones tienen un beneficio social más directo que en occidente. (2) Para presentar al mundo su nuevo cambio de rumbo y la creación del “Think Tank” AMO, plantea una gran exposición en la NeueGallerie de Berlín bajo el título de “Content”, experiencia paralela a la edición del libro con el mismo título, que inicialmente nace como “catálogo de la exposición, pero que internamente siempre se concibió como el documento más trascendente en el estudio desde “SMLXL”. Sin embargo, en muchos aspectos se trata de su opuesto: una publicación con formato revista, de tapa blanda, con paginado muy fino, formato de "folleto de supermercado" y contenido hiperdenso. Es un experimento efímero, fugaz, ligero, barato, de “usar y tirar”. De hecho, está fuera de stock, ya no se edita. Probablemente Rem Koolhaas desaprobaría que se hiciera una investigación que pusiera el foco sobre el mismo, porque diez años después de su publicación seguramente opine que su vigencia ha caducado. Sin embargo, muestra con una claridad meridiana el estado conceptual y vital de OMA en el momento de su publicación y representa, además un verdadero hito en la comunicación arquitectónica, un punto de no retorno, el máximo exponente de lo que hemos denominado “comunicación congestiva”. La presente tesis plantea que “Content” contiene la esencia de la mayor aportación de Rem Koolhaas al mundo de la arquitectura: la transformación profunda y definitiva de la comunicación arquitectónica mediante la convergencia del estado conceptual y la transmisión del mismo. Su legado arquitectónico y conceptual ha marcado a todas las generaciones posteriores de manera indeleble. Sus ensayos, sus teorías, sus proyectos y sus edificaciones ya pertenecen a la historia de la arquitectura, sin ninguna duda. Pero es su revisión del concepto de la comunicación en arquitectura lo que ha tenido y tendrá un reflejo inmediato en las generaciones futuras, no solamente en la comunicación sino en su arquitectura, a través de un intercambio biyectivo. El planteamiento a futuro sería determinar qué sucede tras “Content”, tras la hiperdensidad máxima, tras la cultura de la congestión visual; qué es lo que propone Koolhaas y qué se va a plantear también en el mundo de la comunicación arquitectónica. Para ello, estudiaremos en profundidad sus últimos proyectos relacionados con la comunicación, como su propuesta para la Biennale de Arquitectura de Venecia de 2014, su intensa investigación sobre el “Metabolismo” en “Project Japan: Metabolism Talks...”, o la dirección de sus últimos planteamientos territoriales. En los últimos tiempos Rem Koolhaas habla de “Preservación”, de “Sobriedad”, de “Esencialismo”, de “Performance”... El autor intelectual de la cultura de la congestión habla ahora de la “low density”...como no podía ser de otra manera en la otra cara del espejo. En definitiva, el color blanco como suma de todos los colores, todas las longitudes de onda del espectro visible recibidas al tiempo. ABSTRACT When talking about Rem Koolhaas, the mirror does not only reflect one but numerous images: it is nothing but a polyhedral prism. His mirror gives us the image of Rem the media celebrity, the intellectual, the conceptualizer, the builder, the analyst, the journalist, the actor... This research sets the spotlight on Rem the COMMUNICATOR. "Rem on both sides of the mirror" belongs to a research on architectural media, its influence on the architectural production and vice versa. It is aimed at getting to discern whether communication and architectural production collide and converge in the case of great communicators such as Rem Koolhaas, and whether the message and transmission media acquire the same features. Focusing on the figure of Rem Koolhaas, this thesis addresses the evolution of his communicative facet and the successive transformations in the field of architectural communication, parallel to the conceptual evolution he underwent throughout his career. Therefore, this research is not so much focused on his theoretical component or on the OMA’s architectural practice, but on the exhibition of his production to the world, especially through his essays and books. "Delirious New York" and "SMLXL" hold up a mirror to the conceptual moment they are part of, and contain a great deal of information about the graphic references that have inevitably influenced his work. Specially, the launch of "SMLXL" was a salutary shock for the architectural communication world, since it set the spotlight on the importance of leaving a linear and unifocal narrative behind in order to face communication considering multiple variables and approaches, based on a process similar to the development of an architectural project. It offers a very innovative design and an extremely careful editing, which deals with parameters much more ambitious than those merely narrative. It explores the need for a global subject and suggests the most appropriate approach for each of the projects described, giving the reader a closer insight to the sensory that goes beyond what’s strictly visual. In addition, its huge international impact and the great interest shown, not only by architects but also by graphic designers, publishers, people from all kinds of artistic trends and the general public, led to the globalisation of the architectural publications phenomenon and brought the importance of communication as a discipline in itself, within the architectural production in the age at hand, to light. Despite the importance of "SMLXL" at all levels, this thesis suggests that the communication experience really culminates in "Content", for it includes new conceptual parameters associated with the container-content conceptual fusion. It is in this book where the purpose of communication and the expression of such become a single element, ruled by similar laws. In this particular case, the fundamental law is to implement the "culture of congestion" to its extreme consequences in both the message and the media, leading to what we have agreed to refer to as "congestive communication”. This concept leads to its inevitable materialisation into an ephemeral, disposable, almost virtual product, because it meets the conditions of a very concrete and specific time, and outside that context it loses its significance. The "culture of congestion" emerged in Koolhaas’ approaches under the guidance of Elia Zenghelis, in the Architectural Association School of Architecture of London. Subsequently, his retroactive manifesto on Manhattan, "Delirious New York" developed it, waging an all-out war against the modern movement urbanism and maintaining that the really contemporary cities are those hyperdense ones that rise as a result of an unplanned development and thanks to the typical technological advances of their time. Finally it began to materialise in the Diploma Unit 9 of the AA, in which he started lecturing in 1975, leaving an indelible mark on subsequent generations of architects who passed that unit. First and foremost, Rem Koolhaas is an intellectual and, therefore, all the theoretical construct in the metropolis began to be reflected in his work through OMA since the beginnings of his production. Broadly speaking, we can say that his career is influenced by two essential historic events, which determine three different stages in his production. In the early years of his career, Koolhaas was still fascinated by the urban metropolis and the implementation of the paranoiac-critical method to his architectural production. He was then a deeply surreal architect. He understood this method as a knowledge strategy and an approach to the world around him: "let the subconscious out but hold it with the crutches of reasonableness”. However, he was actually interested in its implementation on a broad scale, the "Bigness", and therefore, he took part in ambitious projects that led to the accrual of concepts that, beyond being rewarded, left an ideological impression on the evolution of architecture. These projects included his proposal for the Parc de la Villette or the Très Grande Bibliotèque in Paris. The projects he carried out during this period showed a great conceptual background, which evolved into surprising interiors but a sober, or even "povera", exterior appearance, thanks to the use of ephemeral materials that were atypical in the macro-architecture field until that moment. Suddenly, in 1997, the so-called "Bilbao effect" boomed thanks to Frank Gehry (1). The Guggenheim Museum of Bilbao amazed the world with its spectacular nature and its pregnant and impossible shapes. It was the beginning of the era of “The architecture of spectacle”: the transformation of the city through ICONS that would act as nodes of attraction and gathering, around which the economic, cultural and socio-political activity of the city was supposed to be revitalized, as if through a single gesture all internal tissues of the city could be rebuilt. Rem Koolhaas quickly realized that the approach to the city, and especially to the global market, had changed. In the world of globalisation, the only way to get to materialise such "Bigness" was by keeping his intellectual exercises in pregnant, beautiful, iconic and spectacular shapes. Koolhaas found his personal brand in the Stealth aesthetic, resulting from the eighties American combat aircrafts whose shape was faceted in order to avoid radars. Projects such as the Casa da Música in Porto or the Seattle Library date from this period; both buildings are faceted icons of pregnant beauty that left an indelible mark on the city and caused, like the Guggenheim, some degree of recovery and revitalization on the environment in which they were based, at least temporarily. In any case, Koolhaas never gave the merely theoretical exercises up, but he segregated his work in two: OMA produced what was destined to be built and ruled by the parameters of the global market and AMO, Rem’s other side of the mirror, applied the architectural thought in unexplored fields, notwithstanding external agents and being able to work as a purely experimental laboratory. In light of this backdrop, September 11th 2001 came and the attacks on the Twin Towers in New York had devastating effects at all levels, leading to a change in the world order, in a surprisingly short period of time. Rem Koolhaas made a 180° turn directing his vision towards China, where he believed his contributions would have a more direct social benefit than in the Western world. (2) In order to introduce his new course of direction and the creation of the AMO "Think Tank", he planned a major exhibition in the Neue Nationalgalerie of Berlin under the title "Content", in parallel with edition of the book with the same title, which was at first the "exhibition catalog” but, deep down, was always conceived as the most important document of the Office since "SMLXL". However, in many ways it was just the opposite: a publication characterised by its magazine format, soft cover, very fine paging, "supermarket brochure" form and hyperdense content. It was an ephemeral, brief, light, cheap and "disposable" experiment. In fact, it is currently out of stock and out of print. Rem Koolhaas would probably disapprove of a research that sets the spotlight on him, for he would probably say that his validity has expired given that it has been ten years since its publication. However, it shows OMA’s conceptual and vital status at the time of its publication with crystalline clarity and it is also a true milestone in architectural communication. A point of no return. The epitome of the so-called "congestive communication ". This thesis suggests that "Content" contains the essence of Rem Koolhaas’ greatest contribution to the world of architecture: the deep and definitive transformation of architectural communication through the convergence of the conceptual state and the transmission thereof. His architectural and conceptual legacy has left an indelible mark on all subsequent generations. There is no doubt his essays, theories, projects and buildings already belong to the history of architecture. But it is his review on the concept of communication in architecture that has had and shall have an immediate influence on future generations, not only in their communication but also in their architecture, through a bijective exchange. Future approaches should try to determine what happens after "Content", after the maximum hyperdensity, after the visual culture of congestion; what shall Koolhaas suggest as well as what shall happen in the world of architectural communication. To this end, we shall study his latest communication-related projects, such as the design of the Venetian Architecture Biennale in 2014, his intensive research on the "Metabolism" in "Project Japan: Metabolism Talks ...", or the course of his latest territorial approaches in depth. Most recently, Rem Koolhaas has talked about "Preservation", "Sobriety" of "Essentialism", "Performance", etc. The mastermind of the culture of congestion now speaks of the "low density"... as it could not be otherwise, on the other side of the mirror. Summarizing, the white color as the sum of all colors; all wavelengths of the visible spectrum received at the same time.

GroEL-GroES-mediated protein folding requires an intact central cavity

The chaperonin GroEL is an oligomeric double ring structure that, together with the cochaperonin GroES, assists protein folding. Biochemical analyses indicate that folding occurs in a cis ternary complex in which substrate is sequestered within the GroEL central cavity underneath GroES. Recently, however, studies of GroEL “minichaperones” containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. Here we compare directly the folding activity of minichaperones with that of the full GroEL-GroES system. In agreement with earlier studies, minichaperones assist folding of some proteins. However, this effect is observed only under conditions where substantial spontaneous folding is also observed and is indistinguishable from that resulting from addition of the nonchaperone protein α-casein. By contrast, the full GroE system efficiently promotes folding of several substrates under conditions where essentially no spontaneous folding is observed. These data argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.

Human argininosuccinate lyase: A structural basis for intragenic complementation

Intragenic complementation has been observed at the argininosuccinate lyase (ASL) locus. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits enzymatic activity that is greater than that found in the oligomeric proteins produced by each mutant allele alone. The mutations involved in the most successful complementation event observed in ASL deficiency were found to be an aspartate to glycine mutation at codon 87 of one allele (D87G) coupled with a glutamine to arginine mutation at codon 286 of the other (Q286R). To understand the structural basis of the Q286R:D87G intragenic complementation event at the ASL locus, we have determined the x-ray crystal structure of recombinant human ASL at 4.0 Å resolution. The structure has been refined to an R factor of 18.8%. Two monomers related by a noncrystallographic 2-fold axis comprise the asymmetric unit, and a crystallographic 2-fold axis of space group P3121 completes the tetramer. Each of the four active sites is composed of residues from three monomers. Structural mapping of the Q286R and D87G mutations indicate that both are near the active site and each is contributed by a different monomer. Thus when mutant monomers combine randomly such that one active site contains both mutations, it is required by molecular symmetry that another active site exists with no mutations. These “native” active sites give rise to the observed partial recovery of enzymatic activity.

cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme

Methyl chloride transferase catalyzes the synthesis of methyl chloride from S-adenosine-l-methionine and chloride ion. This enzyme has been purified 2,700-fold to homogeneity from Batis maritima, a halophytic plant that grows abundantly in salt marshes. The purification of the enzyme was accomplished by a combination of ammonium sulfate fractionation, column chromatography on Sephadex G100 and adenosine-agarose, and TSK-250 size-exclusion HPLC. The purified enzyme exhibits a single band on SDS/PAGE with a molecular mass of approximately 22.5 kDa. The molecular mass of the purified enzyme was 22,474 Da as determined by matrix-associated laser desorption ionization mass spectrometry. The methylase can function in either a monomeric or oligomeric form. A 32-aa sequence of an internal fragment of the methylase was determined (GLVPGCGGGYDVVAMANPER FMVGLDIXENAL, where X represents unknown residue) by Edman degradation, and a full-length cDNA of the enzyme was obtained by rapid amplification of cDNA ends–PCR amplification of cDNA oligonucleotides. The cDNA gene contains an ORF of 690 bp encoding an enzyme of 230 aa residues having a predicted molecular mass of 25,761 Da. The disparity between the observed and calculated molecular mass suggests that the methylase undergoes posttranslational cleavage, possibly during purification. Sequence homologies suggest that the B. maritima methylase defines a new family of plant methyl transferases. A possible function for this novel methylase in halophytic plants is discussed.

Homogeneous immunoconjugates for boron neutron-capture therapy: Design, synthesis, and preliminary characterization

The application of immunoprotein-based targeting strategies to the boron neutron-capture therapy of cancer poses an exceptional challenge, because viable boron neutron-capture therapy by this method will require the efficient delivery of 103 boron-10 atoms by each antigen-binding protein. Our recent investigations in this area have been focused on the development of efficient methods for the assembly of homogeneous immunoprotein conjugates containing the requisite boron load. In this regard, engineered immunoproteins fitted with unique, exposed cysteine residues provide attractive vehicles for site-specific modification. Additionally, homogeneous oligomeric boron-rich phosphodiesters (oligophosphates) have been identified as promising conjugation reagents. The coupling of two such boron-rich oligophosphates to sulfhydryls introduced to the CH2 domain of a chimeric IgG3 has been demonstrated. The resulting boron-rich immunoconjugates are formed efficiently, are readily purified, and have promising in vitro and in vivo characteristics. Encouragingly, these studies showed subtle differences in the properties of the conjugates derived from the two oligophosphate molecules studied, providing a basis for the application of rational design to future work. Such subtle details would not have been as readily discernible in heterogeneous conjugates, thus validating the rigorous experimental design employed here.

DAD1, the defender against apoptotic cell death, is a subunit of the mammalian oligosaccharyltransferase

DAD1, the defender against apoptotic cell death, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line. Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST). Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported. Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme. Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48. Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits. DAD1 can be crosslinked to OST48 in intact microsomes with dithiobis(succinimidylpropionate). Crosslinked ribophorin II–OST48 heterodimers, DAD1–ribophorin II–OST48 heterotrimers and DAD1–ribophorin I–ribophorin II–OST48 heterotetramers also were detected. The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.

Localization and Recycling of gp27 (hp24γ3): Complex Formation with Other p24 Family Members

We report here the characterization of gp27 (hp24γ3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15°C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24α2), p24 (hp24β1), and p23 (hp24δ1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24γ4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.