Distributed control strategies for photovoltaic systems in urban environments

Este trabajo es una contribución a los sistemas fotovoltaicos (FV) con seguimiento distribuido del punto de máxima potencia (DMPPT), una topología que se caracteriza porque lleva a cabo el MPPT a nivel de módulo, al contrario de las topologías más tradicionales que llevan a cabo el MPPT para un número más elevado de módulos, pudiendo ser hasta cientos de módulos. Las dos tecnologías DMPPT que existen en el mercado son conocidos como microinversores y optimizadores de potencia, y ofrecen ciertas ventajas sobre sistemas de MPPT central como: mayor producción en situaciones de mismatch, monitorización individual de cada módulo, flexibilidad de diseño, mayor seguridad del sistema, etc. Aunque los sistemas DMPPT no están limitados a los entornos urbanos, se ha enfatizado en el título ya que es su mercado natural, siendo difícil una justificación de su sobrecoste en grandes huertas solares en suelo. Desde el año 2010 el mercado de estos sistemas ha incrementado notablemente y sigue creciendo de una forma continuada. Sin embargo, todavía falta un conocimiento profundo de cómo funcionan estos sistemas, especialmente en el caso de los optimizadores de potencia, de las ganancias energéticas esperables en condiciones de mismatch y de las posibilidades avanzadas de diagnóstico de fallos. El principal objetivo de esta tesis es presentar un estudio completo de cómo funcionan los sistemas DMPPT, sus límites y sus ventajas, así como experimentos varios que verifican la teoría y el desarrollo de herramientas para valorar las ventajas de utilizar DMPPT en cada instalación. Las ecuaciones que modelan el funcionamiento de los sistemas FVs con optimizadores de potencia se han desarrollado y utilizado para resaltar los límites de los mismos a la hora de resolver ciertas situaciones de mismatch. Se presenta un estudio profundo sobre el efecto de las sombras en los sistemas FVs: en la curva I-V y en los algoritmos MPPT. Se han llevado a cabo experimentos sobre el funcionamiento de los algoritmos MPPT en situaciones de sombreado, señalando su ineficiencia en estas situaciones. Un análisis de la ventaja del uso de DMPPT frente a los puntos calientes es presentado y verificado. También se presenta un análisis sobre las posibles ganancias en potencia y energía con el uso de DMPPT en condiciones de sombreado y este también es verificado experimentalmente, así como un breve estudio de su viabilidad económica. Para ayudar a llevar a cabo todos los análisis y experimentos descritos previamente se han desarrollado una serie de herramientas software. Una siendo un programa en LabView para controlar un simulador solar y almacenar las medidas. También se ha desarrollado un programa que simula curvas I-V de módulos y generador FVs afectados por sombras y este se ha verificado experimentalmente. Este mismo programa se ha utilizado para desarrollar un programa todavía más completo que estima las pérdidas anuales y las ganancias obtenidas con DMPPT en instalaciones FVs afectadas por sombras. Finalmente, se han desarrollado y verificado unos algoritmos para diagnosticar fallos en sistemas FVs con DMPPT. Esta herramienta puede diagnosticar los siguientes fallos: sombras debido a objetos fijos (con estimación de la distancia al objeto), suciedad localizada, suciedad general, posible punto caliente, degradación de módulos y pérdidas en el cableado de DC. Además, alerta al usuario de las pérdidas producidas por cada fallo y no requiere del uso de sensores de irradiancia y temperatura. ABSTRACT This work is a contribution to photovoltaic (PV) systems with distributed maximum power point tracking (DMPPT), a system topology characterized by performing the MPPT at module level, instead of the more traditional topologies which perform MPPT for a larger number of modules. The two DMPPT technologies available at the moment are known as microinverters and power optimizers, also known as module level power electronics (MLPE), and they provide certain advantages over central MPPT systems like: higher energy production in mismatch situations, monitoring of each individual module, system design flexibility, higher system safety, etc. Although DMPPT is not limited to urban environments, it has been emphasized in the title as it is their natural market, since in large ground-mounted PV plants the extra cost is difficult to justify. Since 2010 MLPE have increased their market share steadily and continuing to grow steadily. However, there still lacks a profound understanding of how they work, especially in the case of power optimizers, the achievable energy gains with their use and the possibilities in failure diagnosis. The main objective of this thesis is to provide a complete understanding of DMPPT technologies: how they function, their limitations and their advantages. A series of equations used to model PV arrays with power optimizers have been derived and used to point out limitations in solving certain mismatch situation. Because one of the most emphasized benefits of DMPPT is their ability to mitigate shading losses, an extensive study on the effects of shadows on PV systems is presented; both on the I-V curve and on MPPT algorithms. Experimental tests have been performed on the MPPT algorithms of central inverters and MLPE, highlighting their inefficiency in I-V curves with local maxima. An analysis of the possible mitigation of hot-spots with DMPPT is discussed and experimentally verified. And a theoretical analysis of the possible power and energy gains is presented as well as experiments in real PV systems. A short economic analysis of the benefits of DMPPT has also been performed. In order to aide in the previous task, a program which simulates I-V curves under shaded conditions has been developed and experimentally verified. This same program has been used to develop a software tool especially designed for PV systems affected by shading, which estimates the losses due to shading and the energy gains obtained with DMPPT. Finally, a set of algorithms for diagnosing system faults in PV systems with DMPPT has been developed and experimentally verified. The tool can diagnose the following failures: fixed object shading (with distance estimation), localized dirt, generalized dirt, possible hot-spots, module degradation and excessive losses in DC cables. In addition, it alerts the user of the power losses produced by each failure and classifies the failures by their severity and it does not require the use of irradiance or temperature sensors.

Distributed control strategies for photovoltaic systems in urban environments : Versión definitiva

Este trabajo es una contribución a los sistemas fotovoltaicos (FV) con seguimiento distribuido del punto de máxima potencia (DMPPT), una topología que se caracteriza porque lleva a cabo el MPPT a nivel de módulo, al contrario de las topologías más tradicionales que llevan a cabo el MPPT para un número más elevado de módulos, pudiendo ser hasta cientos de módulos. Las dos tecnologías DMPPT que existen en el mercado son conocidos como microinversores y optimizadores de potencia, y ofrecen ciertas ventajas sobre sistemas de MPPT central como: mayor producción en situaciones de mismatch, monitorización individual de cada módulo, flexibilidad de diseño, mayor seguridad del sistema, etc. Aunque los sistemas DMPPT no están limitados a los entornos urbanos, se ha enfatizado en el título ya que es su mercado natural, siendo difícil una justificación de su sobrecoste en grandes huertas solares en suelo. Desde el año 2010 el mercado de estos sistemas ha incrementado notablemente y sigue creciendo de una forma continuada. Sin embargo, todavía falta un conocimiento profundo de cómo funcionan estos sistemas, especialmente en el caso de los optimizadores de potencia, de las ganancias energéticas esperables en condiciones de mismatch y de las posibilidades avanzadas de diagnóstico de fallos. El principal objetivo de esta tesis es presentar un estudio completo de cómo funcionan los sistemas DMPPT, sus límites y sus ventajas, así como experimentos varios que verifican la teoría y el desarrollo de herramientas para valorar las ventajas de utilizar DMPPT en cada instalación. Las ecuaciones que modelan el funcionamiento de los sistemas FVs con optimizadores de potencia se han desarrollado y utilizado para resaltar los límites de los mismos a la hora de resolver ciertas situaciones de mismatch. Se presenta un estudio profundo sobre el efecto de las sombras en los sistemas FVs: en la curva I-V y en los algoritmos MPPT. Se han llevado a cabo experimentos sobre el funcionamiento de los algoritmos MPPT en situaciones de sombreado, señalando su ineficiencia en estas situaciones. Un análisis de la ventaja del uso de DMPPT frente a los puntos calientes es presentado y verificado. También se presenta un análisis sobre las posibles ganancias en potencia y energía con el uso de DMPPT en condiciones de sombreado y este también es verificado experimentalmente, así como un breve estudio de su viabilidad económica. Para ayudar a llevar a cabo todos los análisis y experimentos descritos previamente se han desarrollado una serie de herramientas software. Una siendo un programa en LabView para controlar un simulador solar y almacenar las medidas. También se ha desarrollado un programa que simula curvas I-V de módulos y generador FVs afectados por sombras y este se ha verificado experimentalmente. Este mismo programa se ha utilizado para desarrollar un programa todavía más completo que estima las pérdidas anuales y las ganancias obtenidas con DMPPT en instalaciones FVs afectadas por sombras. Finalmente, se han desarrollado y verificado unos algoritmos para diagnosticar fallos en sistemas FVs con DMPPT. Esta herramienta puede diagnosticar los siguientes fallos: sombras debido a objetos fijos (con estimación de la distancia al objeto), suciedad localizada, suciedad general, posible punto caliente, degradación de módulos y pérdidas en el cableado de DC. Además, alerta al usuario de las pérdidas producidas por cada fallo y no requiere del uso de sensores de irradiancia y temperatura. ABSTRACT This work is a contribution to photovoltaic (PV) systems with distributed maximum power point tracking (DMPPT), a system topology characterized by performing the MPPT at module level, instead of the more traditional topologies which perform MPPT for a larger number of modules. The two DMPPT technologies available at the moment are known as microinverters and power optimizers, also known as module level power electronics (MLPE), and they provide certain advantages over central MPPT systems like: higher energy production in mismatch situations, monitoring of each individual module, system design flexibility, higher system safety, etc. Although DMPPT is not limited to urban environments, it has been emphasized in the title as it is their natural market, since in large ground-mounted PV plants the extra cost is difficult to justify. Since 2010 MLPE have increased their market share steadily and continuing to grow steadily. However, there still lacks a profound understanding of how they work, especially in the case of power optimizers, the achievable energy gains with their use and the possibilities in failure diagnosis. The main objective of this thesis is to provide a complete understanding of DMPPT technologies: how they function, their limitations and their advantages. A series of equations used to model PV arrays with power optimizers have been derived and used to point out limitations in solving certain mismatch situation. Because one of the most emphasized benefits of DMPPT is their ability to mitigate shading losses, an extensive study on the effects of shadows on PV systems is presented; both on the I-V curve and on MPPT algorithms. Experimental tests have been performed on the MPPT algorithms of central inverters and MLPE, highlighting their inefficiency in I-V curves with local maxima. An analysis of the possible mitigation of hot-spots with DMPPT is discussed and experimentally verified. And a theoretical analysis of the possible power and energy gains is presented as well as experiments in real PV systems. A short economic analysis of the benefits of DMPPT has also been performed. In order to aide in the previous task, a program which simulates I-V curves under shaded conditions has been developed and experimentally verified. This same program has been used to develop a software tool especially designed for PV systems affected by shading, which estimates the losses due to shading and the energy gains obtained with DMPPT. Finally, a set of algorithms for diagnosing system faults in PV systems with DMPPT has been developed and experimentally verified. The tool can diagnose the following failures: fixed object shading (with distance estimation), localized dirt, generalized dirt, possible hot-spots, module degradation and excessive losses in DC cables. In addition, it alerts the user of the power losses produced by each failure and classifies the failures by their severity and it does not require the use of irradiance or temperature sensors.

Toward mass production of microtextured microdevices: linking rapid prototyping with microinjection molding

The possibility of manufacturing textured materials and devices, with surface properties controlled from the design stage, instead of being the result of machining processes or chemical attacks, is a key factor for the incorporation of advanced functionalities to a wide set of micro and nanosystems. Recently developed high-precision additive manufacturing technologies, together with the use of fractal models linked to computer-aided design tools, allow for a precise definition and control of final surface properties for a wide set of applications, although the production of larger series based on these resources is still an unsolved challenge. However, rapid prototypes, with controlled surface topography, can be used as original masters for obtaining micromold inserts for final large-scale series manufacture of replicas using microinjection molding. In this study, an original procedure is presented, aimed at connecting rapid prototyping with microinjection molding, for the mass production of two different microtextured microsystems, linked to tissue engineering tasks, using different thermoplastics as ultimate materials.

Dust suppressant treatments. Quality control = Tratamientos supresores de polvo. Control de calidad

La emisión de polvo por efecto del viento desde depósitos de residuos mineros o industriales y el paso de vehículos en vías no pavimentadas, es un problema que afecta las actividades productivas; el ambiente y la salud de las personas que permanecen en el área contaminada. En Chile, en los últimos años la sensibilidad social y las exigencias ambientales han aumentado, así como la oferta de diferentes supresores y tecnologías de aplicación. Se han revisado las causas que provocan emisión de polvo y las tecnologías disponibles en Chile para la supresión de polvo, además de las metodologías y normativa para evaluar el desempeño de los materiales tratados con diferentes supresores. En algunos casos no es posible comparar propiedades de desempeño, como durabilidad, dosis a aplicar y frecuencia de las aplicaciones, entre otros aspectos. Los procedimientos descritos en la norma NCh3266-2012 permiten evaluar la erosión eólica en depósitos de residuos, sitios eriazos y caminos no pavimentados, entre otros, junto con evaluar el desempeño de diferentes tipos de supresores de polvo a partir de datos objetivos comparables. Esto permite seleccionar el supresor más adecuado, mejorar la eficiencia de los tratamientos, optimizar los costos y mejorar los procesos productivos. Palabras clave: Erosión-eólica, supresor de polvo, residuos-mineros, caminos-no pavimentados. Dust emissions by wind effect from mining deposits or industrial waste and passing vehicles on unpaved roads, is a problem that affects the productive activities; the environment and the health of those who remain in the contaminated area. The social sensitivity and environmental requirements on this issue in Chile have increased, as well as offering different suppressors and application technologies. Have been reviewed the causes of dust emission and technologies available in Chile for dust suppression, plus methodologies and standards for assessing the performance of the treated materials with different suppressors. In some cases it is not possible to compare performance properties such as durability, application dose and frequency of applications, among others aspects. The procedures described in the NCh 3266-2012 standard allows the assessment of wind erosion in waste deposits, vacant lots and unpaved roads, among others, along with evaluating the performance of different types of dust suppressants from comparable objective data. This allows selecting the most suitable suppressor, improve efficiency of treatments, optimize costs and improve production processes. Keywords: Wind-erosion, dust-suppressor, mining-waste, unpavedroads

Analytical models for the power subsystem and the attitude control subsystem of a microsatellite

El trabajo realizado en la presente tesis doctoral se debe considerar parte del proyecto UPMSat-2, que se enmarca dentro del ámbito de la tecnología aeroespacial. El UPMSat-2 es un microsatélite (de bajo coste y pequeño tamaño) diseñado, construido, probado e integrado por la Universidad Politécnica de Madrid (España), para fines de demostración tecnológica y educación. El objetivo de la presente tesis doctoral es presentar nuevos modelos analíticos para estudiar la interdependencia energética entre los subsistemas de potencia y de control de actitud de un satélite. En primer lugar, se estudia la simulación del subsistema de potencia de un microsatélite, prestando especial atención a la simulación de la fuente de potencia, esto es, los paneles solares. En la tesis se presentan métodos sencillos pero precisos para simular la producción de energía de los paneles en condiciones ambientales variables a través de su circuito equivalente. Los métodos propuestos para el cálculo de los parámetros del circuito equivalente son explícitos (o al menos, con las variables desacopladas), no iterativos y directos; no se necesitan iteraciones o valores iniciales para calcular los parámetros. La precisión de este método se prueba y se compara con métodos similares de la literatura disponible, demostrando una precisión similar para mayor simplicidad. En segundo lugar, se presenta la simulación del subsistema de control de actitud de un microsatélite, prestando especial atención a la nueva ley de control propuesta. La tesis presenta un nuevo tipo de control magnético es aplicable a la órbita baja terrestre (LEO). La ley de control propuesta es capaz de ajustar la velocidad de rotación del satélite alrededor de su eje principal de inercia máximo o mínimo. Además, en el caso de órbitas de alta inclinación, la ley de control favorece la alineación del eje de rotación con la dirección normal al plano orbital. El algoritmo de control propuesto es simple, sólo se requieren magnetopares como actuadores; sólo se requieren magnetómetros como sensores; no hace falta estimar la velocidad angular; no incluye un modelo de campo magnético de la Tierra; no tiene por qué ser externamente activado con información sobre las características orbitales y permite el rearme automático después de un apagado total del subsistema de control de actitud. La viabilidad teórica de la citada ley de control se demuestra a través de análisis de Monte Carlo. Por último, en términos de producción de energía, se demuestra que la actitud propuesto (en eje principal perpendicular al plano de la órbita, y el satélite que gira alrededor de ella con una velocidad controlada) es muy adecuado para la misión UPMSat-2, ya que permite una área superior de los paneles apuntando hacia el sol cuando se compara con otras actitudes estudiadas. En comparación con el control de actitud anterior propuesto para el UPMSat-2 resulta en un incremento de 25% en la potencia disponible. Además, la actitud propuesto mostró mejoras significativas, en comparación con otros, en términos de control térmico, como la tasa de rotación angular por satélite puede seleccionarse para conseguir una homogeneización de la temperatura más alta que apunta satélite y la antena. ABSTRACT The work carried out in the present doctoral dissertation should be considered part of the UPMSat-2 project, falling within the scope of the aerospace technology. The UPMSat-2 is a microsatellite (low cost and small size) designed, constructed integrated and tested for educational and technology demonstration purposes at the Universidad Politécnica de Madrid (Spain). The aim of the present doctoral dissertation is to present new analytical models to study the energy interdependence between the power and the attitude control subsystems of a satellite. First, the simulation of the power subsystem of a microsatellite is studied, paying particular attention to the simulation of the power supply, i.e. the solar panels. Simple but accurate methods for simulate the power production under variable ambient conditions using its equivalent circuit are presented. The proposed methods for calculate the equivalent circuit parameters are explicit (or at least, with decoupled variables), non-iterative and straight forward; no iterations or initial values for the parameters are needed. The accuracy of this method is tested and compared with similar methods from the available literature demonstrating similar precision but higher simplicity. Second, the simulation of the control subsystem of a microsatellite is presented, paying particular attention to the new control law proposed. A new type of magnetic control applied to Low Earth Orbit (LEO) satellites has been presented. The proposed control law is able to set the satellite rotation speed around its maximum or minimum inertia principal axis. Besides, the proposed control law favors the alignment of this axis with the normal direction to the orbital plane for high inclination orbits. The proposed control algorithm is simples, only magnetorquers are required as actuators; only magnetometers are required as sensors; no estimation of the angular velocity is needed; it does not include an in-orbit Earth magnetic field model; it does not need to be externally activated with information about the orbital characteristics and it allows automatic reset after a total shutdown of attitude control subsystem. The theoretical viability of the control law is demonstrated through Monte Carlo analysis. Finally, in terms of power production, it is demonstrated that the proposed attitude (on principal axis perpendicular to the orbit plane, and the satellite rotating around it with a controlled rate) is quite suitable for the UPMSat-2 mission, as it allows a higher area of the panels pointing towards the sun when compared to other studied attitudes. Compared with the previous attitude control proposed for the UPMSat-2 it results in a 25% increment in available power. Besides, the proposed attitude showed significant improvements, when compared to others, in terms of thermal control, as the satellite angular rotation rate can be selected to achieve a higher temperature homogenization of the satellite and antenna pointing.

Effect of level of fiber of the rearing phase diets on egg production, digestive tract traits, and body measurements of brown egg-laying hens fed diets differing in energy concentration

This research studied the effects of additional fiber in the rearing phase diets on egg production, gastrointestinal tract (GIT) traits, and body measurements of brown egg-laying hens fed diets varying in energy concentration from 17 to 46 wk of age. The experiment was completely randomized with 10 treatments arranged as a 5 × 2 factorial with 5 rearing phase diets and 2 laying phase diets. During the rearing phase, treatments consisted of a control diet based on cereals and soybean meal and 4 additional diets with a combination of 2 fiber sources (cereal straw and sugar beet pulp, SBP) at 2 levels (2 and 4%). During the laying phase, diets differed in energy content (2,650 vs. 2,750 kcal AMEn/kg) but had the same amino acid content per unit of energy. The rearing diet did not affect any production trait except egg production that was lower in birds fed SBP than in birds fed straw (91.6 and 94.1%, respectively; P < 0.05). Laying hens fed the high energy diet had lower feed intake (P < 0.001), better feed conversion (P < 0.01), and greater BW gain (P < 0.05) than hens fed the low energy diet but egg production and egg weight were not affected. At 46 wk of age, none of the GIT traits was affected by previous dietary treatment. At this age, hen BW was positively related with body length (r = 0.500; P < 0.01), tarsus length (r = 0.758; P < 0.001), and body mass index (r = 0.762; P < 0.001) but no effects of type of diet on these traits were detected. In summary, the inclusion of up to 4% of a fiber source in the rearing diets did not affect GIT development of the hens but SBP reduced egg production. An increase in the energy content of the laying phase diet reduced ADFI and improved feed efficiency but did not affect any of the other traits studied.

Effect of nano-Si2O and nano-Al2O3 on cement mortars for use in agriculture and livestock production

The effect of nano-silica, nano-alumina and binary combinations on surface hardness, resistance to abrasion and freeze-thaw cycle resistance in cement mortars was investigated. The Vickers hardness, the Los Angeles coefficient (LA) and the loss of mass in each of the freeze–thaw cycles to which the samples were subjected were measured. Four cement mortars CEM I 52.5R were prepared, one as control, and the other three with the additions: 5% nano-Si, 5% nano-Al and mix 2.5% n-Si and 2.5% n-Al. Mortars were tested at 7, 28 and 90 d of curing to determine compression strength, total porosity and pore distribution by mercury intrusion porosimetry (MIP) and the relationship between the CSH gel and Portlandite total by thermal gravimetric analysis (TGA). The capillary suction coefficient and an analysis by a scanning electron microscope (SEM) was made. There was a large increase in Vickers surface hardness for 5% n-Si mortar and a slight increase in resistance to abrasion. No significant difference was found between the mortars with nano-particles, whose LA was about 10.8, classifying them as materials with good resistance to abrasion. The microstructure shows that the addition of n-Si in mortars refines their porous matrix, increases the amount of hydrated gels and generates significant changes in both Portlandite and Ettringite. This produced a significant improvement in freeze–thaw cycle resistance. The effect of n-Al on mortar was null or negative with respect to freeze–thaw cycle resistance.

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2α kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2α phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2α kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

ADD1/SREBP1 activates PPARγ through the production of endogenous ligand

Adipose differentiation is an important part of the energy homeostasis system of higher organisms. Recent data have suggested that this process is controlled by an interplay of transcription factors including PPARγ, the C/EBPs, and ADD1/SREBP1. Although these factors interact functionally to initiate the program of differentiation, there are no data concerning specific mechanisms of interaction. We show here that the expression of ADD1/SREBP1 specifically increases the activity of PPARγ but not other isoforms, PPARα, or PPARδ. This activation occurs through the ligand-binding domain of PPARγ when it is fused to the DNA-binding domain of Gal4. The stimulation of PPARγ by ADD1/SREBP1 does not require coexpression in the same cells; supernatants from cultures that express ADD1/SREBP1 augment the transcriptional activity of PPARγ. Finally, we demonstrate directly that cells expressing ADD1/SREBP1 produce and secrete lipid molecule(s) that bind directly to PPARγ, displacing the binding of radioactive thiazolidinedione ligands. These data establish that ADD1/SREBP1 can control the production of endogenous ligand(s) for PPARγ and suggest a mechanism for coordinating the actions of these adipogenic factors.

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Doxycycline control of prion protein transgene expression modulates prion disease in mice

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at ≈50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

The production of recombinant proteins in transgenic barley grains

The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3–1, 4)-β-glucanase with the D hordein gene (Hor3–1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an α-amylase promoter and the code for the export signal peptide yielded ≈1 μg⋅mg−1 soluble protein, whereas 54 μg⋅mg−1 soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3–1, 4)-β-glucanase under the control of the Hor3–1 promoter.

A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

The p53 tumor suppressor protein and the MDM2 oncoprotein form a feedback-control loop that up-regulates cellular MDM2 production, blocks p53 activity, and promotes p53 decay. tsg101 was discovered as a gene whose deficiency results in neoplastic transformation of NIH 3T3 cells and the ability to generate metastatic tumors in nude mice. Its protein product contains a domain, Ubc, characteristic of the catalytic domain of ubiquitin conjugase (E2) enzymes but lacking an active-site cysteine crucial for ubiquitin conjugase activity. Here we report that TSG101 participates with MDM2 in an autoregulatory loop that modulates the cellular levels of both proteins, and also of p53, by affecting protein decay. We show that the Ubc domain of TSG101 interferes with ubiquitination of MDM2, that TSG101 inhibits MDM2 decay and elevates its steady-state level, and that these events are associated with down-regulation of p53 protein. Conversely, pulse–chase and Western blot experiments in wild-type and mutant fibroblasts indicate that elevation of MDM2 by overexpression of wild-type p53, by amplification of the endogenous MDM2 gene, or by transfection of MDM2-expressing constructs promotes TSG101 loss, which we show occurs by 26S proteasome-dependent decay. Our results identify TSG101 as both a regulator of, and target of, MDM2/p53 circuitry.

Translational control of human p53 expression in yeast mediated by 5′-UTR–ORF structural interaction

We have expressed human p53 cDNA in the yeast Saccharomyces cerevisiae and shown that the level of production and the length of the p53 protein depends on the presence of untranslated mRNA regions (UTRs). The expression of the ORF alone leads to a p53 protein of correct size (53 kDa) that accumulates to high levels, concomitantly with the presence of a small amount of a p40 protein (40 kDa). However, when either the entire 5′-UTR and a part of the 3′- or 5′-UTR alone is used, this leads to the production of small amounts of the 40 kDa truncated form only. The p40 protein corresponds to a truncated form of p53 at the C-terminal extremity since it reacts only with a monoclonal antibody recognising the N-terminal epitope. This effect on the amount and length of p53 protein had no correlation at the mRNA level, suggesting that translational control probably occurs through the 5′-UTR. We propose a model of structural interaction between this UTR and a part of the ORF mRNA for the regulation of p53 expression in this heterologous context.