A procedure for testing padding materials in fruit packing lines.

Padding materials are commonly used in fruit packing lines with the objective of diminishing impact damage in postharvest handling. Two sensors, instrumented sphere IS 100 and impact tester, have been compared to analyze the performance of six different padding materials used in Spanish fruit packing lines. Padding materials tested have been classified according to their capability to decrease impact intensities inflicted to fruit in packing lines. A procedure to test padding materials has been developed for "Golden" apples. Its basis is a logistic regression to predict bruise probability in fruit. The model combines two kinds of parameters: padding material parameters measured with IS, and fruit properties.

A new procedure for race analysis in swimming based on individual distance measurements

biomecanica de la natación

LED bulb lamps technical specification and testing procedure for solar home systems.

With the consolidation of the new solid state lighting LEOs devices, te5t1n9 the compliance 01 lamps based on this technology lor Solar Home Systems (SHS) have been analyzed. The definition of the laboratory procedures to be used with final products 15 a necessary step in arder to be able to assure the quality of the lamps prior to be installed [1]. As well as with CFL technology. particular attention has been given to simplicity and technical affordability in arder to facilitate the implementation of the test with basie and simple laboratory too15 even on the same SHS electrification program locations. The block of test procedures has been applied to a set of 14 low-cost lamps. They apply to lamp resistance, reliability and performance under normal, extreme and abnormal operating conditions as a simple but complete quality meter tool 01 any LEO bulb.

Quantification of impacts in artistic gymnastics with accelerometry: an approximation

Intensity and volume of training in Artisti Gymnastics are increasing as the sooner athlete's age of incorporation creating some disturbance in them.

LED bulbs technical specification and testing procedure for solar home systems

The definition of technical specifications and the corresponding laboratory procedures are necessary steps in order to assure the quality of the devices prior to be installed in Solar Home Systems (SHS). To clarify and unify criteria a European project supported the development of the Universal Technical Standard for Solar Home Systems (UTSfSHS). Its principles were to generate simple and affordable technical requirements to be optimized in order to facilitate the implementation of tests with basic and simple laboratory tools even on the same SHS electrification program countries. These requirements cover the main aspects of this type of installations and its lighting chapter was developed based on the most used technology at that time: fluorescent tubes and CFLs. However, with the consolidation of the new LED solid state lighting devices, particular attention is being given to this matter and new procedures are required. In this work we develop a complete set of technical specifications and test procedures that have been designed within the frame of the UTSfSHS, based on an intense review of the scientific and technical publications related to LED lighting and their practical application. They apply to lamp reliability, performance and safety under normal, extreme and abnormal operating conditions as a simple but complete quality meter tool for any LED bulb.

Contextualización teórica del acto pedagógico en la enseñanza y el aprendizaje del proyecto arquitectónico: el caso de la E.T.S.A.M.

El taller de proyectos constituye el núcleo de la enseñanza arquitectónica. Estudiar sus procesos educativos en la actualidad implica la contextualización teórica del acto educativo y la revisión histórica de la evolución de su estructura social. Esta estructura, es heredera de la larga tradición del taller donde los maestros de obra, artesanos, arquitectos y artistas, se ocupaban desde siempre de la enseñanza de la construcción, de la artesanía, del diseño arquitectónico y del arte. Los aprendices se sometían a la autoridad de sus maestros y pasaban horas practicando, produciendo y aprendiendo junto a ellos. Con la aparición de las primeras Academias de arte y posteriormente de arquitectura, se evidenció un progresivo interés de los Estados hacía los productos artísticos y arquitectónicos. La consideración de las artes y de la arquitectura como proyecto estatal, supuso la construcción lenta, pero consolidable, de un proyecto educativo paralelo, coexistiendo simultáneamente con los talleres de los maestros, pero sin posibilidad de integración, hasta mucho más tarde. La “teoría”, que es de lo que se ocupaba la academia, con la “práctica” que es lo que se desarrollaba en el taller, no encontraban fácilmente la manera de encajarse y complementarse mutuamente en un proyecto educativo común. Las concepciones educativas de ambos, afrontaban la enseñanza y el aprendizaje desde puntos de vista también diferentes; mientras la Academia representaba el conocimiento validado y explicitado, en el taller se trabajaba con un conocimiento tácito e implícito. En la práctica artística del taller era donde se producía el aprendizaje mientras que en la Academia es donde se validaba. Esta estructura llegó en muchas ocasiones a situaciones extremas, no siendo casual que las más grandes crisis registradas en la historia de la enseñanza de las artes, coincidieran con un aumento de la distancia entre estas dos “instituciones”, talleres y academias. Por otra parte, parece que cualquier proyecto o concepto innovador, se ha fundado sobre la redistribución de estos equilibrios perdidos. En dicho contexto, en el campo de la educación y especialmente en el siglo XX, surge un debate que se estructura en base a los fines de la educación, contemplando dos posturas bien diferenciadas. Una de ellas sostiene como fin primordial de la educación, el desarrollo de la conciencia y la reciprocidad social del individuo. La otra, fija como fin el desarrollo de su singularidad. La búsqueda del equilibrio entre ambas, parte del interés por fomentar el crecimiento de lo que cada ser humano posee de individual, armonizándolo con la unidad orgánica del grupo social al que pertenece (Read 2010, 33). Sobre esta tensión se han basado muchos de los discursos pedagógicos y especialmente los aquí usados. La estructura social en los talleres de proyectos arquitectónicos, presenta hoy día una máxima integración entre las dos instituciones, el taller y la Academia, tanto a nivel del espacio, donde tiene lugar la enseñanza, como a nivel conceptual y pedagógico. Los talleres de proyectos poseen un formato de enseñanza y aprendizaje que constituye un paradigma (Schön, 2008) no solo dentro, sino también fuera del campo arquitectónico. Bajo este formato se complementa el aprendizaje práctico con el teórico y la producción, con la validación del conocimiento. Aunque tal estructura pedagógica presenta importantes variaciones entre unas escuelas de arquitectura y otras, los principales procesos que tienen lugar, son lo suficientemente similares, como para poder ser examinados desde una perspectiva común. Esta investigación, estudia el taller de proyectos desde un aspecto pedagógico, que contempla tanto los discursos educativos, como la historia de la evolución del taller como constructo social. El análisis se estructura sobre los elementos fundantes del acto didáctico: un sujeto que aprende, un sujeto que enseña, un método, la estrategia o procedimiento a través del que se enseña, un contenido y el propio acto docente (Sánchez Cerezo, 1994, 530). Además, se han añadido otros dos elementos que se consideran fundamentales para llevar a cabo el estudio: el contexto de la enseñanza, tanto el tangible como el intangible y la evaluación de la enseñanza y del aprendizaje. El caso de estudio de la presente investigación se sitúa en la Escuela de Arquitectura de Madrid en la actualidad. Sin embargo, no se pretende generar un retrato exacto de esta institución sino utilizarla como ejemplo principal en el desarrollo de los capítulos del método, contenido, acto docente y contexto, en los que también se introducen ejemplos de otras escuelas de arquitectura que amplían los argumentos presentados que constituyen la contextualización teórica del acto pedagógico en los talleres de proyectos arquitectónicos. ABSTRACT Design studio constitutes the core of architectural education. To study its current educational processes involves a theoretical approach of its educational praxis and an historic revision of how its social structure evolved. This structure is inherited from the long tradition of the workshop in which master masons, craftsmen, architects and artists have always been in charge of teaching construction, crafts, architectural design and art. Apprentices were subjected to the authority of their teachers and spent hours practicing, producing and learning along with them. With the establishment of the first Academies of Art and later of Architecture, the interest of the State in artistic and architectural products started growing. The understanding of arts and architecture as a state project entailed the slow, but robust development of a parallel education project. This project coexisted with the masters’ workshops, without the possibility of integration between two, until much later. It was difficult to find a way to synthesize academic “theory” with workshop “practice”. The workshops’ and the Academy's conception about teaching and learning differed significantly. While the Academy represented a verified and explicit knowledge, the workshop worked with a tacit and implicit knowledge. The workshops produced education through artistic practice, while the the Academy organized and verified knowledge. This dual framework has on occasions reached extremes. It is no accident that the biggest known crises in the history of arts education coincide with an increase in the distance between these two "institutions", the workshops and Academies. Furthermore, it seems that most innovative concepts or projects have been founded on restoring the lost balance between the two. In this context, in the field of education, and especially during the 20th century, a debate that contemplated the purpose of education and resulted in two quite differentiated approaches, emerged,. One position claims as the primary purpose of education the development of social awareness and mutuality in individuals. The other approach sets as a purpose developing each student's uniqueness. The quest for the right balance between these two positions is based on the assumption that the general purpose of education is to foster the growth of what is individual in each human being, at the same time harmonizing the individuality thus educed with the organic unity of the social group to which the individual belongs (Read, 2010, 33). This tension forms the basis for many pedagogical discourses, especially the ones utilized in this dissertation. The social structure of architecture studios today demonstrates a very high level of integration between the two institutions, the workshop and the Academy, both in terms of space —where the teaching takes place— as well as on a conceptual and pedagogical level. Architecture studios today have developed a format for teaching and learning that has established a paradigm (Schön, 2008) , not only in architecture, but also in other fields. Under this paradigm, practical and theoretical learning, as well as production and verification of knowledge, complement each other. And although this pedagogical structure presents important variations among different schools of architecture, the principal processes that take place in the studio are sufficiently similar so as to be examined from a common perspective. This research examines the architecture studio from a pedagogical point of view, that takes into account both the educational discourses, as well as the historical evolution of the workshop as a social structure. The analysis presented here is structured on the fundamentals of the teaching act: an individual learning, an individual teaching, a method, strategy or procedure for teaching and learning, the content and the teaching act itself (Sánchez Cerezo, 1994, 530). Two extra elements that were considered essential for carrying out this study have also been added: the context in which teaching takes place, tangible as well as intangible, and the evaluation of teaching and learning. The Madrid School of Architecture in the present day served as a case study. However the aim is not to generate an accurate portrayal of this school but to use it as the principal example for the development of the chapters of method, content, teaching act and context. In addition to that, examples from other schools of architecture are introduced in order to further the presented arguments that constitute the theoretical contextualization of the pedagogical act in architecture studios.

Evaluation of the surface free energy of plant surfaces: toward standardizing the procedure

Plant surfaces have been found to have a major chemical and physical heterogeneity and play a key protecting role against multiple stress factors. During the last decade, there is a raising interest in examining plant surface properties for the development of biomimetic materials. Contact angle measurement of different liquids is a common tool for characterizing synthetic materials, which is just beginning to be applied to plant surfaces. However, some studies performed with polymers and other materials showed that for the same surface, different surface free energy values may be obtained depending on the number and nature of the test liquids analyzed, materials' properties, and surface free energy calculation methods employed. For 3 rough and 3 rather smooth plant materials, we calculated their surface free energy using 2 or 3 test liquids and 3 different calculation methods. Regardless of the degree of surface roughness, the methods based on 2 test liquids often led to the under- or over-estimation of surface free energies as compared to the results derived from the 3-Liquids method. Given the major chemical and structural diversity of plant surfaces, it is concluded that 3 different liquids must be considered for characterizing materials of unknown physico-chemical properties, which may significantly differ in terms of polar and dispersive interactions. Since there are just few surface free energy data of plant surfaces with the aim of standardizing the calculation procedure and interpretation of the results among for instance, different species, organs, or phenological states, we suggest the use of 3 liquids and the mean surface tension values provided in this study.

Haptically assisted connection procedure for the reconstruction of dendritic spines

Dendritic spines are thin protrusions that cover the dendritic surface of numerous neurons in the brain and whose function seems to play a key role in neural circuits. The correct segmentation of those structures is difficult due to their small size and the resulting spines can appear incomplete. This paper presents a four-step procedure for the complete reconstruction of dendritic spines. The haptically driven procedure is intended to work as an image processing stage before the automatic segmentation step giving the final representation of the dendritic spines. The procedure is designed to allow both the navigation and the volume image editing to be carried out using a haptic device. A use case employing our procedure together with a commercial software package for the segmentation stage is illustrated. Finally, the haptic editing is evaluated in two experiments; the first experiment concerns the benefits of the force feedback and the second checks the suitability of the use of a haptic device as input. In both cases, the results shows that the procedure improves the editing accuracy.

A procedure to track vibration modes under changing external factors: application to a pedestrian bridge

A low-cost vibration monitoring system has been developed and installed on an urban steel- plated stress-ribbon footbridge. The system continuously measures: the acceleration (using 18 triaxial MEMS accelerometers distributed along the structure), the ambient temperature and the wind velocity and direction. Automated output-only modal parameter estimation based on the Stochastic Subspace Identification (SSI) is carried out in order to extract the modal parameters, i.e., the natural frequencies, damping ratios and modal shapes. Thus, this paper analyzes the time evolution of the modal parameters over a whole-year data monitoring. Firstly, for similar environmental/operational factors, the uncertainties associated to the time window size used are studied and quantified. Secondly, a methodology to track the vibration modes has been established since several of them with closely-spaced natural frequencies are identified. Thirdly, the modal parameters have been correlated against external factors. It has been shown that this stress-ribbon structure is highly sensitive to temperature variation (frequency changes of more than 20%) with strongly seasonal and daily trends

Criterios de restauración, intervención y revitalización del patrimonio industrial : la fábrica de gas de San Paolo en Roma

La tesis está centrada en el patrimonio industrial, y trata de ahondar en los temas enunciados en el título, Criterios de restauración, intervención y revitalización del patrimonio industrial (capítulo 1). La fábrica de gas de San Paolo en Roma (capítulo 2): En el primer capítulo, se profundiza en los criterios institucionalizados de actuación en el patrimonio, pero también se habla sobre la singularidad, los valores originales, estratificados en cada construcción y su contexto. Ello resume la intención de la doctoranda al entender la necesidad del análisis de cada obra en modo profundo, concreto y abierto a novedosos acercamientos, antes de su transformación. Al hablar de intervención frente a restauración, se aborda la conocida polémica que plantea el modo de acercarse al legado histórico. Parece necesario enunciar cómo se consideran en el trabajo estos términos. Mientras la restauración (como diría Cesare Brandi, constituye el momento metodológico del reconocimiento de la obra de arte en su consistencia física y en su doble polaridad estética e histórica, en orden a su transmisión al futuro) es una acción más restrictiva, que implica atención suma a la recuperación y conservación de los valores significativos y únicos del bien, en comunicación con un (posible) equipo interdisciplinar; por intervención se entiende aquí un desarrollo más personal y libre por parte del proyectista, en el que se produce un proceso dialéctico entre la preexistencia y el posicionamiento crítico adoptado a la hora de dar un obligado nuevo uso a ese bien. Es decir, el contraste está en el diferente reconocimiento de la materialidad física y sus significados. En torno a la restauración/intervención, se analizan las perspectivas de actuación en España e Italia, contrastando factores como la formación del arquitecto o el entorno laboral-cultural, y utilizando el método analítico comparativo de casos. Habría un tercer concepto: el de revitalización del patrimonio industrial, Éste hace referencia a su transformación, a sus usos más comunes y compatibles, a la redimensión artística que ha experimentado la arquitectura industrial gracias al arte contemporáneo y a su relación con la Modernidad. Por último, se enuncia en el título el caso de estudio, la Fábrica, que ocupa el segundo capítulo. Se trata de un enorme conjunto industrial singular semi abandonado en el confín del centro histórico, cuyo protagonista es un gran gasómetro, que la autora ha tenido la oportunidad de descubrir en su estancia en la Ciudad Eterna. Este barrio constituyó el primer y único sector industrial de la Roma moderna a inicios del siglo XX. Por varias causas históricas y voluntad política, la ciudad prácticamente no tuvo más desarrollo industrial que el área mencionada, donde se implantaron servicios y actividades de gran transformación (Matadero, Mercados Generales, Almacenes Fluviales, Central Montemartini o esta fábrica). A partir de 1920, se empezó a construir un tejido productivo que la llevó de la situación de posguerra al milagro económico. Frente al mito de la Roma monumental o de la Roma cotidiana, aparece el aspecto industrial como mito de la contemporaneidad. Sin embargo, esta ciudad es demasiado antigua como para convertirse en moderna, y su proceso industrial en contraste con su larga historia resulta breve. La inmediata relación de la fase industrial romana con el resultado de este desarrollo viene identificada con las instalaciones señaladas, más significativas por su excepcionalidad que por la duración del momento irrepetible que representan. Su presencia física perdura a pesar de su abandono, aisladas tras su muro perimetral y casi desconocidas. Esto las hace apetecibles para la especulación inmobiliaria y resultan un caso de estudio ideal para realizar un Plan de Intervención Global, en el que determinar una serie de intervenciones necesarias, usos compatibles y directrices para la ordenación del conjunto, coherentes con los conceptos manejados en el primer capítulo. ABSTRACT The thesis focuses on industrial heritage, delving into the topics listed in its title, Criteria for the restoration, intervention and revitalisation of industrial heritage (chapter 1). The San Paolo gas factory in Rome (chapter 2): Chapter one elaborates on the official criteria to take action on heritage, but also deals with the singularity and original values unique to each construction and its context. This underlines the PhD candidate’s intentions by understanding the need for an in-depth and specific analysis, open to novel approaches to each site before its transformation. When speaking of intervention, as opposed to restoration, we address the well-known controversy that stems from the different ways of approaching historical heritage. It seems necessary to explain how these terms are interpreted in this piece of work. Whereas restoration (as Cesare Brandi would say, constitutes the methodological moment in which the work of art is appreciated in its material form and in its historical and aesthetic duality, with a view to transmitting it to the future) is a more restrictive action, demanding the utmost attention to the recovery and preservation of the piece of heritage’s defining and exceptional values, (ideally) in communication with a interdisciplinary team. By intervention, we hereby understand a more personal and free procedure carried out by the designer in which a dialectical process takes place between preexistence and the critical stance taken in order to give that good a much needed new use. That is, contrast lies in the different recognition of physical materiality and its meanings. As for the restoration/intervention dilemma, the different courses of action followed in Spain and Italy will be analysed, contrasting the architects’ training, the labourcultural environment and taking an analytical comparative case study method. There is a third concept: the revitalisation of industrial heritage, which refers to its transformation, its most common and compatible uses, the artistic redimensioning that industrial architecture has experienced thanks to contemporary art, and its relationship with Modernity. Lastly, the title refers to the case study, i.e., the analysis of the Factory, which makes up chapter 2. It is an enormous and semiabandoned peerless industrial complex on the edge of the historical city centre whose main landmark is a huge gasometer, which the author had the chance to discover during her stay in the Eternal City. This district made up the first and only industrial sector in the modern Rome of the early 20th century. Due to a number of reasons, both historical and political, barely no further industrial development took place in the city beyond the mentioned area, where processing services and activities were set up (Slaughterhouse, General Markets, Riverside Warehouses, Montemartini Power Plant or the said factory). In the 1920s began the construction of the productive fabric that raised the city from its postwar plight to the economic miracle. As opposed to the myth of monumental Rome and daily-life Rome, this industrial Rome rose as contemporaneity’s myth. However, this city is too ancient to become modern, and its industrial process strikes us as brief when confronted with its long history. The close relationship between Rome’s industrial stage and the consequences of this development is best represented by the aforementioned facilities, more relevant due to their uniqueness than the length of the once-in-a-lifetime moment they represent. Their physical presence lives on despite abandonment and isolation, as they stand vastly unknown behind their perimeter walls. This renders them appealing to property speculators, becoming an ideal case study for the creation of a Global Intervention Plan in which to determine a series of necessary actions, compatible uses and directives, all of them consistent with the concepts dealt with in chapter 1. RIASSUNTO La tesi è focalizzata sul patrimonio industriale e cerca di approfondire le tematiche contenute nel titolo, Criteri di restauro, intervento e rivitalizzazione del patrimonio industriale (capitolo 1). La Fabbrica del gas di San Paolo a Roma (capitolo 2): Nel primo capitolo vengono presi in esame i criteri istituzionalizzati di attuazione nel patrimonio, ma si parla anche della unicità, i valori originali, stratificati in ogni costruzione ed il suo contesto. Questo riassume l’intenzione della dottoranda di capire la necessità dell’analisi di ogni opera in modo profondo, concreto e aperto a nuovi approcci, prima della sua trasformazione. Nel parlare di intervento versus restauro, si affronta la famosa polemica che considera il modo di avicinarsi all’eredità storica. Appare necessario precisare come questi termini sono considerati in questo lavoro. Mentre il restauro (come direbbe Cesare Brandi, costituisce il momento metodologico del riconoscimento dell’opera d’arte nella sua consistenza fisica e nella duplice polarità estetica e storica, in vista della sua trasmissione nel futuro) è un’azione più restrittiva, e quindi implica massima attenzione al recupero e conservazione dei valori significativi ed unici del bene, in comunicazione con una (eventuale) squadra interdisciplinare; per intervento si intende qui un processo più personale e libero del progettista, nel quale si produce uno sviluppo dialettico tra la preesistenza e la posizione critica adottata quando si deve dare un nuovo uso di quel bene. Vale a dire che il contrasto risiede nel diverso riconoscimento della materialità fisica e i loro significati. Intorno al restauro / intervento, si analizzano le prospettive di attuazione in Spagna e Italia, contrastando diversi fattori come la formazione dell’architetto o l’ambiente lavorativo-culturale, ed impiegando il metodo analitico comparativo dei casi. Ci sarebbe un terzo concetto: quello della rivitalizzazione del patrimonio industriale. Questo fa riferimento alla sua trasformazione, ai suoi usi più comuni e compatibili, al ridimensionamento artistico vissuto dall’archeologia industriale grazie all’arte contemporanea ed al suo rapporto con la Modernità. In ultimo, si enuncia nel titolo il caso di studio, ovvero l’analisi della Fabbrica, che occupa il secondo capitolo. Si tratta di un enorme complesso industriale semi-abbandonato al confine con il centro storico, il cui protagonista è un grande gasometro, che l’autrice ha avuto modo di scoprire durante il suo soggiorno nella Città Eterna. Questo quartiere costituì il primo e unico moderno settore industriale della Roma moderna all’inizio del Novecento. Per diverse ragioni storiche e volontà politiche, la città praticamente non ha subito ulteriore sviluppo industriale oltre l’area citata, dove si impiantarono servizi e attività di grande trasformazione (Mattatoio, Mercati Generali, Magazzini Generali, Centrale Montemartini o questa fabbrica). Dal 1920, si inizió a costruire un tessuto prodottivo che portò la città dalla situazione post-bellica al miracolo economico. Di fronte al mito della Roma monumentale o della Roma quotidiana, appare questa Roma industriale come mito della contemporaneità. Tuttavia, questa città è troppo antica per diventare moderna, ed il suo processo industriale in contrasto con la sua lunga storia risulta breve. Il rapporto diretto della fase industriale romana con il risultato di questo sviluppo viene identificato con gli impianti segnalati, più significativi per la loro unicità che per la durata del momento irripetibile che rappresentano. La loro presenza fisica perdura nonostante l’abbandono, isolati dietro il loro muro di cinta e quasi sconosciuti. Questo li rende auspicabile per la speculazione immobiliare e sono un caso ideale per eseguire un Piano di Intervento Globale, che determini una serie di interventi necessari, usi compatibili e linee guida per la pianificazione del tutto, in linea con i concetti utilizzati nel primo capitolo.

The deferral option in the collective dismissal procedure. La opción de aplazamiento en los expedientes de regulación de empleo.

The work in this paper focuses on the integration of the real options theory for organizational projects in the management of Human Resources, and particularly on the inclusion of the deferral option in collective dismissal procedures. This option has been studied and developed to be applied to ?Expediente de regulación de empleo?, which is the legal form existing in Spain for the collective termination of employment contracts and which organizations turn to when confronted with a negative financial situation, as a way of maintaining their viability. Two main issues which it is hoped to resolve are examined: the search for a source of uncertainty to make the deferral option viable for this type of projects, and the development of a procedure to obtain the value of the option and therefore facilitate decision making. The analysis performed has enabled us to state that the volatility of demand is the source of uncertainty that makes the option viable. The procedure developed by the binomial tree, which is determined by the evolution of demand, is the tool that enables the value of the option to be found.

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens

Reciprocal subtraction differential RNA display: An efficient and rapid procedure for isolating differentially expressed gene sequences

A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired 125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

A general procedure for locating and analyzing protein-binding sequence motifs in nucleic acids

In the last decade, two tools, one drawn from information theory and the other from artificial neural networks, have proven particularly useful in many different areas of sequence analysis. The work presented herein indicates that these two approaches can be joined in a general fashion to produce a very powerful search engine that is capable of locating members of a given nucleic acid sequence family in either local or global sequence searches. This program can, in turn, be queried for its definition of the motif under investigation, ranking each base in context for its contribution to membership in the motif family. In principle, the method used can be applied to any binding motif, including both DNA and RNA sequence families, given sufficient family size.