Diseño de una red de acceso mediante fibra óptica

Con el objetivo de consolidar los conocimientos en Sistemas de Telecomunicación, se pretende diseñar una red para la distribución de servicios avanzados, como televisión, internet de banda ancha y telefonía, mediante el uso de la tecnología FTTH. La red será totalmente pasiva, óptica, y de gran ancho de banda, Tipo PON (Passive Optical Network), de manera que el haz de luz del emisor se distribuye hacia múltiples fibras siguiendo diferentes direcciones, o las confina en el sentido opuesto usando técnicas WDM y TDMA. En primer lugar, es preciso realizar un estudio teórico sobre las características de la fibra óptica, junto con las propiedades de los elementos activos y pasivos que interactúan con ella, para poder comprender la tecnología en la que se basa y así ofrecer una solución final acorde a las necesidades que se presenten. Tras un vistazo general a la tecnología de fibra óptica, se procede a estudiar las características y topologías de las redes de acceso basadas en la misma, junto con los requisitos de cara al diseño de la redes y a la gestión de proyectos. Se definirán los objetivos de la red, ya que la complejidad de la red depende de factores como la extensión y cobertura de la misma, o el ancho de banda. Posteriormente se irá diseñando la red en una arquitectura descendente hasta destinar una única fibra óptica para cada hogar dentro de la cobertura de la red. Se detallarán todas las especificaciones necesarias para definir la red, (potencias, modulaciones y tipos de fibra) así como el instrumental y demás medios necesarios para operar con la misma. Aunque el diseño de una red FTTH contempla muchos más aspectos de los estudiados en el presente proyecto, se tomó como objetivo el diseño y planificación correspondientes a una sección de un área de escasa población donde la instalación se tomó como viable, obviando así pequeños detalles y centrándose en el despliegue de fibra óptica. Se pretende así ofrecer una solución de infraestructura doméstica de telecomunicaciones, apta para las futuras necesidades que implican la creciente demanda de servicios como internet de banda ancha o almacenamiento y procesado de aplicaciones en nube. La fibra óptica tiene mucho que ofrecer a las telecomunicaciones, y cada vez es más frecuente encontrar redes de fibra óptica como parte de un servicio de telecomunicaciones, tanto como para particulares como empresas. ABSTRACT. Aiming to strengthen knowledge in Telecommunications Systems, is intended to design a network for advanced services broadcasting, including digital TV, broadband internet and telephony, by using FTTH technology. This network will be entirely passive optic, and high bandwidth, PON type (Passive Optical Network) so that the transmitter beam is broadcasting to multiple optical fibers, branching out in different ways or joining them in the opposite. First, it is necessary to perform a theoretical study on the characteristics of the optical fiber, along with the properties of the active and passive elements that interact with it, to understand the technology that is based and offer a final solution according needs that arise. After an overview of the optical fiber technology, we proceed to study the characteristics and topologies access networks based on that, together with the requirements to face the network design and project management. System objectives will be determined, since the complexity of the network depends on factors such as the size and scope of it, or bandwidth. Later, the network will be designing in downstream architecture to deliver a single optical fiber to each household within the network area. All necessary specifications to define the network (power, modulation and fiber types) will be determined, as instruments and other means to operate it. Although FTTH network design includes many more aspects of those studied in this project, it was taken as objective the network design and planning corresponding to a section of a sparsely populated area where the facility was taken as feasible thus obviating small details and focus on the deployment of optical fiber. This is intended to provide a solution of household telecommunications infrastructure suitable for future needs involving the growing demand for services such as broadband internet or processing storage and cloud applications. The optical fiber has much to offer to telecommunications, and it is increasingly common to find fiber optic networks as part of a telecommunications service, both to individuals and businesses.

Metodología para la evaluación de productos innovadores de construcción : (preassessment-preevaluación DIT)

The specific research described in this dissertation is based on the author’s work over the years as head of the Eduardo Torroja Institute for Construction Science’s4 Technical Unit for the Assessment of Innovative Construction Products. The hypothesis proposed is the feasibility of developing methodology able to deliver a preliminary estimate (in just 1‐3 months’ time) of the technical suitability of certain innovative construction products for their intended use. The product families studied are the object of technical assessment guides or guidelines authored by two European organisations, the Union Européenne pour l'agrément technique dans la construction (UEAtc) and the European Organization for Technical Assessment (EOTA). Such families are eligible for or have been awarded technical approvals (TAs, also referred to in English under the French term “agréments”) or European Technical Approvals (ETAs), which is presently an 11‐month process. The proposed methodology would consist of a pre‐assessment procedure referred to hereunder as DIT PREASSESSMENT/PREEVALUACIÓN (Spanish initials, PPD). Based on the experience obtained from trials, calculations and studies undertaken in the framework of this research, the proposed procedure would identify the performance factors (primarily identification and fitness for use, although also durability where ascertainable via shortterm testing) that predict construction product or system suitability in a more immediate time frame.

Social development benefits of hydroelectricity CDM projects in Brazil

In recent years, the concept of sustainable development has become increasingly recognized and important. Within organizations, sustainable development is often portrayed as a balancing act, and requires a combination of three elements to be considered: economy, environment, and society. Traditionally, organizational management research has been focused on economical and environmental fronts. However, social aspects are also important for organizations, especially those in emerging and developing countries. The goal of this paper is to investigate the potential of Clean Development Mechanism (CDM) projects to deliver social benefits in Brazil?s hydroelectricity sector. The investigation involved the assessment of 46 registered hydro CDM projects under the Kyoto Protocol in terms of their potential impact on the envisaged social development goals. Two case studies were also examined. Results indicate that organizations managing hydroelectric initiatives in Brazil can provide the pathway towards achieving a number of important social benefits. Successful projects were found to have good community involvement and were managed by both cooperative ventures and money making corporations. The research also identified several challenges that are hindering hydro CDM projects from delivering more social benefits, and enabled a number of recommendations to be extracted for the organizations facing these challenges.

Why neutron guides may end up breaking down? Some results on the macroscopic behaviour of alkali-borosilicate glass support plates under neutron irradiation

In this paper we report on a first part of a study on the mechanisms leading to brittle fracture in neutron guides made of glass as structural element. Such devices are widely used to deliver thermal and cold neu tron beams to experimental lines in most large neutron research facilities. We present results on macroscopic properties of samples of guide glass substrates which are subjected to neutron irradiation at relatively large fluences. The results show a striking dependence of some of the macroscopic properties such as density, shape or surface curvature upon the specific chemical composition of a given glass. The relevance of the present findings for the installation of either replacement guides at the existing facilities or for the deployment of instruments for ongoing projects such as the European Spallation Source is briefly discussed.

User experience with immersive and interactive media = Experiencia de usuario en entornos 3D inmersivos e interactivos

Estudios recientes promueven la integración de estímulos multisensoriales en activos multimedia con el fin de mejorar la experiencia de usuario mediante la estimulación de nuevos sentidos, más allá de la tradicional experiencia audiovisual. Del mismo modo, varios trabajos proponen la introducción de componentes de interacción capaces de complementar con nuevas características, funcionalidades y/o información la experiencia multimedia. Efectos sensoriales basados en el uso de nuevas técnicas de audio, olores, viento, vibraciones y control de la iluminación, han demostrado tener un impacto favorable en la sensación de Presencia, en el disfrute de la experiencia multimedia y en la calidad, relevancia y realismo de la misma percibidos por el usuario. Asimismo, los servicios basados en dos pantallas y la manipulación directa de (elementos en) la escena de video tienen el potencial de mejorar la comprensión, la concentración y la implicación proactiva del usuario en la experiencia multimedia. El deporte se encuentra entre los géneros con mayor potencial para integrar y explotar éstas soluciones tecnológicas. Trabajos previos han demostrado asimismo la viabilidad técnica de integrar éstas tecnologías con los estándares actualmente adoptados a lo largo de toda la cadena de transmisión de televisión. De este modo, los sistemas multimedia enriquecidos con efectos sensoriales, los servicios interactivos multiplataforma y un mayor control del usuario sobre la escena de vídeo emergen como nuevas formas de llevar la multimedia immersiva e interactiva al mercado de consumo de forma no disruptiva. Sin embargo, existen numerosas interrogantes relativas a los efectos sensoriales y/o soluciones interactivas más adecuadas para complementar un contenido audiovisual determinado o a la mejor manera de de integrar y combinar dichos componentes para mejorar la experiencia de usuario de un segmento de audiencia objetivo. Además, la evidencia científica sobre el impacto de factores humanos en la experiencia de usuario con estas nuevas formas de immersión e interacción en el contexto multimedia es aún insuficiente y en ocasiones, contradictoria. Así, el papel de éstos factores en el potencial de adopción de éstas tecnologías ha sido amplia-mente ignorado. La presente tesis analiza el impacto del audio binaural, efectos sensoriales (de iluminación y olfativos), interacción con objetos 3D integrados en la escena de vídeo e interacción con contenido adicional utilizando una segunda pantalla en la experiencia de usuario con contenidos de deporte. La posible influencia de dichos componentes en las variables dependientes se explora tanto a nivel global (efecto promedio) como en función de las características de los usuarios (efectos heterogéneos). Para ello, se ha llevado a cabo un experimento con usuarios orientado a explorar la influencia de éstos componentes immersivos e interactivos en dos grandes dimensiones de la experiencia multimedia: calidad y Presencia. La calidad de la experiencia multimedia se analiza en términos de las posibles variaciones asociadas a la calidad global y a la calidad del contenido, la imagen, el audio, los efectos sensoriales, la interacción con objetos 3D y la interacción con la segunda pantalla. El posible impacto en la Presencia considera dos de las dimensiones definidas por el cuestionario ITC-SOPI: Presencia Espacial (Spatial Presence) e Implicación (Engagement). Por último, los individuos son caracterizados teniendo en cuenta los siguientes atributos afectivos, cognitivos y conductuales: preferencias y hábitos en relación con el contenido, grado de conocimiento de las tecnologías integradas en el sistema, tendencia a involucrarse emocionalmente, tendencia a concentrarse en una actividad bloqueando estímulos externos y los cinco grandes rasgos de la personalidad: extroversión, amabilidad, responsabilidad, inestabilidad emocional y apertura a nuevas experiencias. A nivel global, nuestro estudio revela que los participantes prefieren el audio binaural frente al sistema estéreo y que los efectos sensoriales generan un aumento significativo del nivel de Presencia Espacial percibido por los usuarios. Además, las manipulaciones experimentales realizadas permitieron identificar una gran variedad de efectos heterogéneos. Un resultado interesante es que dichos efectos no se encuentran distribuidos de forma equitativa entre las medidas de calidad y Presencia. Nuestros datos revelan un impacto generalizado del audio binaural en la mayoría de las medidas de calidad y Presencia analizadas. En cambio, la influencia de los efectos sensoriales y de la interacción con la segunda pantalla se concentran en las medidas de Presencia y calidad, respectivamente. La magnitud de los efectos heterogéneos identificados está modulada por las siguientes características personales: preferencias en relación con el contenido, frecuencia con la que el usuario suele ver contenido similar, conocimiento de las tecnologías integradas en el demostrador, sexo, tendencia a involucrarse emocionalmente, tendencia a a concentrarse en una actividad bloqueando estímulos externos y niveles de amabilidad, responsabilidad y apertura a nuevas experiencias. Las características personales consideradas en nuestro experimento explicaron la mayor parte de la variación en las variables dependientes, confirmando así el importante (y frecuentemente ignorado) papel de las diferencias individuales en la experiencia multimedia. Entre las características de los usuarios con un impacto más generalizado se encuentran las preferencias en relación con el contenido, el grado de conocimiento de las tecnologías integradas en el sistema y la tendencia a involucrarse emocionalmente. En particular, los primeros dos factores parecen generar un conflicto de atención hacia el contenido versus las características/elementos técnicos del sistema, respectivamente. Asimismo, la experiencia multimedia de los fans del fútbol parece estar modulada por procesos emociona-les, mientras que para los no-fans predominan los procesos cognitivos, en particular aquellos directamente relacionados con la percepción de calidad. Abstract Recent studies encourage the integration of multi-sensorial stimuli into multimedia assets to enhance the user experience by stimulating other senses beyond sight and hearing. Similarly, the introduction of multi-modal interaction components complementing with new features, functionalities and/or information the multimedia experience is promoted. Sensory effects as odor, wind, vibration and light effects, as well as an enhanced audio quality, have been found to favour media enjoyment and to have a positive influence on the sense of Presence and on the perceived quality, relevance and reality of a multimedia experience. Two-screen services and a direct manipulation of (elements in) the video scene have the potential to enhance user comprehension, engagement and proactive involvement of/in the media experience. Sports is among the genres that could benefit the most from these solutions. Previous works have demonstrated the technical feasibility of implementing and deploying end-to-end solutions integrating these technologies into legacy systems. Thus, sensorially-enhanced media, two-screen services and an increased user control over the displayed scene emerge as means to deliver a new form of immersive and interactive media experiences to the mass market in a non-disruptive manner. However, many questions remain concerning issues as the specific interactive solutions or sensory effects that can better complement a given audiovisual content or the best way in which to integrate and combine them to enhance the user experience of a target audience segment. Furthermore, scientific evidence on the impact of human factors on the user experience with these new forms of immersive and interactive media is still insufficient and sometimes, contradictory. Thus, the role of these factors on the potential adoption of these technologies has been widely ignored. This thesis analyzes the impact of binaural audio, sensory (light and olfactory) effects, interaction with 3D objects integrated into the video scene and interaction with additional content using a second screen on the sports media experience. The potential influence of these components on the dependent variables is explored both at the overall level (average effect) and as a function of users’ characteristics (heterogeneous effects). To these aims, we conducted an experimental study exploring the influence of these immersive and interactive elements on the quality and Presence dimensions of the media experience. Along the quality dimension, we look for possible variations on the quality scores as-signed to the overall media experience and to the media components content, image, audio, sensory effects, interaction with 3D objects and interaction using the tablet device. The potential impact on Presence is analyzed by looking at two of the four dimensions defined by the ITC-SOPI questionnaire, namely Spatial Presence and Engagement. The users’ characteristics considered encompass the following personal affective, cognitive and behavioral attributes: preferences and habits in relation to the content, knowledge of the involved technologies, tendency to get emotionally involved and tendency to get absorbed in an activity and block out external distractors and the big five personality traits extraversion, agreeableness, conscientiousness, neuroticism and openness to experience. At the overall level, we found that participants preferred binaural audio than standard stereo audio and that sensory effects increase significantly the level of Spatial Presence. Several heterogeneous effects were also revealed as a result of our experimental manipulations. Interestingly, these effects were not equally distributed across the quality and Presence measures analyzed. Whereas binaural audio was foud to have an influence on the majority of the quality and Presence measures considered, the effects of sensory effects and of interaction with additional content through the tablet device concentrate mainly on the dimensions of Presence and on quality measures, respectively. The magnitude of these effects was modulated by individual’s characteristics, such as: preferences in relation to the content, frequency of viewing similar content, knowledge of involved technologies, gender, tendency to get emotionally involved, tendency to absorption and levels of agreeableness, conscientiousness and openness to experience. The personal characteristics collected in our experiment explained most of the variation in the dependent variables, confirming the frequently neglected role of individual differences on the media experience. Preferences in relation to the content, knowledge of involved technologies and tendency to get emotionally involved were among the user variables with the most generalized influence. In particular, the former two features seem to present a conflict in the allocation of attentional resources towards the media content versus the technical features of the system, respectively. Additionally, football fans’ experience seems to be modulated by emotional processes whereas for not fans, cognitive processes (and in particular those related to quality judgment) prevail.

EDUCAÇÃO E HISTÓRIA A Escola Estadual Maria Iracema Munhoz, em São Bernardo do Campo, desde 1890 a 1930

Esta dissertação focaliza a trajetória do surgimento da Escola Estadual Maria Iracema Munhoz, apresentando o seu percurso desde 1890 até 1930. Para isso foi necessário fazer uma retrospectiva da educação brasileira, lembrar como surgiu São Bernardo, município onde está localizada a escola e, o surgimento da educação na cidade. O objetivo de reflexão e de pesquisa da presente dissertação foi buscar entender, pela história do passado, os problemas do presente, reconstruindo os avanços e os insucessos no processo da escolarização e das políticas públicas educacionais assumidas nesse período. A hipótese apresentada é que, nosso atraso educativo não vem tanto do que deixamos de fazer nas últimas décadas, mas do que fizemos nos séculos anteriores, em se tratando da educação.(AU)

EDUCAÇÃO E HISTÓRIA A Escola Estadual Maria Iracema Munhoz, em São Bernardo do Campo, desde 1890 a 1930

Esta dissertação focaliza a trajetória do surgimento da Escola Estadual Maria Iracema Munhoz, apresentando o seu percurso desde 1890 até 1930. Para isso foi necessário fazer uma retrospectiva da educação brasileira, lembrar como surgiu São Bernardo, município onde está localizada a escola e, o surgimento da educação na cidade. O objetivo de reflexão e de pesquisa da presente dissertação foi buscar entender, pela história do passado, os problemas do presente, reconstruindo os avanços e os insucessos no processo da escolarização e das políticas públicas educacionais assumidas nesse período. A hipótese apresentada é que, nosso atraso educativo não vem tanto do que deixamos de fazer nas últimas décadas, mas do que fizemos nos séculos anteriores, em se tratando da educação.(AU)

QUALIDADE DE VIDA NO TRABALHO - QVT E RESPONSABILIDADE SOCIAL EMPRESARIAL - RSE: UM ESTUDO SOBRE A SATISFAÇÃO DE QVT COM FUNCIONÁRIOS VOLUNTÁRIOS EM PROGRAMAS RSE

As organizações se encontram em meio a uma série de transformações na sociedade e no mundo empresarial de natureza estrutural e tecnológica. Essas mudanças relacionadas ao processo de globalização, cada vez mais constantes na vida organizacional implicam as organizações uma postura cada vez mais responsável tanto a nível organizacional quanto social. Em se tratando de adaptações organizacionais, o funcionário poderá ser mais exigido. Se a empresa precisa estar mais competitiva para continuar no mercado e resistir, há expectativa de uma maior capacidade produtiva. Nesse contexto de pressões constantes a todos da organização, a Qualidade de Vida no Trabalho passa ser destaque como um diferencial competitivo para organizações, que programam e usufruem de seus resultados. Porém, não se pode deixar de pensar que além de produzir, as empresas devem estar preocupadas no seu papel perante a sociedade, nesse item, entra a Responsabilidade Social Empresarial. As empresas são cobradas tanto como estas podem influir na degradação dos recursos naturais, como na sua ação de fazer algo a mais que contribua para sociedade. Esse assunto vem se expandindo devido à sua importante aplicabilidade e atualmente tem pautado algumas discussões no campo organizacional. Com base neste conceito, este estudo buscou analisar as possíveis relações existentes entre os programas de Qualidade de Vida no Trabalho QVT e Responsabilidade Social Empresarial - RSE considerando a satisfação de QVT dos indivíduos que participam dos programas de RSE nas empresas que adotam ambos os programas QVT e RSE. Optou-se em adotar um estudo de caso em associação com métodos quantitativos para aplicação do instrumento BPSO (96) e métodos qualitativos, entrevistas e observação do pesquisador. Concluiu-se que os Envolvidos em programas de RSE não apresentaram maior satisfação de QVT em relação ao grupo Não Participa . O fato da participação do funcionário em RSE não deve ser considerado como um indicador de QVT. O que pode ter influenciado nos resultados é o perfil da amostra, que se mostrou muito heterogêneo, onde estão presentes vários tipo de pessoas e cada um com uma expectativa diferente do empenho da empresa com sua Qualidade de Vida no Trabalho.

QUALIDADE DE VIDA NO TRABALHO - QVT E RESPONSABILIDADE SOCIAL EMPRESARIAL - RSE: UM ESTUDO SOBRE A SATISFAÇÃO DE QVT COM FUNCIONÁRIOS VOLUNTÁRIOS EM PROGRAMAS RSE

As organizações se encontram em meio a uma série de transformações na sociedade e no mundo empresarial de natureza estrutural e tecnológica. Essas mudanças relacionadas ao processo de globalização, cada vez mais constantes na vida organizacional implicam as organizações uma postura cada vez mais responsável tanto a nível organizacional quanto social. Em se tratando de adaptações organizacionais, o funcionário poderá ser mais exigido. Se a empresa precisa estar mais competitiva para continuar no mercado e resistir, há expectativa de uma maior capacidade produtiva. Nesse contexto de pressões constantes a todos da organização, a Qualidade de Vida no Trabalho passa ser destaque como um diferencial competitivo para organizações, que programam e usufruem de seus resultados. Porém, não se pode deixar de pensar que além de produzir, as empresas devem estar preocupadas no seu papel perante a sociedade, nesse item, entra a Responsabilidade Social Empresarial. As empresas são cobradas tanto como estas podem influir na degradação dos recursos naturais, como na sua ação de fazer algo a mais que contribua para sociedade. Esse assunto vem se expandindo devido à sua importante aplicabilidade e atualmente tem pautado algumas discussões no campo organizacional. Com base neste conceito, este estudo buscou analisar as possíveis relações existentes entre os programas de Qualidade de Vida no Trabalho QVT e Responsabilidade Social Empresarial - RSE considerando a satisfação de QVT dos indivíduos que participam dos programas de RSE nas empresas que adotam ambos os programas QVT e RSE. Optou-se em adotar um estudo de caso em associação com métodos quantitativos para aplicação do instrumento BPSO (96) e métodos qualitativos, entrevistas e observação do pesquisador. Concluiu-se que os Envolvidos em programas de RSE não apresentaram maior satisfação de QVT em relação ao grupo Não Participa . O fato da participação do funcionário em RSE não deve ser considerado como um indicador de QVT. O que pode ter influenciado nos resultados é o perfil da amostra, que se mostrou muito heterogêneo, onde estão presentes vários tipo de pessoas e cada um com uma expectativa diferente do empenho da empresa com sua Qualidade de Vida no Trabalho.

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: Implication for the in vivo delivery of CD8+ T cell epitopes into antigen-presenting cells

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8+ cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11–25 amino acids, corresponding to defined CD8+ T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (−1 or −2) decreased or completely blocked (charge of −4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system

The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ–TTC gene fusion retains the biological functions of both proteins in vivo—i.e., retrograde transynaptic transport of the TTC fragment and β-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.

T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes

We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20–35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.

Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles

The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactive vpr, vpu, and nef coding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 107 colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+ cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1 stage of the cell cycle by density-dependent inhibition of growth. Human CD34+ cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.