On the harmonic analysis of cup anemometer rotation speed: A principle to monitor performance and maintenance status of rotating meteorological sensors

The calibration results of one anemometer equipped with several rotors, varying their size, were analyzed. In each case, the 30-pulses pert turn output signal of the anemometer was studied using Fourier series decomposition and correlated with the anemometer factor (i.e., the anemometer transfer function). Also, a 3-cup analytical model was correlated to the data resulting from the wind tunnel measurements. Results indicate good correlation between the post-processed output signal and the working condition of the cup anemometer. This correlation was also reflected in the results from the proposed analytical model. With the present work the possibility of remotely checking cup anemometer status, indicating the presence of anomalies and, therefore, a decrease on the wind sensor reliability is revealed.

Application of PZT and FBG impedance sensors for structural health monitoring of reinforced concrete beams strengthened with FRP composite materials

Esta Tesis tiene como objetivo principal el desarrollo de métodos de identificación del daño que sean robustos y fiables, enfocados a sistemas estructurales experimentales, fundamentalmente a las estructuras de hormigón armado reforzadas externamente con bandas fibras de polímeros reforzados (FRP). El modo de fallo de este tipo de sistema estructural es crítico, pues generalmente es debido a un despegue repentino y frágil de la banda del refuerzo FRP originado en grietas intermedias causadas por la flexión. La detección de este despegue en su fase inicial es fundamental para prevenir fallos futuros, que pueden ser catastróficos. Inicialmente, se lleva a cabo una revisión del método de la Impedancia Electro-Mecánica (EMI), de cara a exponer sus capacidades para la detección de daño. Una vez la tecnología apropiada es seleccionada, lo que incluye un analizador de impedancias así como novedosos sensores PZT para monitorización inteligente, se ha diseñado un procedimiento automático basado en los registros de impedancias de distintas estructuras de laboratorio. Basándonos en el hecho de que las mediciones de impedancias son posibles gracias a una colocación adecuada de una red de sensores PZT, la estimación de la presencia de daño se realiza analizando los resultados de distintos indicadores de daño obtenidos de la literatura. Para que este proceso sea automático y que no sean necesarios conocimientos previos sobre el método EMI para realizar un experimento, se ha diseñado e implementado un Interfaz Gráfico de Usuario, transformando la medición de impedancias en un proceso fácil e intuitivo. Se evalúa entonces el daño a través de los correspondientes índices de daño, intentando estimar no sólo su severidad, sino también su localización aproximada. El desarrollo de estos experimentos en cualquier estructura genera grandes cantidades de datos que han de ser procesados, y algunas veces los índices de daño no son suficientes para una evaluación completa de la integridad de una estructura. En la mayoría de los casos se pueden encontrar patrones de daño en los datos, pero no se tiene información a priori del estado de la estructura. En este punto, se ha hecho una importante investigación en técnicas de reconocimiento de patrones particularmente en aprendizaje no supervisado, encontrando aplicaciones interesantes en el campo de la medicina. De ahí surge una idea creativa e innovadora: detectar y seguir la evolución del daño en distintas estructuras como si se tratase de un cáncer propagándose por el cuerpo humano. En ese sentido, las lecturas de impedancias se emplean como información intrínseca de la salud de la propia estructura, de forma que se pueden aplicar las mismas técnicas que las empleadas en la investigación del cáncer. En este caso, se ha aplicado un algoritmo de clasificación jerárquica dado que ilustra además la clasificación de los datos de forma gráfica, incluyendo información cualitativa y cuantitativa sobre el daño. Se ha investigado la efectividad de este procedimiento a través de tres estructuras de laboratorio, como son una viga de aluminio, una unión atornillada de aluminio y un bloque de hormigón reforzado con FRP. La primera ayuda a mostrar la efectividad del método en sencillos escenarios de daño simple y múltiple, de forma que las conclusiones extraídas se aplican sobre los otros dos, diseñados para simular condiciones de despegue en distintas estructuras. Demostrada la efectividad del método de clasificación jerárquica de lecturas de impedancias, se aplica el procedimiento sobre las estructuras de hormigón armado reforzadas con bandas de FRP objeto de esta tesis, detectando y clasificando cada estado de daño. Finalmente, y como alternativa al anterior procedimiento, se propone un método para la monitorización continua de la interfase FRP-Hormigón, a través de una red de sensores FBG permanentemente instalados en dicha interfase. De esta forma, se obtienen medidas de deformación de la interfase en condiciones de carga continua, para ser implementadas en un modelo de optimización multiobjetivo, cuya solución se haya por medio de una expansión multiobjetivo del método Particle Swarm Optimization (PSO). La fiabilidad de este último método de detección se investiga a través de sendos ejemplos tanto numéricos como experimentales. ABSTRACT This thesis aims to develop robust and reliable damage identification methods focused on experimental structural systems, in particular Reinforced Concrete (RC) structures externally strengthened with Fiber Reinforced Polymers (FRP) strips. The failure mode of this type of structural system is critical, since it is usually due to sudden and brittle debonding of the FRP reinforcement originating from intermediate flexural cracks. Detection of the debonding in its initial stage is essential thus to prevent future failure, which might be catastrophic. Initially, a revision of the Electro-Mechanical Impedance (EMI) method is carried out, in order to expose its capabilities for local damage detection. Once the appropriate technology is selected, which includes impedance analyzer as well as novel PZT sensors for smart monitoring, an automated procedure has been design based on the impedance signatures of several lab-scale structures. On the basis that capturing impedance measurements is possible thanks to an adequately deployed PZT sensor network, the estimation of damage presence is done by analyzing the results of different damage indices obtained from the literature. In order to make this process automatic so that it is not necessary a priori knowledge of the EMI method to carry out an experimental test, a Graphical User Interface has been designed, turning the impedance measurements into an easy and intuitive procedure. Damage is then assessed through the analysis of the corresponding damage indices, trying to estimate not only the damage severity, but also its approximate location. The development of these tests on any kind of structure generates large amounts of data to be processed, and sometimes the information provided by damage indices is not enough to achieve a complete analysis of the structural health condition. In most of the cases, some damage patterns can be found in the data, but none a priori knowledge of the health condition is given for any structure. At this point, an important research on pattern recognition techniques has been carried out, particularly on unsupervised learning techniques, finding interesting applications in the medicine field. From this investigation, a creative and innovative idea arose: to detect and track the evolution of damage in different structures, as if it were a cancer propagating through a human body. In that sense, the impedance signatures are used to give intrinsic information of the health condition of the structure, so that the same clustering algorithms applied in the cancer research can be applied to the problem addressed in this dissertation. Hierarchical clustering is then applied since it also provides a graphical display of the clustered data, including quantitative and qualitative information about damage. The performance of this approach is firstly investigated using three lab-scale structures, such as a simple aluminium beam, a bolt-jointed aluminium beam and an FRP-strengthened concrete specimen. The first one shows the performance of the method on simple single and multiple damage scenarios, so that the first conclusions can be extracted and applied to the other two experimental tests, which are designed to simulate a debonding condition on different structures. Once the performance of the impedance-based hierarchical clustering method is proven to be successful, it is then applied to the structural system studied in this dissertation, the RC structures externally strengthened with FRP strips, where the debonding failure in the interface between the FRP and the concrete is successfully detected and classified, proving thus the feasibility of this method. Finally, as an alternative to the previous approach, a continuous monitoring procedure of the FRP-Concrete interface is proposed, based on an FBGsensors Network permanently deployed within that interface. In this way, strain measurements can be obtained under controlled loading conditions, and then they are used in order to implement a multi-objective model updating method solved by a multi-objective expansion of the Particle Swarm Optimization (PSO) method. The feasibility of this last proposal is investigated and successfully proven on both numerical and experimental RC beams strengthened with FRP.

Evaluating the quality of riparian forest vegetation: The Riparian Forest Evaluation (RFV) index

Aim of study: This paper presents a novel index, the Riparian Forest Evaluation (RFV) index, for assessing the ecological condition of riparian forests. The status of riparian ecosystems has global importance due to the ecological and social benefits and services they provide. The initiation of the European Water Framework Directive (2000/60/CE) requires the assessment of the hydromorphological quality of natural channels. The Directive describes riparian forests as one of the fundamental components that determine the structure of riverine areas. The RFV index was developed to meet the aim of the Directive and to complement the existing methodologies for the evaluation of riparian forests. Area of study: The RFV index was applied to a wide range of streams and rivers (170 water bodies) inSpain. Materials and methods: The calculation of the RFV index is based on the assessment of both the spatial continuity of the forest (in its three core dimensions: longitudinal, transversal and vertical) and the regeneration capacity of the forest, in a sampling area related to the river hydromorphological pattern. This index enables an evaluation of the quality and degree of alteration of riparian forests. In addition, it helps to determine the scenarios that are necessary to improve the status of riparian forests and to develop processes for restoring their structure and composition. Main results: The results were compared with some previous tools for the assessment of riparian vegetation. The RFV index got the highest average scores in the basins of northernSpain, which suffer lower human influence. The forests in central and southern rivers got worse scores. The bigger differences with other tools were found in complex and partially altered streams and rivers. Research highlights: The study showed the index’s applicability under diverse hydromorphological and ecological conditions and the main advantages of its application. The utilization of the index allows a better understanding of the status of riparian forests, and enhances improvements in the conservation and management of riparian areas.

Influence of new integrated pest management strategies on aphidophagous predators in horticultural crops

El 1 de enero de 2014 entró en vigor la Directiva Europea 2009/128/CE sobre uso sostenible de plaguicidas y el Real Decreto 1311/2012 por el cual se traspone dicha normativa comunitaria al ámbito nacional. Estos reglamentos establecen el marco legal por el que las explotaciones agrícolas deben cumplir los principios generales de la Gestión Integrada de Plagas (GIP). Los principios de la GIP dan preferencia a aquellos métodos de control que sean sostenibles y respetuosos con el medio ambiente, dando prioridad al control biológico, al físico y a otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos con los enemigos naturales es necesario en ocasiones para el adecuado manejo de las plagas en cultivos hortícolas. Por ello, el objetivo general de esta Tesis ha sido aportar conocimientos para la mejora del control de plagas en cultivos hortícolas, mediante la integración de estrategias de lucha biológica, física y química. La primera de las líneas de investigación de esta Tesis se centró en el estudio del efecto de la presencia dos depredadores, larvas Chrysoperla carnea y adultos de Adalia bipunctata, en la dispersión del virus de transmisión no persistente Cucumber mosaic virus (CMV) y del virus de transmisión persistente Cucurbit aphid-borne yellows virus (CABYV), transmitidos por el pulgón Aphis gosypii en cultivo de pepino. La tasa de transmisión de CMV fue baja para los dos tiempos de evaluación ensayados (1 y 5 días), debido al limitado movimiento de su vector A. gossypii. Las plantas que resultaron infectadas se localizaron próximas a la fuente de inóculo central y la presencia de ambos enemigos naturales no incrementó significativamente el porcentaje de plantas ocupadas por pulgones ni la tasa de transmisión de CMV. Los patrones de distribución de A. gossypii y de CMV tan solo fueron coincidentes en las proximidades de la planta central infectada en la que se liberaron los insectos. En los ensayos con CABYV, la presencia de C. carnea y de A. bipunctata respectivamente provocó un incremento significativo de la dispersión de A. gossypii tras 14 días, pero no tras 7 días desde la liberación de los insectos. La reducción en el número inicial de pulgones en la planta central infectada con CABYV fue siempre mayor tras la liberación de C. carnea en comparación con A. bipunctata. Sin embargo, la tasa de transmisión de CABYV y su distribución espacial no se vieron significativamente modificadas por la presencia de ninguno de los depredadores, ni tras 7 días ni tras 14 días desde el inicio de los ensayos. Al igual que se estudió el efecto de la presencia de enemigos naturales en el comportamiento de las plagas y en la epidemiología de las virosis que transmiten, en una segunda línea de investigación se evaluó el posible efecto del consumo de pulgones portadores de virus por parte de los enemigos naturales. Este trabajo se llevó a cabo en el Laboratorio de Ecotoxicología del Departamento de Entomología de la Universidade Federal de Lavras (UFLA) (Brasil). En él se evaluó la influencia en los parámetros biológicos del enemigo natural Chrysoperla externa al alimentarse de Myzus persicae contaminados con el virus de transmisión persistente Potato leafroll virus (PLRV). El consumo de M. persicae contaminados con PLRV incrementó significativamente la duración de la fase larvaria, reduciendo también la supervivencia en comparación a otras dos dietas a base de M. persicae no contaminados con el virus y huevos del lepidóptero Ephestia kuehniella. La duración de la fase de pupa de C. externa no difirió significativamente entre las dietas a base de pulgones contaminados con PLRV y pulgones no contaminados, pero ambas fueron menores que con la dieta con huevos de E. kuehniella. Sin embargo, ni la supervivencia en la fase de pupa ni los parámetros reproductivos de los adultos emergidos mostraron diferencias significativas entre las dietas evaluadas. Por el contrario, la supervivencia de los adultos durante los 30 primeros días desde su emergencia sí se vio significativamente afectada por la dieta, siendo al término de este periodo del 54% para aquellos adultos de C. externa que durante su fase larvaria consumieron pulgones con PLRV. Dentro de la GIP, una de las estrategias de carácter físico que se emplean para el control de plagas y enfermedades en cultivos hortícolas protegidos es el uso de plásticos con propiedades fotoselectivas de absorción de la radiación ultravioleta (UV). Por ello, la tercera línea de investigación de la Tesis se centró en el estudio de los efectos directos e indirectos (mediados por la planta) de condiciones especiales de baja radiación UV sobre el crecimiento poblacional del pulgón A. gossypii y los parámetros biológicos del enemigo natural C. carnea, así como sobre las plantas de pepino en las que se liberaron los insectos. Los ensayos se realizaron en jaulones dentro de invernadero, utilizándose en el primero de ellos plantas de pepino sanas, mientras que en el segundo las plantas de pepino fueron previamente infectadas con CABYV para estudiar de qué manera afectaba la incidencia del virus en las mismas condiciones. Las condiciones de baja radiación UV (bajo plástico Térmico Antivirus®) ejercieron un efecto directo en las fases iniciales del cultivo de pepino, promoviendo su crecimiento, mientras que en fases más avanzadas del cultivo indujeron un aumento en el contenido en nitrógeno de las plantas. Las plantas de pepino que fueron sometidas a mayor intensidad de radiación UV (bajo plástico Térmico Blanco®) al inicio del cultivo mostraron un engrosamiento significativo de las paredes de las células epidérmicas del haz de las hojas, así como de la cutícula. El uso del plástico Térmico Antivirus®, utilizado como barrera fotoselectiva para crear condiciones de baja radiación UV, no alteró con respecto al plástico Térmico Blanco® (utilizado como control) el desarrollo poblacional del pulgón A. gossypii ni los parámetros biológicos evaluados en el depredador C. carnea. En el segundo experimento, realizado con plantas infectadas con CABYV, la incidencia de la virosis enmascaró las diferencias encontradas en experimento con plantas sanas, reduciendo aparentemente la influencia de las distintas condiciones de radiación UV. Por último, para el desarrollo de las estrategias de GIP es importante estudiar los posibles efectos secundarios que los plaguicidas pueden tener en los enemigos naturales de las plagas. Es por ello que en la Tesis se evaluaron la toxicidad y los efectos subletales (fecundidad y fertilidad) de flonicamida, flubendiamida, metaflumizona, spirotetramat, sulfoxaflor y deltametrina en los enemigos naturales C. carnea y A. bipunctata. Los efectos secundarios fueron evaluados por contacto residual tanto para larvas como para adultos de ambos enemigos naturales en condiciones de laboratorio. Flonicamida, flubendiamida, metaflumizona y spirotetramat fueron inocuos para larvas de último estadio y adultos de C. carnea y A. bipunctata. Por este motivo, estos insecticidas se presentan como buenos candidatos para ser incorporados dentro de programas de GIP en combinación con estos enemigos naturales para el control de plagas de cultivos hortícolas. Sulfoxaflor fue ligeramente tóxico para adultos de C. carnea y altamente tóxico para larvas de último estadio de A. bipunctata. Para A. bipunctata, sulfoxaflor y deltametrina fueron los compuestos más dañinos. Deltametrina fue también el compuesto más tóxico para larvas y adultos de C. carnea. Por tanto, el uso de deltametrina y sulfoxaflor en programas de GIP debería tomarse en consideración cuando se liberasen cualquiera de estos dos enemigos naturales debido al comportamiento tóxico que mostraron en condiciones de laboratorio. ABSTRACT On 1 January 2014 came into effect the Directive 2009/128/EC of the European Parliament about sustainable use of pesticides and the Royal Decree 1311/2012 that transposes the regulation to the Spanish level. These regulations establish the legal framework that agricultural holdings must adhere to in order to accomplish the general principles of Integrated Pest Management (IPM). The guidelines of IPM give priority to sustainable and eco-friendly pest control techniques, such as biological and physical measures. Nevertheless, the use of pesticides that are selective to natural enemies is sometimes a necessary strategy to implement accurate pest management programs in horticultural protected crops. Therefore, the general objective of this Thesis was to contribute to the improvement of pest management strategies in horticultural crops, by means of the integration of biological, physical and chemical techniques. The first research line of this Thesis was focused on the evaluation of the effects of two aphidophagous predators, Chrysoperla carnea larvae and Adalia bipunctata adults, on the spread of the non-persistently transmitted Cucumber mosaic virus (CMV, Cucumovirus) and the persistently transmitted Cucurbit aphid-borne yellows virus (CABYV, Polerovirus), by the aphid vector Aphis gossypii in a cucumber crop under greenhouse conditions. The CMV transmission rate was generally low, both after 1 and 5 days, due to the limited movement of its aphid vector A. gossypii. Infected plants were mainly located around the central virusinfected source plant, and the percentage of aphid occupation and CMV-infected plants did not differ significantly in absence and presence of natural enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the central plant where insects were released. In the CABYV experiments, the presence of C. carnea larvae and A. bipunctata adults induced significant A. gossypii dispersal after 14 days but not after 7 days. The reduction in the initial aphid population established in the central plant was always higher for C. carnea than for A. bipunctata. Nevertheless, CABYV spread was not significantly modified by the presence of each predator either in the short term (7 days) or in the long term (14 days). Furthermore, the percentage of CABYV-infected plants did not significantly differ when each natural enemy was present in any evaluation period. It is important to evaluate the influence that natural enemies have on pest dynamics and on the spread of viral diseases, but it should be also taken into account the possible effect on the performance of natural enemies when they feed on preys that act as vectors of viruses. Thus, in a second research line developed in the Laboratory of Ecotoxicology, Department of Entomology, of the Universidade Federal de Lavras (UFLA) (Brazil), it was evaluated the performance of Chrysoperla externa under the condition of consuming Myzus persicae acting as vector of Potato leafroll virus (PLRV). The diet composed of PLRV-infected M. persicae significantly increased the length and reduced the survival rate, of the larval period in regard to the other two diets, composed of non-infected M. persicae and Ephestia kuehniella eggs. The lengths of the pupal stage were not significantly different between the aphid diets, but both were significantly shorter than that of E. kuehniella eggs. Neither pupal survival nor reproductive parameters revealed significant differences among the diets. Nevertheless, the adult survival curves during the first 30 days after emergence showed significant differences, reaching at the end of this interval a value of 54% for those C. externa adults fed on PLRVinfected aphids during their larval period. According to the IPM guidelines, one of the physical strategies for the control of pests and diseases in horticultural protected crops is the use of plastic films with photoselective properties that act as ultraviolet (UV) radiation blocking barriers. In this sense, the third research line of the Thesis dealt with the study of the direct and plant-mediated influence of low UV radiation conditions on the performance of the aphid A. gossypii and on the biological parameters of the natural enemy C. carnea, as well as on the cucumber plants where insects were released. The experiments were conducted inside cages under greenhouse conditions, using for the first one healthy cucumber plants, while for the second experiment the cucumber plants were previously infected with CABYV in order to assess the influence of the virus in the same conditions. The low UV radiation conditions (under Térmico Antivirus® plastic film) seemed to exert a direct effect in the early stages of cucumber plants, enhancing their growth, and in an increasing nitrogen content at further developmental stages. The higher UV radiation exposure (under Térmico Blanco® plastic film) in the early stages of the cucumber crop induced the thickening of the adaxial epidermal cell walls and the cuticle of leaves. The use of Térmico Antivirus® plastic film as a photoselective barrier to induce low UV radiation conditions did not modify, in regard to Térmico Blanco® plastic film (used as control), neither the population development of A. gossypii nor the studied biological parameters of the predator C. carnea. In the second experiment, done with CABYV-infected cucumber plants, the incidence of the virus seemed to mask the direct and plant-mediated influence of the different UV radiation conditions. In last term, for the development of IPM strategies it is important to study the potential side effects that pesticides might have on natural enemies. For this reason, in the Thesis were tested the toxicity and sublethal effects (fecundity and fertility) of flonicamid, flubendiamide, metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on the natural enemies C. carnea and A. bipunctata. The side effects of the active ingredients of the insecticides were evaluated with residual contact tests for the larvae and adults of these predators under laboratory conditions. Flonicamid, flubendiamide, metaflumizone and spirotetramat were innocuous to last instar larvae and adults of C. carnea and A. bipunctata. Therefore, these pesticides are promising candidates for being incorporated into IPM programs in combination with these natural enemies for the control of particular greenhouse pests. In contrast, sulfoxaflor was slightly toxic to adults of C. carnea and was highly toxic to last instar larvae of A. bipunctata. For A. bipunctata, sulfoxaflor and deltamethrin were the most damaging compounds. Deltamethrin was also the most toxic compound to larvae and adults of C. carnea. In accordance with this fact, the use of sulfoxaflor and deltamethrin in IPM strategies should be taken into consideration when releasing either of these biological control agents, due to the toxic behavior observed under laboratory conditions.

A clustering technique for partial discharge and noise sources identification in power cables by means of waveform parameters

On-line partial discharge (PD) measurements have become a common technique for assessing the insulation condition of installed high voltage (HV) insulated cables. When on-line tests are performed in noisy environments, or when more than one source of pulse-shaped signals are present in a cable system, it is difficult to perform accurate diagnoses. In these cases, an adequate selection of the non-conventional measuring technique and the implementation of effective signal processing tools are essential for a correct evaluation of the insulation degradation. Once a specific noise rejection filter is applied, many signals can be identified as potential PD pulses, therefore, a classification tool to discriminate the PD sources involved is required. This paper proposes an efficient method for the classification of PD signals and pulse-type noise interferences measured in power cables with HFCT sensors. By using a signal feature generation algorithm, representative parameters associated to the waveform of each pulse acquired are calculated so that they can be separated in different clusters. The efficiency of the clustering technique proposed is demonstrated through an example with three different PD sources and several pulse-shaped interferences measured simultaneously in a cable system with a high frequency current transformer (HFCT).

Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality

The Dld gene product, known as dihydrolipoamide dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine Dld gene (Dldtm1mjp). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the Dld−/− class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The Dld−/− embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides in vivo evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.

Import into and Degradation of Cytosolic Proteins by Isolated Yeast Vacuoles

In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

The satellite tobacco necrosis virus RNA is uncapped and requires a 3′ translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30.

Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors

The proteasome is a large protease complex consisting of multiple catalytic subunits that function simultaneously to digest protein substrates. This complexity has made deciphering the role each subunit plays in the generation of specific protein fragments difficult. Positional scanning libraries of peptide vinyl sulfones were generated in which the amino acid located directly at the site of hydrolysis (P1 residue) was held constant and sequences distal to that residue (P2, P3, and P4 positions) were varied across all natural amino acids (except cysteine and methionine). Binding information for each of the individual catalytic subunits was obtained for each library under a variety of different conditions. The resulting specificity profiles indicated that substrate positions distal to P1 are critical for directing substrates to active subunits in the complex. Furthermore, specificity profiles of IFN-γ-regulated subunits closely matched those of their noninducible counterparts, suggesting that subunit swapping may modulate substrate processing by a mechanism that does require a change in the primary sequence specificity of individual catalytic subunits in the complex. Finally, specificity profiles were used to design specific inhibitors of a single active site in the complex. These reagents can be used to further establish the role of each subunit in substrate processing by the proteasome.

Structure of neurolysin reveals a deep channel that limits substrate access

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded β-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

Behavior of several two-dimensional fluid equations in singular scenarios

We give conditions that rule out formation of sharp fronts for certain two-dimensional incompressible flows. We show that a necessary condition of having a sharp front is that the flow has to have uncontrolled velocity growth. In the case of the quasi-geostrophic equation and two-dimensional Euler equation, we obtain estimates on the formation of semi-uniform fronts.

On the origin of the order Artiodactyla.

The first known members of the order Artiodactyla appeared suddenly throughout the Holarctic region at the beginning of the Eocene. They are characterized by distinctive cursorial skeletal specializations. Owing to their abrupt appearance and the lack of transitional forms, the origin of the order is problematic. Descent from a "condylarth," specifically the arctocyonid Chriacus, has been suggested based on dental resemblances, but until now postcranial anatomy seemed to preclude close relationship between Arctocyonidae and Artiodactyla. A middle Paleocene specimen of a small arctocyonid (?Chriacus) reported here is much more similar to the oldest artiodactyl, Diacodexis, in the derived condition of the hindlimb, reviving the possibility that Artiodactyla evolved from an arctocyonid.

The treatment of chronic progressive multiple sclerosis with cladribine.

A 2-year, placebo-controlled, double-blind, crossover study was started in 1992 to evaluate cladribine, an immunosuppressive drug, in the treatment of chronic progressive multiple sclerosis. In the first year patients were given cladribine 0.10 mg/kg per day for 7 days as four monthly courses for a total of 2.8 mg/kg or placebo. During the second year patients treated with placebo during the first year were given i.v. infusions of 0.10 mg, 0.05 mg, and 0.05 mg of cladribine per kg of body weight per day for 7 consecutive days in three successive monthly courses, for a total dose of 1.4 mg/kg. Patients who had been treated previously with cladribine were crossed over to placebo. Analysis of the results revealed a favorable influence on the neurological performance scores, both in the Kurtze extended disability status and the Scripps neurological rating scale, and on MRI findings in patients treated with cladribine. In the first year the most striking finding was that while clinical deterioration continued in the placebo-treated patients, the condition of patients who received cladribine stabilized or even improved slightly. Toxicity and therapeutic response were dose-related.

Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.

Parkinson's disease and gastrointestinal dysfunctions: morphological, neurochemical and functional modifications of the enteric nervous system associated with central dopaminergic impairment

Parkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, mostly represented by abdominal distension, constipation and defecatory dysfunctions. Despite GI dysfunctions have a major impact on the clinical picture of PD, there is currently a lack of information on the neurochemical, pathological and functional correlates of GI dysmotility associated with PD. Moreover, there is a need of effective and safe pharmacological therapies for managing GI disturbances in PD patients. The present research project has been undertaken to investigate the relationships between PD and related GI dysfunctions by means of investigations in an animal model of PD induced by intranigral injection of 6-hydroxydopamine (6-OHDA). The use of the 6-OHDA experimental model of PD in the present program has allowed to pursue the following goals: 1) to examine the impact of central dopaminergic denervation on colonic excitatory cholinergic and tachykininergic neuromotility by means of molecular, histomorphologic and functional approaches; 2) to elucidate the role of gut inflammation in the onset and progression of colonic dysmotility associated with PD, characterizing the degree of inflammation and oxidative damage in colonic tissues, as well as identifying the immune cells involved in the production of pro-inflammatory cytokines in the gut; 3) to evaluate the impact of chronic treatment with L-DOPA plus benserazide on colonic neuromuscular activity both in control and PD animals. The results suggest that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory cholinergic neurotransmission and an enhanced tachykininergic control, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate. These motor alterations might result from the occurrence of a condition of gut inflammation associated with central intranigral denervation. The treatment with L-DOPA/BE following central dopaminergic neurodegeneration can restore colonic motility, likely through a normalization of the cholinergic enteric neurotransmission, and it can also improve the colonic inflammation associated with central dopaminergic denervation.