Diseño para fabricación digital: definición unívoca entre forma y fabricación en arquitectura

La presente investigación se inicia planteando el objetivo de identificar los parámetros geométricos que son exclusivos del proceso de generación de la Forma y relacionarlos con los invariantes relacionados con la Fabricación digital aplicada a la Arquitectura. Con ello se pretende recuperar la geometría como herramienta principal del proceso de Proyecto ampliando su ámbito de actuación al encontrar una relación con los procesos de fabricación digital. El primer capítulo describe los antecedentes y contexto histórico centrándose especialmente en la influencia de la capacidad de definir geometrías complejas digitalmente mediante la aplicación de algoritmos. En los primeros ejemplos la aproximación del Arquitecto a proyectos con geometrías complejas no euclídeas aún se emplea sin precisión en la comunicación de la geometría ideada para su puesta en obra. Las técnicas constructivas obligan a asumir una tolerancia de desviación entre proyecto y obra y la previsión del comportamiento de esa geometría no permite asegurar su comportamiento final. No será hasta la introducción de herramientas CAD en el proceso de ideación arquitectónica cuando el Arquitecto se capacite para generar geometrías no representables de forma analógica. Sin embargo, la imposibilidad de trasladar la geometría proyectada a la praxis constructiva impedirá la plasmación de un proceso completo, salvo en las contadas ocasiones que se recogen en este texto. “El análisis cronológico de las referencias establece como aspecto esencial para la construcción de geometrías complejas la capacidad primero para definir y comunicar de forma precisa e inequívoca la geometría y después la capacidad de analizar el desempeño prestacional de dicha propuesta geométrica”. La presente investigación se inicia planteando el objetivo de identificar los parámetros geométricos que son exclusivos del proceso de generación de la Forma y relacionarlos con los invariantes relacionados con la Fabricación digital aplicada a la Arquitectura. Con ello se pretende recuperar la geometría como herramienta principal del proceso de Proyecto ampliando su ámbito de actuación al encontrar una relación con los procesos de fabricación digital. El primer capítulo describe los antecedentes y contexto histórico centrándose especialmente en la influencia de la capacidad de definir geometrías complejas digitalmente mediante la aplicación de algoritmos. En los primeros ejemplos la aproximación del Arquitecto a proyectos con geometrías complejas no euclídeas aún se emplea sin precisión en la comunicación de la geometría ideada para su puesta en obra. Las técnicas constructivas obligan a asumir una tolerancia de desviación entre proyecto y obra y la previsión del comportamiento de esa geometría no permite asegurar su comportamiento final. No será hasta la introducción de herramientas CAD en el proceso de ideación arquitectónica cuando el Arquitecto se capacite para generar geometrías no representables de forma analógica. Sin embargo, la imposibilidad de trasladar la geometría proyectada a la praxis constructiva impedirá la plasmación de un proceso completo, salvo en las contadas ocasiones que se recogen en este texto. “El análisis cronológico de las referencias establece como aspecto esencial para la construcción de geometrías complejas la capacidad primero para definir y comunicar de forma precisa e inequívoca la geometría y después la capacidad de analizar el desempeño prestacional de dicha propuesta geométrica”. Establecida la primera conclusión, el capítulo de contexto histórico continúa enfocándose sobre la aplicación de las técnicas digitales en el Proceso de proyecto primero, y en la puesta en obra después. Los casos de estudio identifican claramente como un punto de inflexión para la generación de formas complejas mediante un software CAD el Museo Guggenheim de Bilbao en 1992. El motivo esencial para elegir este proyecto como el primer proyecto digital es el uso de la herramienta de definición digital de la geometría para su reproducción inequívoca en obra. “La revolución digital ha aportado al Arquitecto la posibilidad de abandonar las tipologías arquitectónicas basados en restricciones geométricas-constructivas. La aplicación de técnicas de fabricación digital ha permitido la capacidad de diseñar con independencia del sistema constructivo y libertad formal. En este nuevo contexto las prestaciones suponen los nuevos límites conceptuales, ya que el acceso y disposición de la información del comportamiento de las alternativas que cada geometría conlleva demanda del Arquitecto la jerarquización de los objetivos y la formulación en un conjunto coherente de parámetros”. Los proyectos que emplean herramientas digitales para la resolución de las distintas etapas del proceso proyectual se verán incrementados de forma exponencial desde 1992 hasta nuestros días. A pesar del importante auge de las técnicas de diseño asistido por ordenador el principal desafío sigue siendo la vinculación de las geometrías y materiales propuestos con las capacidades de las técnicas de manufactura y puesta en obra. El proceso de diseño para fabricación en un entorno digital es una tecnología madura en otras industrias como la aeroespacial o la automovilística, incluso la de productos de consumo y decoración, sin embargo en el sector de Construcción es un sistema inmaduro e inconexo. Las particularidades de la industria de la construcción aún no han sido abordadas en su totalidad y las propuestas de investigación realizadas en este ámbito se han centrado hasta 2015 en partes del proceso y no en el proceso total. “El principal obstáculo para la estandarización e implantación globalizada de un proceso digital desde el origen de la forma hasta la construcción es la inexistencia de un protocolo integrado que integre las limitaciones de fabricación, económicas y de puesta en obra junto a la evaluación de desempeño prestacional durante la fases iniciales de proyecto”. En el capítulo número 3 se estudian los distintos procesos de generación de la forma. Se propone una definición específica para el ámbito de la investigación de “forma” en el entendemos que se incluye la envolvente exterior y el conjunto organizativo de espacios interiores conectados. Por lo tanto no es excluyente del interior. El objetivo de este estudio es analizar y clasificar los procesos para la generación digital de formas en los distintos proyectos seleccionados como emblemáticos de cada tipología. Se concluye que la aproximación a este proceso es muy variada y compleja, con aplicación segregada y descoordinada entre los distintos agentes que han intervenir. En un proceso de generación formal analógico los parámetros que intervienen son en parte conscientes y en parte inconscientes o aprendidos. El Arquitecto sólo tiene control sobre la parte consciente de los parámetros a integrar en el diseño, de acuerdo a sus conocimientos y capacidades será capaz de manejar un número limitado de parámetros. La parte aprendida permanece en el inconsciente y dirige el proceso analógico, aportando prejuicios estéticos incorporados durante el proceso formativo y propio del entorno cultural. “El empleo de herramientas digitales basadas en la evaluación prestacional durante el proceso de selección formal permite al Arquitecto conocer “en tiempo real” el desempeño en el conjunto de prestaciones evaluadoras del conjunto de alternativas geométricas a la propuesta previamente definida por la intuición arquitectónica. El proceso definido no persigue identificar una solución óptima sino asistir al Arquitecto en el proceso de generación de la forma mediante la evaluación continua de los vectores direccionales más idóneos que el procedimiento generativo plantea”. La definición de complejidad en generación y producción de formas en relación con el proceso de diseño digital paramétrico global o integrado, es esencial para establecer un protocolo que optimice su gestión. “Se propone como definición de complejidad como factor resultante de multiplicar el número de agentes intervinientes por el número de parámetros e interacciones comunes que intervienen en el proceso de generación de la forma, dividido por la complejidad de intercambio de información digital desde el origen hasta la fase de fabricación y construcción”. Una vez analizados los procesos de generación digital de Arquitectura se propone identificar los parámetros geométricos que definen el proceso de Diseño digital, entendiendose por Diseño el proceso que engloba desde la proposición de una forma inicial basada en la intuición del Arquitecto, la generación y evaluación de variantes y posterior definición digital para producción, tanto de un objeto, un sistema o de la totalidad del Proyecto. En la actualidad el proceso de Diseño es discontinuo y lineal organizandose los parámetros por disciplinas en las que está estructurada las atribuciones profesionales en la industria de la construcción. Para simplificar la identificación y listado se han agrupado siguiendo estos grupos de conocimiento. Entendemos parametros invariables aquellos que son independientes de Tipologías arquitectónicas o que dependen del mismo proceso de generación de la Forma. “El listado de los parámetros que intervienen en un proceso de generación formal es una abstracción de una realidad compleja. La parametrización de las decisiones que intervienen en la selección de una forma determinada mediante “well defined problems” es imposible. El proceso que esta tesis describe entiende esta condición como un elemento que pone en valor el propio procedimiento generativo por la riqueza que la subjetividad que el equipo de diseño aporta”. La segunda parte esencial de esta investigación pretende extraer las restricciones propias del estado del arte de la fabricación digital para posteriormente incorporarlos en los procesos digitales de definición de la Forma arquitectónica. “La integración de las restricciones derivadas de las técnicas de fabricación y construcción digitales en el proceso de generación de formas desde el ámbito de la Arquitectura debe referirse a los condicionantes geométricos asociados a cada sistema constructivo, material y técnica de fabricación. La geometría es además el vínculo que permite asociar el conjunto de parámetros prestacionales seleccionados para un Proyecto con los sistemas de fabricación digital”. A estos condicionantes geométricos obtenidos del análisis de cada sistema de fabricación digital se les ha denominado “invariantes geométricos”. Bajo este término se engloban tanto límites dimensionales de fabricación, como materiales compatibles, tolerancias de manufactura e instalación y cualidades prestacionales asociadas. El objetivo de esta propuesta es emplear la geometría, herramienta fundamental y propia del Arquitecto, como nexo de unión entre el conjunto complejo y heterogéneo de parámetros previamente listados y analizados. Para ello se han simplificado en tablas específicas para cada parámetro prestacional los condicionantes geométricos que se derivan de los Sistemas de fabricación digital compatibles (ver apéndice 1). El estudio y evaluación de las capacidades y objetivos de las distintas plataformas de software disponibles y de las experiencias profesionales evaluadas en los proyectos presentados, permiten concluir que la propuesta de plataforma digital de diseño integral multi-paramétrico de formas arquitectónicas requiere de un protocolo de interoperatibilidad específico aún no universalmente establecido. Actualmente el enfoque de la estrategia para normalizar y universalizar el contexto normativo para regular la interoperatibilidad se centra en figura del gestor denominado “BIM manager”. Las atribuciones y roles de esta figura se enfocan a la gestión del continente y no del contenido (Definición de los formatos de intercambio, niveles de desarrollo (LOD) de los componentes o conjuntos constructivos, detección de interferencias y documentación del propio modelo). Siendo este ámbito un desarrollo necesario para la propuesta de universalización del sistema de diseño para fabricación digital integrado, la presente investigación aporta un organigrama y protocolo asociado. El protocolo: 1. Establece la responsabilidad de identificar y definir la Información que debe determinar el proceso de generación y desarrollo de la forma arquitectónica. 2. Define la forma digital apropiada para generar la geometría del Proyecto, incluyendo la precisión necesaria para cada componente y el nivel de detalle necesario para su exportación inequívoca al proceso de fabricación. 3. Define el tempo de cada etapa de diseño identificando un nivel de detalle acorde. 4. Acopla este organigrama dentro de las estructuras nuevas que se proponen en un entorno BIM para asegurar que no se producen solapes o vacíos con las atribuciones que se identifican para el BIM Manager. “El Arquitecto debe dirigir el protocolo de generación coordinada con los sistemas de producción digital para conseguir que la integración completa. El protocolo debe asistir al proceso de generación de forma mediante la evaluación del desempeño prestacional de cada variante en tiempo real. La comunicación entre herramientas digitales es esencial para permitir una ágil transmisión de información. Es necesario establecer un protocolo adaptado a los objetivos y las necesidades operativas de cada proyecto ya que la estandarización de un protocolo único no es posible”. Una decisión estratégica a la hora de planificar una plataforma de diseño digital común es establecer si vamos a optar por un Modelo digital único o diversos Modelos digitales federados. Cada uno de los modos de trabajo tiene fortalezas y debilidades, no obstante en el ámbito de investigación se ha concluido que un proceso integrado de Diseño que incorpore la evaluación prestacional y conceptual definida en el Capítulo 3, requiere necesariamente de varios modelos de software distintos que han de relacionarse entre sí mediante un protocolo de comunicación automatizado. Una plataforma basada en un modelo federado consiste en establecer un protocolo de comunicación entre los programas informáticos empleados por cada disciplina. En este modelo de operación cada equipo de diseño debe establecer las bases de comunicación en función del número y tipo de programas y procesos digitales a emplear. En esta investigación se propone un protocolo basado en los estándares de intercambio de información que estructura cualquier proceso de generación de forma paramétrico “La investigación establece el empleo de algoritmos evolutivos como el sistema actual óptimo para desarrollar un proceso de generación de formas basadas en la integración y coordinación de invariantes geométricos derivados de un conjunto de objetivos prestacionales y constructivos. No obstante, para la aplicación en el caso práctico realizado se ha podido verificar que la evaluación del desempeño aún no puede realizarse en una única herramienta y por lo tanto el proceso de selección de las variantes genéticas óptimas ha de ejecutarse de forma manual y acumulativa. El proceso debe realizarse de manera federada para la selección evolutiva de los invariantes geométricos dimensionales”. La evaluación del protocolo de integración y los condicionantes geométricos obtenidos como parámetros geométricos que controlan las posibles formas compatibles se realiza mediante su aplicación en un caso práctico. El ejercicio simula la colaboración multidisciplinar con modelos federados de plataformas distintas. La elección del tamaño y complejidad constructiva del proyecto se ha modulado para poder alcanzar un desarrollo completo de cada uno de los parámetros prestacionales seleccionados. Continuando con el mismo objetivo propuesto para los parámetros prestacionales, la tipología constructiva-estructural seleccionada para el ejercicio permite la aplicación la totalidad de invariantes geométricos asociados. El objetivo de este caso práctico es evaluar la capacidad alterar la forma inicialmente propuesta mediante la evaluación del desempeño prestacional de conjunto de variantes geométricas generadas a partir de un parámetro dimensional determinado. Para que este proceso tenga sentido, cada una de las variantes debe ser previamente validada conforme a las limitaciones geométricas propias de cada sistema de fabricación y montaje previstos. El interés de las conclusiones obtenidas es la identificación de una variante geométrica distante a la solución simétrica inicialmente como la solución óptima para el conjunto de parámetros seleccionados. Al tiempo se ha comprobado como la participación de un conjunto de parámetros multi-disciplinares que representan la realidad compleja de los objetivos arquitectónicos favorecen la aparición de variaciones genéticas con prestaciones mejoradas a la intuición inicial. “La herencias tipológicas suponen un límite para la imaginación de variantes formales al proceso de ideación arquitectónica. El ejercicio realizado demuestra que incluso en casos donde aparentemente la solución óptima aparenta ser obvia una variante aleatoria puede mejorar su desempeño global. La posibilidad de conocer las condiciones geométricas de las técnicas de fabricación digital compatibles con el conjunto de parámetros seleccionados por el Arquitecto para dirigir el proceso asegura que los resultados del algoritmo evolutivo empleado sean constructivamente viables. La mejora de imaginación humana con la aportación de geometrías realmente construibles supone el objetivo último de esta tesis”. ABSTRACT Architectural form generation process is shifting from analogical to digital. Digital technology has changed the way we design empowering Architects and Engineers to precisely define any complex geometry envisioned. At the same time, the construction industry, following aeronautical and automotive industries, is implementing digital manufacturing techniques to improve efficiency and quality. Consequently construction complexity will no longer be related to geometry complexity and it is associated to coordination with digital manufacturing capacities. Unfortunately it is agreed that non-standard geometries, even when proposed with performance optimization criteria, are only suitable for projects with non-restricted budgets. Furthemore, the lack of coordinated exportation protocol and geometry management between design and construction is avoiding the globalization of emergence process in built projects Present research first objective is to identify exclusive form-generation parameters related to digital manufacturing geometrical restraints. The intention was to use geometry as the form-generation tool and integrate the digital manufacturing capacities at first stages of the project. The first chapter of this text describes the investigation historical context focusing on the influence between accurate geometry definition at non-standard forms and its construction. At first examples of non-Euclidean geometries built the communication between design and construction were based on analogical partial and imprecise documentation. Deficient communication leads to geometry adaptation on site leaving the final form uncontrolled by the Architect. Computer Aided Design enable Architects to define univocally complex geometries that previously where impossible to communicate. “The univocally definition of the Form, and communication between design and construction is essential for complex geometry Projects”. The second chapter is focused on digital technologies application in form finding process and site construction. The case studies selected identifies a clear inflexion node at 1992 with the Guggenheim Museum in Bilbao. The singularity of this project was the use of Aeronautics software to define digitally the external envelope complex geometry to enable the contractor to build it. “The digital revolution has given the Architect the capacity to design buildings beyond the architectural archetypes driven by geometric-constructive limitations. The application of digital manufacturing techniques has enabled a free-form construction without geometrical limitations. In this new context performance shall be the responsible to set new conceptual boundaries, since the behavior of each possible geometry can be compare and analyze beforehand. The role of the Architect is to prioritize the performance and architectural objectives of each project in a complete and coherent set of parameters”. Projects using digital tools for solving various stages of the design process were increased exponentially since 1992 until today. Despite the significant rise of the techniques of computer-aided design the main challenge remains linking geometries and materials proposed at each design with the capabilities of digital manufacturing techniques. Design for manufacturing in a digital environment is a mature technology in other industries such as aerospace and automotive, including consumer products and decoration, but in the construction sector is an immature and disjointed system. The peculiarities of the construction industry have not yet been addressed in its entirety and research proposals made in this area until 2015 have focused in separate parts of the process and not the total process. “The main obstacle to global standardization and implementation of a complete digital process from the form-finding to construction site is the lack of an integrated protocol that integrates manufacturing, economic and commissioning limitations, together with the performance evaluation of each possible form”. The different form generation processes are studied at chapter number 3. At the introduction of this chapter there is a specific definition of "form" for the research field. Form is identified with the outer envelope geometry, including the organizational set of connected indoor spaces connected to it. Therefore it is not exclusive of the interior. The aim of this study is to analyze and classify the main digital form generation processes using different selected projects as emblematic of each type. The approach to this process is complex, with segregated and uncoordinated different actors have to intervene application. In an analogical form-generation process parameters involved are partly conscious and partly unconscious or learned. The architect has control only over limited part of the parameters to be integrated into the design, according to their knowledge and. There is also a learned aesthetical prejudice that leads the form generation process to a specific geometry leaving the performance and optimization criteria apart from the decision making process. “Using performance evaluation digital tools during form finding process provides real-time comparative information to the Architect enabling geometry selection based on its performance. The generative form generation process described at this document does not ambition to identify the optimum geometry for each set of parameters. The objective is to provide quick information at each generation of what direction is most favorable for the performance parameters selected”. Manufacturing complexity definition in relation to a global and integral process of digital design for manufacture is essential for establishing an efficient managing protocol. “The definition of complexity associated to design for production in Architecture is proposed as the factor between number of different agents involved in the process by the number of interactions required between them, divided by the percentage of the interchange of information that is standardized and proof of information loss”. Design in architecture is a multi-objective process by definition. Therefore, addressing generation process linked to a set of non-coherent parameters requires the selection of adequate generative algorithm and the interaction of the architect. During the second half of the twentieth century and early twenty-first century it have been developed various mathematical algorithms for multi-parametric digital design. Heuristic algorithms are the most adequate algorithms for architectural projects due to its nature. The advantage of such algorithms is the ability to efficiently handle large scale optimization cases where a large number of design objectives and variables are involved. These generative processes do not pursue the optimum solution, in fact it will be impossible to proof with such algorithm. This is not a problem in architectural design where the final goal is to guide the form finding process towards a better performance within the initial direction provided by the architect. This research has focused on genetic algorithms due to its capacity to generate geometric alternatives in multiple directions and evaluate the fitness against a set of parameters specified in a single process. "Any protocol seeks to achieve standardization. The design to manufacturing protocol aims to provide a coordinated and coherent form generation process between a set of design parameters and the geometrical requirements of manufacturing technique. The protocol also provides an information exchange environment where there is a communication path and the level of information is ensured. The research is focused on the process because it is considered that each project will have its own singularities and parameters but the process will stay the same. Again the development of a specific tool is not a goal for the research, the intention is to provide an open source protocol that is valid for any set of tools”. Once the digital generation processes are being analized and classified, the next step is to identify the geometric parameters that define the digital design process. The definition of design process is including from the initial shape proposal based on the intuition of the architect to the generation, evaluation, selection and production of alternatives, both of an object , system or of the entire project . The current design process in Architecture is discontinuous and linear, dividing the process in disciplines in which the construction industry is structured. The proposal is to unify all relevant parameters in one process. The parameters are listed in groups of knowledge for internal classification but the matrix used for parameter relationship determination are combined. “A multi-parameter determination of the form-finding process is the integration all the measurable decisions laying behind Architect intuition. It is not possible to formulate and solve with an algorithm the design in Architecture. It is not the intention to do so with the proposal of this research. The process aims to integrate in one open protocol a selection of parameters by using geometry as common language. There is no optimum solution for any step of the process, the outcome is an evaluation of performance of all the form variations to assist the Architect for the selection of the preferable solution for the project”. The research follows with the geometrical restrictions of today Digital manufacturing techniques. Once determined it has been integrated in the form-finding process. “Digital manufacturing techniques are integrated in the form-finding process using geometry as common language. Geometric restraints define the boundary for performance parametric form-finding process. Geometrical limitations are classified by material and constructive system”. Choose between one digital model or several federate models is a strategic decision at planning a digital design for manufacturing protocol. Each one of the working models have strengths and weakens, nevertheless for the research purposes federated models are required to manage the different performance evaluation software platforms. A protocol based on federated models shall establish a communication process between software platforms and consultants. The manager shall integrate each discipline requirements defining the communication basis. The proposed protocol is based on standards on information exchange with singularities of the digital manufacturing industry. “The research concludes evolutionary algorithms as current best system to develop a generative form finding process based on the integration and coordination of a set of performance and constructive objectives. However, for application in professional practice and standardize it, the performance evaluation cannot be done in only one tool and therefore the selection of optimal genetic variants must be run in several iterations with a cumulative result. Consequently, the evaluation process within the geometrical restraints shall be carried out with federated models coordinated following the information exchange protocol”. The integration protocol and geometric constraints evaluation is done by applying in a practical case study. The exercise simulates multidisciplinary collaboration across software platforms with federated models. The choice of size and construction complexity of the project has been modulated to achieve the full development of each of the parameters selected. Continuing with the same objective proposed for the performance parameters the constructive and structural type selected for the exercise allows the application all geometric invariants associated to the set of parameters selected. The main goal of the case study is to proof the capacity of the manufacturing integrated form finding process to generate geometric alternatives to initial form with performance improved and following the restrictions determined by the compatible digital manufacturing technologies. The process is to be divided in consecutive analysis each one limited by the geometrical conditions and integrated in a overall evaluation. The interest of this process is the result of a non-intuitive form that performs better than a double symmetrical form. The second conclusion is that one parameter evaluation alone will not justify the exploration of complex geometry variations, but when there is a set of parameters with multidisciplinary approach then the less obvious solution emerge as the better performing form. “Architectural typologies impose limitation for Architects capacity to imagine formal variations. The case study and the research conclusions proof that even in situations where the intuitive solution apparently is the optimum solution, random variations can perform better when integrating all parameters evaluation. The capacity of foreseing the geometrical properties linking each design parameter with compatible manufacturing technologies ensure the result of the form-finding process to be constructively viable. Finally, the propose of a complete process where the geometry alternatives are generated beyond the Architect intuition and performance evaluated by a set of parameters previously selected and coordinated with the manufacturing requirements is the final objective of the Thesis”.

Regulation of T cell receptor (TCR) β gene expression by CD3 complex signaling in immature thymocytes: Implications for TCRβ allelic exclusion

During αβ thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRβ genes. On expression of a TCRβ polypeptide chain, the pre-TCR is assembled, and TCRβ locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRβ locus allelic exclusion in mice single-deficient or double-deficient for CD3ζ/η and/or p56lck. These mice display defects in the expression of endogenous TCRβ genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRβ VDJ (variable, diversity, joining) rearrangements by a functional TCRβ transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25+ double-negative (DN) thymocytes of the mutant mice failed to express the TCRβ transgene, suggesting defective expression of the TCRβ transgene similar to endogenous TCRβ genes. In the mutant mice, a proportion of CD25+ DN thymocytes that failed to express the transgene expressed endogenous TCRβ polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRβ chains or more than one endogenous TCRβ chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRβ locus by inducing the expression of rearranged TCRβ genes in CD25+ DN thymocytes.

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR−) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR− MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR− nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR− MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of ≈45 Å and a length of ≈200 Å. Because of the pleomorphic shape and paracrystalline packing of the Gag–RNA complexes, we raise the possibility that assembly of MuLV is driven by protein–RNA, as well as protein–protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest

In pre-B lymphocytes, productive rearrangement of Ig light chain genes allows assembly of the B cell receptor (BCR), which selectively promotes further developmental maturation through poorly defined transmembrane signaling events. Using a novel in vitro system to study immune tolerance during development, we find that BCR reactivity to auto-antigen blocks this positive selection, preventing down-regulation of light chain gene recombination and promoting secondary light chain gene rearrangements that often alter BCR specificity, a process called receptor editing. Under these experimental conditions, self-antigen induces secondary light chain gene rearrangements in at least two-thirds of autoreactive immature B cells, but fails to accelerate cell death at this stage. These data suggest that in these cells the mechanism of immune tolerance is receptor selection rather than clonal selection.

Posttranscriptional modification of retroviral primers is required for late stages of DNA replication

During reverse transcription of retroviral RNA, synthesis of (−) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5′ long terminal repeat. For (+) strand synthesis using a (−) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3′ terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (−) strand linked to natural modified or synthetic unmodified tRNA3Lys, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA3Lys, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA3Lys. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA3Lys template. Because all retroviral tRNA primers contain the same 1-methyl-A58 modification, our results suggest that 1-methyl-A58 is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A58 may provide a target for HIV chemotherapy is discussed.

An in vivo membrane fusion assay implicates SpoIIIE in the final stages of engulfment during Bacillus subtilis sporulation

Shortly after the synthesis of the two cells required for sporulation in Bacillus subtilis, the membranes of the larger mother cell begin to migrate around and engulf the smaller forespore cell. At the completion of this process the leading edges of the migrating membrane meet and fuse, releasing the forespore into the mother cell cytoplasm. We developed a fluorescent membrane stain-based assay for this membrane fusion event, and we isolated mutants defective in the final stages of engulfment or membrane fusion. All had defects in spoIIIE, which is required for translocation of the forespore chromosome across the polar septum. We isolated one spoIIIE mutant severely defective in chromosome translocation, but not in membrane fusion; this mutation disrupts the ATP/GTP-binding site of SpoIIIE, suggesting that ATP binding and hydrolysis are required for DNA translocation but not for the late engulfment function of SpoIIIE. We also correlated relocalization of SpoIIIE-green fluorescent protein from the sporulation septum to the forespore pole with the completion of membrane fusion and engulfment. We suggest that SpoIIIE is required for the final steps of engulfment and that it may regulate or catalyze membrane fusion events.

Mature monocytic cells enter tissues and engraft

The goal of this study was to identify the circulating cell that is the immediate precursor of tissue macrophages. ROSA 26 marrow mononuclear cells (containing the β-geo transgene that encodes β-galactosidase and neomycin resistance activities) were cultured in the presence of macrophage colony-stimulating factor and flt3 Ligand for 6 days to generate monocytic cells at all stages of maturation. Expanded monocyte cells (EMC), the immature (ER-MP12+) and more mature (ER-MP20+) subpopulations, were transplanted into irradiated B6/129 F2 mice. β-gal staining of tissue sections from animals 15 min after transplantation demonstrated that the donor cells landed randomly. By 3 h, donor cells in lung and liver were more frequent in animals transplanted with ER-MP20+ (more mature) EMC than in animals transplanted with unseparated EMC or fresh marrow mononuclear cells, a pattern that persisted at 3 and 7 days. At 3 days, donor cells were found in spleen, liver, lung, and brain (rarely) as clusters as well as individual cells. By 7 and 14 days, the clusters had increased in size, and the cells expressed the macrophage antigen F4/80, suggesting that further replication and differentiation had occurred. PCR for the neogene was used to quantitate the amount of donor DNA in tissues from transplanted animals and confirmed that ER-MP20+ EMC preferentially engrafted. These data demonstrate that a mature monocytic cell gives rise to tissue macrophages. Because these cells can be expanded and manipulated in vitro, they may be a suitable target population for gene therapy of lysosomal storage diseases.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

Abundant empty class II MHC molecules on the surface of immature dendritic cells

A monoclonal antibody specific for the empty conformation of class II MHC molecules revealed the presence of abundant empty molecules on the surface of spleen- and bone marrow-derived dendritic cells (DC) among various types of antigen-presenting cells. The empty class II MHC molecules are developmentally regulated and expressed predominantly on immature DC. They can capture peptide antigens directly from the extracellular medium and present bound peptides to antigen-specific T lymphocytes. The ability of the empty cell-surface class II MHC proteins to bind peptides and present them to T cells without intracellular processing can serve to extend the spectrum of antigens able to be presented by DC, consistent with their role as sentinels in the immune system.

Extracellular antigen processing and presentation by immature dendritic cells

In antigen presentation to CD4+ T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded onto class II MHC proteins for subsequent transport to the surface and presentation to T cells. Here, we provide evidence for an additional antigen-processing pathway in immature dendritic cells (DC). Immature DC express at the cell surface empty or peptide-receptive class II MHC molecules, as well as H-2M or HLA-DM. Secreted DC proteases act extracellularly to process intact proteins into antigenic peptides. Peptides produced by such activity are efficiently loaded onto cell surface class II MHC molecules. Together these elements comprise an unusual extracellular presentation pathway in which antigen processing and peptide loading can occur entirely outside of the cell.

Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

Role of Syk in B-cell development and antigen-receptor signaling

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles—promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79α/β BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.