Herramienta de partes de actividad

Para entender el proyecto de una manera muy simplificada lo podríamos resumir de la siguiente forma: Se trata de una aplicación que permite a los usuarios imputar las horas de las tareas que realizan de una forma sencilla, y que los gestores de estos usuarios, mediante informes ejecutivos, puedan analizar las dedicaciones y costes de los proyectos que éstos gestionan. Además se dispondrá de pantallas para administrar todas las entidades que están implicadas en las imputaciones. Las imputaciones se realizarán agrupándolas por el concepto “Parte”. Los Partes dividen los meses en grupos de 10 días, así cada usuario tendrá que imputar las horas que realiza cada 10 días, enviando a su supervisor el parte para que este pueda revisarlo y analizar los resultados de las imputaciones a través de los informes. Vamos a resumir las entidades que intervienen en la aplicación y sus relaciones de la siguiente forma: - Los usuarios tendrán asignados una serie de cargos, los cuales definirán las posibles tareas que podrán realizar categorizadas en los conceptos Proceso y Subproceso. Un ejemplo de esta relación podría ser el cargo “Programador” podrá realizar tareas de tipo Proceso “Desarrollo” y tipos de Subproceso “Programación” y “Pruebas unitarias” - También se asignarán a los usuarios los proyectos en los que trabajan. Estos proyectos pertenecerán a una unidad (se podría equiparar el concepto unidad por ejemplo a clientes, departamentos, etc). - También se podrán asignar a los usuarios las peticiones concretas de proyectos en los que intervienen. Una petición podría ser una incidencia detectada en un proyecto, una mejora concreta en un proyecto de mantenimiento, un soporte realizado para arreglar unos datos incorrectos, etc. - A los usuarios se le asignarán unos costes que podrán ser distintos por periodos de tiempo con el fin de conocer el coste exacto de una hora realizada en la fecha en la que fue realizada. - A los usuarios se les asignará un Perfil de usuario. Este perfil de usuario definirá los permisos sobre los módulos disponibles de la herramienta. Habrá un permiso por cada entidad, dividiéndolos en Alta, Baja, Modificación y Consulta. Además de estos permisos habrá permisos especiales sobre módulos que no se refieran al manteniendo de entidades pero que sí requieran de un permiso especial para poder ejecutarse como por ejemplo poder ejecutar los informes de costes, poder realizar y consultar partes de otros usuarios además de los propios del usuario, etc. Los perfiles serán dinámicos. Se podrán crear tantos perfiles como se requieran, cada uno con los permisos que se le asignen. - Los usuarios imputarán las horas que realizan categorizando por tarea los siguientes conceptos: Proyecto, Petición (opcional), Proceso, Subproceso, Descripción de la tarea y horas invertidas en los días de la decena. La aplicación permite poder adaptarse a las necesidades de diseño de la organización que la utilice y pueden crearse tantas instancias de la aplicación como se necesiten, compartiendo ono la base de datos entre varias de las instancias, sin necesidad de duplicar el código en el servidor, lo que facilita el mantenimiento de la aplicación cuando se añaden mejoras y se solucionan incidencias. También facilita el poder vender licencias de uso de la aplicación a otras entidades teniendo una sola versión de la aplicación en la nube, lo que hace muy bajo el coste de la venta de licencias aumentando así los beneficios por venta. ABSTRACT To understand the project in a very simplified way we could summarize as follows: Its an application that allows users to charge the hours of tasks that they realize in a easy way, and that the managers of these users, by executive reports, can analyze dedications and costs of projects that they manage. Furthermore screens will be available to manage all entities that are involved in the charge of hours. The charge of hours will be grouped by the concept "Timesheet". The Timesheets divided the months in groups of 10 days, so each user will have to charge the hours every 10 days by sending his supervisor the thimesheet so that it can review it and analyze the results of the charges of hours by reports.Let's summarize the entities involved in the application and their relationships as follows: - Users will be assigned to one or more positions, which define the possible tasks that can perform categorized into concepts Process and Subprocess. An example of this relationship could be the position "Developer" may do tasks of process type "Development" and subprocess types "Programming" and "Unit Testing" - Users will also be assigned to projects in which they work. These projects belongs to a unit (Some possible examples of unit could be customers, departments, ...). - You can also assign users to specific requests for projects thar they are involved. A request could be an issue detected in a project, a concrete improvement in a maintenance project, a support made to fix some incorrect data, ... - Users will be assigned to one or more costs that may be different for periods of time in order to know the exact cost of an hour on the date on which it was made. - Users will be assigned to a User Profile. This user profile define the available permissions on modules of tool. There will be a permission for each entity, dividing them into Insert, Delete, Modify and Read. In addition to these permissions will be special permissions on modules that are not related to maintaining entities but if require special permission to execute such as to execute the cost reports, to make and read Timesheets of others users in addition to themselve user, etc. The profiles will be dynamic. We can create many profiles as we required, each with the permissions that are assigned. - Users charge the hours that they work categorizing per task the following: Project, Request (optional), Process, Subprocess, task description and the hours that they used in the ten days period. The application allows to adapt to the needs of design of the organization that use the application and allows create many instances of the application as needed, yes or not sharing the database among multiple instances without duplicating the code on the server, which facilitates maintenance of the application when improvements are added and incidents are resolved. It also facilitates to sell licenses to use the application to other organizations having a single version of the application in the cloud, making it very low cost from the sale of licenses and increasing profits per sale.

El vidrio y sus máscaras : el sueño de la arquitectura de cristal

Según Heidegger, la esencia de la arquitectura, de la construcción, descansa en un no espacio: en la materia con que se construyen las fronteras que otorgan espacios, irradiando sobre ellos aquello que los caracteriza. Si hay alguna materia, de las utilizadas por la arquitectura a lo largo de su historia para construir fronteras, que haya mantenido una especial relación con la luz y la visión, dando un carácter inconfundible a los espacios aviados por ellas, esta es el vidrio; algunas de las etimologías de su nombre: zakû (ser claro), hyalos (diáfano) o vitrum (ver), así lo evidencian. Posiblemente, sea la pregnancia de este modo fascinante de relacionarse con la luz, la que ha hecho del vidrio, a lo largo del tiempo que lleva siendo usado en arquitectura, y aún antes, el material que ha provocado en el imaginario humano la ilusión de ser aquel en que, en último término, podrían llegar a sublimarse todos los demás, dando lugar con ello a lo que en la tesis hemos denominado el sueño de la arquitectura de cristal. Siendo la luz, siempre, energía, consideraremos en la tesis luz-energía, a aquella que ilumina y calienta; es una luz científica y mesurable. Cuando la luz se “hace visible”, desvelando un mensaje “contenido” en el vidrio, hablaremos de luz-información. Esta luz, no puede medirse científicamente. La luz-energía y la luz-información, se manifiestan al conjuro de la arquitectura de vidrio. Es la segunda la que ha conformado las fronteras de vidrio enmascarado, y la que se estudia con más detenimiento en la tesis. Los distintos modos de usar en arquitectura la infinita combinatoria de las propiedades de absortancia, reflectancia, transmitancia y translucencia del vidrio, ha condicionado al hombre en su manera de “ver” el mundo. Unas veces, “inmerso” en él, puesto que solo lo separa del mismo, una frontera transparente, y “deseadamente” invisible: ese modo de usar el vidrio, ha sido el sueño imposible de una parte importante de la arquitectura del siglo XX. Otras veces, para “aislarse” de él, el hombre ha manipulado la luz y el vidrio para construir mundos diferentes. Las fronteras de vidrio enmascarado de color, mosaicos, vidrieras, pantallas y lo que hemos llamado vidrios complejos (con un cometido similar al que Schiller atribuía al coro en la tragedia griega, aislar a esta del “mundo real”, para mantener su libertad poética), son las fronteras que han construido el sueño posible de la arquitectura de cristal. Ambas actitudes, en distintos momentos de la historia de la arquitectura, han sido dos formas de querer materializar un mismo sueño. La capacidad del vidrio para adaptarse a tantos modos de presentarse ante nosotros, y a poder ser interpretado de tantas formas diferentes, es la que ha servido para dar título a la tesis, pues hasta en su faceta más transparente, el vidrio, de una forma o de otra, se ha mostrado siempre como un material enmascarado en el más amplio sentido de la palabra: se enmascara, incluso cuando apela a la transparencia o se convierte en espejo, para hacernos caer en la ilusión de que no está presente. Cuando el hombre construyó fronteras de vidrio e incluso antes, cuando soñó que con él podría llegar a construirlas, condensó en ellas toda la mítica, la mística y la epistemología en torno a la luz y la visión, dando lugar a una serie de arquetipos arquitectónicos. En la iglesia bizantina, la luz sobre, o la luz desde, los mosaicos, construyó una frontera titilante; y en la catedral gótica, la luz a través de las vidrieras construyó una frontera radiante; en ambos casos con el fin de alcanzar anagógicamente lo Inteligible. En el siglo XIX, con el descubrimiento de la electricidad y su incorporación a la arquitectura, las fronteras se vuelven fulgurantes, aviando, en este caso, el espacio urbano. Poco antes, en este mismo siglo, el espíritu del gótico tiene un efímero resurgir del que se nutrirá, a comienzos del siglo XX, el expresionismo cristalino, en el que la luz anagógica se hace laica. El espacio urbano fulgurante prefigurado por este movimiento y presente en las ciudades desde principios del siglo XX, fue potenciado a mediados de ese siglo con la aparición de las pantallas, extendiéndose desde entonces, imparable, por todo el planeta. La reciente emergencia de los vidrios complejos, ha abierto la posibilidad de construir fronteras a la carta (de vidrios de propiedades múltiples, seleccionadas de forma voluntaria y variable en cada momento). En principio, se pensó que, el uso de estos vidrios como cerramiento, podría llegar a constituirse como la panacea de los problemas del material relacionados con la luz-energía, sin necesidad de recurrir a “prótesis”, y manteniendo por tanto la seductora tersura de la fachada; aunque parece que, por ahora, esa posibilidad es, cuando menos, lejana. Sin embargo, en el campo de las megapantallas urbanas (y ,en general, en el de las pantallas de información), ubicuas actualmente en nuestras vidas, los vidrios complejos ayudan a construir los espesos velos de ilusión, que según Lefebvre sirven para mantener el capitalismo, siendo el último estadio de un desarrollo tecnológico, impuesto por el principio de economía del hombre, que como un metrónomo inexorable, y a modo de contrapunto, ha acompañado siempre (de nuevo en palabras de Lefebvre), a la necesidad del gasto, del juego, de la lucha, del arte, de la fiesta. La tecnología y el arte forman parte de la cultura producida por la sociedad y como señala Lévi-Strauss, esa cultura imprime orden; por el contrario, la sociedad, entendida como el conjunto de relaciones que los hombres mantienen entre sí, produce desorden. Del equilibrio entre esos extremos, surge el progreso, incluido el de la arquitectura. Las fronteras de vidrio que analizamos en la tesis –que avían espacios para la espiritualidad, el fasto y el espectáculo o, desde otro punto de vista, para las distintas manifestaciones del poder: la iglesia, la monarquía, el estado o el mercado– también han surgido de esa concomitancia entre el desorden y el orden; y forma parte de ese desorden, la aventura que ha impulsado al genio individual de místicos, alquimistas, geómetras, abades, reyes, inventores, poetas y arquitectos, a explorar, como escribe Apollinaire, vastos y extraños territorios donde el misterio en flor, se ofrece a quien quiera cogerlo, hogueras nuevas de colores nunca vistos, mil fantasmas imponderables a los que dar cuerpo. ABSTRACT According to Heidegger, the essence of architecture, building, lies in a non-space: the material that creates the boundaries from which something begins its presencing, radiating onto them that which characterizes them. If there is any single material amongst all those used throughout the history of architecture to build boundaries which has maintained a special relationship with light and vision, which has bestowed a distinctive character on spaces avid for them, it is glass. This is evidenced in some of its etymologies: zakû (to be clear), hyalos (transparent), vitrum (see). The rich potential of this fascinating way of relating to light in the history of the architectural use of glass, and even before, is possibly what has triggered the illusion in human imagination of being something that can ultimately sublimate all others, giving rise to what in this thesis we call The Dream of Crystal Architecture. Given that light is always energy, in this thesis we consider energy-light to be that which illuminates and warms. This is scientific, measurable light. When light "becomes visible" and reveals a message “contained” in glass, we speak of information-light. This light cannot be measured scientifically. Energy-light and information-light are manifested under the spell of glass architecture. The latter is what has shaped the boundaries of coloured glass, which is studied in this thesis. Architecture's different ways of using the infinite combinations of the absorptance, reflectance, transmittance and translucency of glass has affected the way we humans "see" the world. Sometimes we are "immersed" in it, since only an invisible, transparent boundary separates us from it: this use of glass has characterized a considerable part of 20th century architecture. In other cases, in order to "isolate" us from it, we have manipulated light and glass to build different worlds: the boundaries of glass "masked" by colour, mosaics, stained glass, screens and what we have called complex glazing, which plays a similar role to what Schiller attributed to the chorus in Greek tragedy, isolating it from the "real world" in order to maintain its poetic license. These are the boundaries that have built the viable dream of crystal architecture. These two approaches have been different ways of making same dream come true at different times in the history of architecture. The ability of glass to adapt to so many forms of manifestation, and interpretation, is what has given rise to the title of the thesis. Even in its most transparent facet, glass has one way or another always been a masking material in the broadest sense of the word: it is masked even when it invites transparency or becomes a mirror, triggering the illusion that it is not present. When man began to build glass boundaries, and even before, when he dreamed that he could build them, he condensed in them all the mythology, mysticism and epistemology concerning light and vision, which gave rise to a series of architectural archetypes. In the Byzantine church, light on or from mosaics created tenuous boundaries. In Gothic cathedrals, the light through the stained glass windows constructed radiant boundaries. In both cases the aim was to achieve, in an anagogical way, the Intelligible. In the 19th, the discovery of electricity and its use in architecture led to the production of dazzling boundaries, in this case employed in urban spaces. Earlier in the same century, the Gothic spirit had a short-lived revival, which in the early 20th century drew from crystalline expressionism in which anagogic light became secular. The dazzling urban space prefigured by this movement, present in cities since the early 20th century, was intensified in the mid-century with the emergence of screens, and since then it has spread unstoppably across the world. The recent emergence of complex glasses has made it possible to build boundaries on demand in glass with multiple properties, chosen at will and at whim at any time. Initially it was thought that the use of this glass as a wall could eventually become the panacea for the material problems related to energy-light, without needing to resort to "prosthesis" and thereby maintain the seductive smoothness of the facade. For now, that possibility seems remote, to say the least. In the realm of urban megascreens and information screens in general, now ubiquitous in our lives, complex glasses help to build the thick veils of illusion which, according to Lefebvre, serve to maintain capitalism. Like an inexorable metronome, in counterpoint, this ultimate state of technological development imposed by man's principle of economy has in fact always accompanied (again in the words of Lefebvre), the need to spend, play, fight, art, and party. Technology and art are part of the culture produced by society. As Levi-Strauss says, this culture imposes order. On the contrary, society, understood as a set of relationships amongst people, produces disorder. Progress, including that of architecture, arises from the balance between these two extremes. The glass boundaries analyzed in this thesis, which propitiate spaces for spirituality, pomp and spectacle or, from a different perspective, for the various manifestations of power: the church, the monarchy, the state and the market, have also emerged from the concomitance of order and disorder. One aspect of this disorder is the adventure that has inspired the individual genius of mystics, alchemists, surveyors, abbots, kings, inventors, poets and architects to explore, as Apollinaire says, vast, strange domains where flowering mystery offers itself to whoever wishes to pluck it, new fires, colours you have never seen before, a thousand intangible phantasms still awaiting reality.

VHDL-based system design of a cognitive sensorimotor loop (CSL) for haptic Human-Machine Interaction (HMI)

This document is a summary of the Bachelor thesis titled “VHDL-Based System Design of a Cognitive Sensorimotor Loop (CSL) for Haptic Human-Machine Interaction (HMI)” written by Pablo de Miguel Morales, Electronics Engineering student at the Universidad Politécnica de Madrid (UPM Madrid, Spain) during an Erasmus+ Exchange Program at the Beuth Hochschule für Technik (BHT Berlin, Germany). The tutor of this project is Dr. Prof. Hild. This project has been developed inside the Neurobotics Research Laboratory (NRL) in close collaboration with Benjamin Panreck, a member of the NRL, and another exchange student from the UPM Pablo Gabriel Lezcano. For a deeper comprehension of the content of the thesis, a deeper look in the document is needed as well as the viewing of the videos and the VHDL design. In the growing field of automation, a large amount of workforce is dedicated to improve, adapt and design motor controllers for a wide variety of applications. In the specific field of robotics or other machinery designed to interact with humans or their environment, new needs and technological solutions are often being discovered due to the existing, relatively unexplored new scenario it is. The project consisted of three main parts: Two VHDL-based systems and one short experiment on the haptic perception. Both VHDL systems are based on a Cognitive Sensorimotor Loop (CSL) which is a control loop designed by the NRL and mainly developed by Dr. Prof. Hild. The CSL is a control loop whose main characteristic is the fact that it does not use any external sensor to measure the speed or position of the motor but the motor itself. The motor always generates a voltage that is proportional to its angular speed so it does not need calibration. This method is energy efficient and simplifies control loops in complex systems. The first system, named CSL Stay In Touch (SIT), consists in a one DC motor system controller by a FPGA Board (Zynq ZYBO 7000) whose aim is to keep contact with any external object that touches its Sensing Platform in both directions. Apart from the main behavior, three features (Search Mode, Inertia Mode and Return Mode) have been designed to enhance the haptic interaction experience. Additionally, a VGA-Screen is also controlled by the FPGA Board for the monitoring of the whole system. This system has been completely developed, tested and improved; analyzing its timing and consumption properties. The second system, named CSL Fingerlike Mechanism (FM), consists in a fingerlike mechanical system controlled by two DC motors (Each controlling one part of the finger). The behavior is similar to the first system but in a more complex structure. This system was optional and not part of the original objectives of the thesis and it could not be properly finished and tested due to the lack of time. The haptic perception experiment was an experiment conducted to have an insight into the complexity of human haptic perception in order to implement this knowledge into technological applications. The experiment consisted in testing the capability of the subjects to recognize different objects and shapes while being blindfolded and with their ears covered. Two groups were done, one had full haptic perception while the other had to explore the environment with a plastic piece attached to their finger to create a haptic handicap. The conclusion of the thesis was that a haptic system based only on a CSL-based system is not enough to retrieve valuable information from the environment and that other sensors are needed (temperature, pressure, etc.) but that a CSL-based system is very useful to control the force applied by the system to interact with haptic sensible surfaces such as skin or tactile screens. RESUMEN. Este documento es un resumen del proyecto fin de grado titulado “VHDL-Based System Design of a Cognitive Sensorimotor Loop (CSL) for Haptic Human-Machine Interaction (HMI)” escrito por Pablo de Miguel, estudiante de Ingeniería Electrónica de Comunicaciones en la Universidad Politécnica de Madrid (UPM Madrid, España) durante un programa de intercambio Erasmus+ en la Beuth Hochschule für Technik (BHT Berlin, Alemania). El tutor de este proyecto ha sido Dr. Prof. Hild. Este proyecto se ha desarrollado dentro del Neurorobotics Research Laboratory (NRL) en estrecha colaboración con Benjamin Panreck (un miembro del NRL) y con Pablo Lezcano (Otro estudiante de intercambio de la UPM). Para una comprensión completa del trabajo es necesaria una lectura detenida de todo el documento y el visionado de los videos y análisis del diseño VHDL incluidos en el CD adjunto. En el creciente sector de la automatización, una gran cantidad de esfuerzo está dedicada a mejorar, adaptar y diseñar controladores de motor para un gran rango de aplicaciones. En el campo específico de la robótica u otra maquinaria diseñada para interactuar con los humanos o con su entorno, nuevas necesidades y soluciones tecnológicas se siguen desarrollado debido al relativamente inexplorado y nuevo escenario que supone. El proyecto consta de tres partes principales: Dos sistemas basados en VHDL y un pequeño experimento sobre la percepción háptica. Ambos sistemas VHDL están basados en el Cognitive Sesnorimotor Loop (CSL) que es un lazo de control creado por el NRL y cuyo desarrollador principal ha sido Dr. Prof. Hild. El CSL es un lazo de control cuya principal característica es la ausencia de sensores externos para medir la velocidad o la posición del motor, usando el propio motor como sensor. El motor siempre genera un voltaje proporcional a su velocidad angular de modo que no es necesaria calibración. Este método es eficiente en términos energéticos y simplifica los lazos de control en sistemas complejos. El primer sistema, llamado CSL Stay In Touch (SIT), consiste en un sistema formado por un motor DC controlado por una FPGA Board (Zynq ZYBO 7000) cuyo objetivo es mantener contacto con cualquier objeto externo que toque su plataforma sensible en ambas direcciones. Aparte del funcionamiento básico, tres modos (Search Mode, Inertia Mode y Return Mode) han sido diseñados para mejorar la interacción. Adicionalmente, se ha diseñado el control a través de la FPGA Board de una pantalla VGA para la monitorización de todo el sistema. El sistema ha sido totalmente desarrollado, testeado y mejorado; analizando su propiedades de timing y consumo energético. El segundo sistema, llamado CSL Fingerlike Mechanism (FM), consiste en un mecanismo similar a un dedo controlado por dos motores DC (Cada uno controlando una falange). Su comportamiento es similar al del primer sistema pero con una estructura más compleja. Este sistema no formaba parte de los objetivos iniciales del proyecto y por lo tanto era opcional. No pudo ser plenamente desarrollado debido a la falta de tiempo. El experimento de percepción háptica fue diseñado para profundizar en la percepción háptica humana con el objetivo de aplicar este conocimiento en aplicaciones tecnológicas. El experimento consistía en testear la capacidad de los sujetos para reconocer diferentes objetos, formas y texturas en condiciones de privación del sentido del oído y la vista. Se crearon dos grupos, en uno los sujetos tenían plena percepción háptica mientras que en el otro debían interactuar con los objetos a través de una pieza de plástico para generar un hándicap háptico. La conclusión del proyecto fue que un sistema háptico basado solo en sistemas CSL no es suficiente para recopilar información valiosa del entorno y que debe hacer uso de otros sensores (temperatura, presión, etc.). En cambio, un sistema basado en CSL es idóneo para el control de la fuerza aplicada por el sistema durante la interacción con superficies hápticas sensibles tales como la piel o pantallas táctiles.

O PAPEL DA COMUNICAÇÃO FACE A FACE NAS ORGANIZAÇÕES NO CONTEXTO DA SOCIEDADE MIDIATIZADA

Este estudo trata da comunicação face a face nas organizações sob diferentes abordagens teóricas. Considera a perspectiva da simultaneidade dos meios, já que as empresas utilizam diversos canais para dialogar com seus públicos de interesse. Leva em conta o fenômeno da midiatização, que reestrutura o modo como as pessoas se relacionam na sociedade contemporânea. O objetivo geral da pesquisa é sistematizar papeis potencialmente exercidos pela interação face a face e conhecer algumas circunstâncias que envolvem sua prática nas organizações. Por se tratar de uma tese teórica, a pesquisa bibliográfica se apresenta como um dos principais procedimentos metodológicos; análises de casos empíricos e um estudo de caso desenvolvido na Embrapa Pantanal constituem situações ilustrativas. Conclui-se que a comunicação face a face nas empresas ocorre de forma simultânea e combinada a outros canais de comunicação, porém, ela proporciona resultados práticos e filosóficos ainda pouco explorados. É rara a utilização estratégica de contatos presenciais como mecanismo para estabelecer relacionamentos, conhecer as reações alheias e ajustar a comunicação, aliar o discurso corporativo às práticas empresariais e avaliar o contexto onde se desenvolvem as interações, o que pode ser decisivo para a comunicação organizacional.

A phenotype-based screen for embryonic lethal mutations in the mouse

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP–TAF complexes perform distinct functions during development.

Synthesis and screening of small molecule libraries active in binding to DNA

Five synthetic combinatorial libraries of 2,080 components each were screened as mixtures for inhibition of DNA binding to two transcription factors. Rapid, solution-phase synthesis coupled to a gel-shift assay led to the identification of two compounds active at a 5- to 10-μM concentration level. The likely mode of inhibition is intercalation between DNA base pairs. The efficient deconvolution through sublibrary synthesis augurs well for the use of large mixtures of small, nonpeptide molecules in biological screens.

Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency. Because of the absence of poly(A)+ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.

Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events

Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1Δ null and skp1-4 mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.

Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice

Understanding nuclear receptor signaling in vivo would be facilitated by an efficient methodology to determine where a nuclear receptor is active. Herein, we present a feedback-inducible expression system in transgenic mice to detect activated nuclear receptor effector proteins by using an inducible reporter gene. With this approach, reporter gene induction is not limited to a particular tissue, and, thus, this approach provides the opportunity for whole-animal screens. Furthermore, the effector and reporter genes are combined to generate a single strain of transgenic mice, which enables direct and rapid analysis of the offspring. The system was applied to localize sites where the retinoic acid receptor ligand-binding domain is activated in vivo. The results identify previously discovered sources of retinoids in the embryo and indicate the existence of previously undiscovered regions of retinoic acid receptor signaling in vivo. Notably, the feedback-inducible nuclear-receptor-driven assay, combined with an independent in vitro assay, provides evidence for a site of retinoid synthesis in the isthmic mesenchyme. These data illustrate the potential of feedback-inducible nuclear-receptor-driven analyses for assessing in vivo activation patterns of nuclear receptors and for analyzing pharmacological properties of natural and synthetic ligands of potential therapeutic value.

Circadian clock-controlled genes isolated from Neurospora crassa are late night- to early morning-specific

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs preferentially accumulate during the late night to early morning, although they vary with respect to steady-state mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper metallothionein, providing the suggestion that not all clock-regulated genes in N. crassa are specifically involved in the development of conidia. This was supported by finding that half of the new ccgs, including cmt(ccg-12), are not transcriptionally induced by developmental or light signals. These data suggest a major role for the clock in the regulation of biological processes distinct from development.

Ol-Prx 3, a member of an additional class of homeobox genes, is unimodally expressed in several domains of the developing and adult central nervous system of the medaka (Oryzias latipes)

Large-scale genetic screens for mutations affecting early neurogenesis of vertebrates have recently been performed with an aquarium fish, the zebrafish. Later stages of neural morphogenesis have attracted less attention in small fish species, partly because of the lack of molecular markers of developing structures that may facilitate the detection of discrete structural alterations. In this context, we report the characterization of Ol-Prx 3 (Oryzias latipes-Prx 3). This gene was isolated in the course of a large-scale screen for brain cDNAs containing a highly conserved DNA binding region, the homeobox helix-three. Sequence analysis revealed that this gene belongs to another class of homeobox genes, together with a previously isolated mouse ortholog, called OG-12 [Rovescalli, A. C., Asoh, S. & Nirenberg, M. (1996) Proc. Natl. Acad. Sci. USA 93, 10691–10696] and with the human SHOX gene [Rao, E., Weiss, B., Fukami, M., Rump, A., Niesler, B., et al. (1997) Nat. Genet. 16, 54–62], thought to be involved in the short-stature phenotype of Turner syndrome patients. These three genes exhibit a moderate level of identity in the homeobox with the other genes of the paired-related (PRX) gene family. Ol-Prx 3, as well as the PRX genes, are expressed in various cartilaginous structures of head and limbs. These genes might thus be involved in common regulatory pathways during the morphogenesis of these structures. Moreover, this paper reports a complex and monophasic pattern of Ol-Prx 3 expression in the central nervous system, which differs markedly from the patterns reported for the PRX genes, Prx 3 excluded: this gene begins to be expressed in a variety of central nervous system territories at late neurula stage. Strikingly, it remains turned on in some of the derivatives of each territory during the entire life of the fish. We hope this work will thus help identify common features for the PRX 3 family of homeobox genes.

Genetically targeted cell disruption in Caenorhabditis elegans

The elimination of identified cells is a powerful tool for investigating development and system function. Here we report on genetically mediated cell disruption effected by the toxic Caenorhabditis elegans mec-4(d) allele. We found that ectopic expression of mec-4(d) in the nematode causes dysfunction of a wide range of nerve, muscle, and hypodermal cells. mec-4(d)-mediated toxicity is dependent on the activity of a second gene, mec-6, rendering cell disruption conditionally dependent on genetic background. We describe a set of mec-4(d) vectors that facilitate construction of cell-specific disruption reagents and note that genetic cell disruption can be used for functional analyses of specific neurons or neuronal classes, for confirmation of neuronal circuitry, for generation of nematode populations lacking defined classes of functional cells, and for genetic screens. We suggest that mec-4(d) and/or related genes may be effective general tools for cell inactivation that could be used toward similar purposes in higher organisms.

P element insertion-dependent gene activation in the Drosophila eye

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene’s function when other approaches are not illuminating. Here we report that an eye-specific, enhancer–promoter present in the P element expression vector pGMR is able to drive high level expression in the eye of genes near the site of P element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea we carried out two overexpression screens for genes that function to regulate cell death. We screened for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a reaper overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5′ to hid, a potent inducer of apoptosis, and insertions 5′ to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the P element insertion new misexpression vectors were created. A screen with one of these vectors identified eagle as a suppressor of a rough eye phenotype associated with overexpression of an activated Ras1 gene.

Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.