Advanced Control Strategies for a 6 DoF Hydraulic Parallel Robot Based on the Dynamic Model

Nowadays robots have made their way into real applications that were prohibitive and unthinkable thirty years ago. This is mainly due to the increase in power computations and the evolution in the theoretical field of robotics and control. Even though there is plenty of information in the current literature on this topics, it is not easy to find clear concepts of how to proceed in order to design and implement a controller for a robot. In general, the design of a controller requires of a complete understanding and knowledge of the system to be controlled. Therefore, for advanced control techniques the systems must be first identified. Once again this particular objective is cumbersome and is never straight forward requiring of great expertise and some criteria must be adopted. On the other hand, the particular problem of designing a controller is even more complex when dealing with Parallel Manipulators (PM), since their closed-loop structures give rise to a highly nonlinear system. Under this basis the current work is developed, which intends to resume and gather all the concepts and experiences involve for the control of an Hydraulic Parallel Manipulator. The main objective of this thesis is to provide a guide remarking all the steps involve in the designing of advanced control technique for PMs. The analysis of the PM under study is minced up to the core of the mechanism: the hydraulic actuators. The actuators are modeled and experimental identified. Additionally, some consideration regarding traditional PID controllers are presented and an adaptive controller is finally implemented. From a macro perspective the kinematic and dynamic model of the PM are presented. Based on the model of the system and extending the adaptive controller of the actuator, a control strategy for the PM is developed and its performance is analyzed with simulation.

Multiphase Design and Control Techniques Applied to a Forward Micro-Inverter

En la última década la potencia instalada de energía solar fotovoltaica ha crecido una media de un 49% anual y se espera que alcance el 16%del consumo energético mundial en el año 2050. La mayor parte de estas instalaciones se corresponden con sistemas conectados a la red eléctrica y un amplio porcentaje de ellas son instalaciones domésticas o en edificios. En el mercado ya existen diferentes arquitecturas para este tipo de instalaciones, entre las que se encuentras los módulos AC. Un módulo AC consiste en un inversor, también conocido como micro-inversor, que se monta en la parte trasera de un panel o módulo fotovoltaico. Esta tecnología ofrece modularidad, redundancia y la extracción de la máxima potencia de cada panel solar de la instalación. Además, la expansión de esta tecnología posibilitará una reducción de costes asociados a las economías de escala y a la posibilidad de que el propio usuario pueda componer su propio sistema. Sin embargo, el micro-inversor debe ser capaz de proporcionar una ganancia de tensión adecuada para conectar el panel solar directamente a la red, mientras mantiene un rendimiento aceptable en un amplio rango de potencias. Asimismo, los estándares de conexión a red deber ser satisfechos y el tamaño y el tiempo de vida del micro-inversor son factores que han de tenerse siempre en cuenta. En esta tesis se propone un micro-inversor derivado de la topología “forward” controlado en el límite entre los modos de conducción continuo y discontinuo (BCM por sus siglas en inglés). El transformador de la topología propuesta mantiene la misma estructura que en el convertidor “forward” clásico y la utilización de interruptores bidireccionales en el secundario permite la conexión directa del inversor a la red. Asimismo el método de control elegido permite obtener factor de potencia cercano a la unidad con una implementación sencilla. En la tesis se presenta el principio de funcionamiento y los principales aspectos del diseño del micro-inversor propuesto. Con la idea de mantener una solución sencilla y de bajo coste, se ha seleccionado un controlador analógico que está originalmente pensado para controlar un corrector del factor de potencia en el mismo modo de conducción que el micro-inversor “forward”. La tesis presenta las principales modificaciones necesarias, con especial atención a la detección del cruce por cero de la corriente (ZCD por sus siglas en inglés) y la compatibilidad del controlador con la inclusión de un algoritmo de búsqueda del punto de máxima potencia (MPPT por sus siglas en inglés). Los resultados experimentales muestran las limitaciones de la implementación elegida e identifican al transformador como el principal contribuyente a las pérdidas del micro-inversor. El principal objetivo de esta tesis es contribuir a la aplicación de técnicas de control y diseño de sistemas multifase en micro-inversores fotovoltaicos. En esta tesis se van a considerar dos configuraciones multifase diferentes aplicadas al micro-inversor “forward” propuesto. La primera consiste en una variación con conexión paralelo-serie que permite la utilización de transformadores con una relación de vueltas baja, y por tanto bien acoplados, para conseguir una ganancia de tensión adecuada con un mejor rendimiento. Esta configuración emplea el mismo control BCM cuando la potencia extraída del panel solar es máxima. Este método de control implica que la frecuencia de conmutación se incrementa considerablemente cuando la potencia decrece, lo que compromete el rendimiento. Por lo tanto y con la intención de mantener unos bueno niveles de rendimiento ponderado, el micro-inversor funciona en modo de conducción discontinuo (DCM, por sus siglas en inglés) cuando la potencia extraía del panel solar es menor que la máxima. La segunda configuración multifase considerada en esta tesis es la aplicación de la técnica de paralelo con entrelazado. Además se han considerado dos técnicas diferentes para decidir el número de fases activas: dependiendo de la potencia continua extraída del panel solar y dependiendo de la potencia instantánea demandada por el micro-inversor. La aplicación de estas técnicas es interesante en los sistemas fotovoltaicos conectados a la red eléctrica por la posibilidad que brindan de obtener un rendimiento prácticamente plano en un amplio rango de potencia. Las configuraciones con entrelazado se controlan en DCM para evitar la necesidad de un control de corriente, lo que es importante cuando el número de fases es alto. Los núcleos adecuados para todas las configuraciones multifase consideradas se seleccionan usando el producto de áreas. Una vez seleccionados los núcleos se ha realizado un diseño detallado de cada uno de los transformadores. Con la información obtenida de los diseños y los resultados de simulación, se puede analizar el impacto que el número de transformadores utilizados tiene en el tamaño y el rendimiento de las distintas configuraciones. Los resultados de este análisis, presentado en esta tesis, se utilizan posteriormente para comparar las distintas configuraciones. Muchas otras topologías se han presentado en la literatura para abordar los diferentes aspectos a considerar en los micro-inversores, que han sido presentados anteriormente. La mayoría de estas topologías utilizan un transformador de alta frecuencia para solventar el salto de tensión y evitar problemas de seguridad y de puesta a tierra. En cualquier caso, es interesante evaluar si topologías sin aislamiento galvánico son aptas para su utilización como micro-inversores. En esta tesis se presenta una revisión de inversores con capacidad de elevar tensión, que se comparan bajo las mismas especificaciones. El objetivo es proporcionar la información necesaria para valorar si estas topologías son aplicables en los módulos AC. Las principales contribuciones de esta tesis son: • La aplicación del control BCM a un convertidor “forward” para obtener un micro-inversor de una etapa sencillo y de bajo coste. • La modificación de dicho micro-inversor con conexión paralelo-series de transformadores que permite reducir la corriente de los semiconductores y una ganancia de tensión adecuada con transformadores altamente acoplados. • La aplicación de técnicas de entrelazado y decisión de apagado de fases en la puesta en paralelo del micro-inversor “forward”. • El análisis y la comparación del efecto en el tamaño y el rendimiento del incremento del número de transformadores en las diferentes configuraciones multifase. • La eliminación de las medidas y los lazos de control de corriente en las topologías multifase con la utilización del modo de conducción discontinuo y un algoritmo MPPT sin necesidad de medida de corriente. • La recopilación y comparación bajo las mismas especificaciones de topologías inversoras con capacidad de elevar tensión, que pueden ser adecuadas para la utilización como micro-inversores. Esta tesis está estructurada en seis capítulos. El capítulo 1 presenta el marco en que se desarrolla la tesis así como el alcance de la misma. En el capítulo 2 se recopilan las topologías existentes de micro-invesores con aislamiento y aquellas sin aislamiento cuya implementación en un módulo AC es factible. Asimismo se presenta la comparación entre estas topologías bajo las mismas especificaciones. El capítulo 3 se centra en el micro-inversor “forward” que se propone originalmente en esta tesis. La aplicación de las técnicas multifase se aborda en los capítulos 4 y 5, en los que se presentan los análisis en función del número de transformadores. El capítulo está orientado a la propuesta paralelo-serie mientras que la configuración con entrelazado se analiza en el capítulo 5. Por último, en el capítulo 6 se presentan las contribuciones de esta tesis y los trabajos futuros. ABSTRACT In the last decade the photovoltaic (PV) installed power increased with an average growth of 49% per year and it is expected to cover the 16% of the global electricity consumption by 2050. Most of the installed PV power corresponds to grid-connected systems, with a significant percentage of residential installations. In these PV systems, the inverter is essential since it is the responsible of transferring into the grid the extracted power from the PV modules. Several architectures have been proposed for grid-connected residential PV systems, including the AC-module technology. An AC-module consists of an inverter, also known as micro-inverter, which is attached to a PV module. The AC-module technology offers modularity, redundancy and individual MPPT of each module. In addition, the expansion of this technology will enable the possibility of economies of scale of mass market and “plug and play” for the user, thus reducing the overall cost of the installation. However, the micro-inverter must be able to provide the required voltage boost to interface a low voltage PV module to the grid while keeping an acceptable efficiency in a wide power range. Furthermore, the quality standards must be satisfied and size and lifetime of the solutions must be always considered. In this thesis a single-stage forward micro-inverter with boundary mode operation is proposed to address the micro-inverter requirements. The transformer in the proposed topology remains as in the classic forward converter and bidirectional switches in the secondary side allows direct connection to the grid. In addition the selected control strategy allows high power factor current with a simple implementation. The operation of the topology is presented and the main design issues are introduced. With the intention to propose a simple and low-cost solution, an analog controller for a PFC operated in boundary mode is utilized. The main necessary modifications are discussed, with the focus on the zero current detection (ZCD) and the compatibility of the controller with a MPPT algorithm. The experimental results show the limitations of the selected analog controller implementation and the transformer is identified as a main losses contributor. The main objective of this thesis is to contribute in the application of control and design multiphase techniques to the PV micro-inverters. Two different multiphase configurations have been applied to the forward micro-inverter proposed in this thesis. The first one consists of a parallel-series connected variation which enables the use of low turns ratio, i.e. well coupled, transformers to achieve a proper voltage boost with an improved performance. This multiphase configuration implements BCM control at maximum load however. With this control method the switching frequency increases significantly for light load operation, thus jeopardizing the efficiency. Therefore, in order to keep acceptable weighted efficiency levels, DCM operation is selected for low power conditions. The second multiphase variation considered in this thesis is the interleaved configuration with two different phase shedding techniques: depending on the DC power extracted from the PV panel, and depending on the demanded instantaneous power. The application of interleaving techniques is interesting in PV grid-connected inverters for the possibility of flat efficiency behavior in a wide power range. The interleaved variations of the proposed forward micro-inverter are operated in DCM to avoid the current loop, which is important when the number of phases is large. The adequate transformer cores for all the multiphase configurations are selected according to the area product parameter and a detailed design of each required transformer is developed. With this information and simulation results, the impact in size and efficiency of the number of transformer used can be assessed. The considered multiphase topologies are compared in this thesis according to the results of the introduced analysis. Several other topological solutions have been proposed to solve the mentioned concerns in AC-module application. The most of these solutions use a high frequency transformer to boost the voltage and avoid grounding and safety issues. However, it is of interest to assess if the non-isolated topologies are suitable for AC-module application. In this thesis a review of transformerless step-up inverters is presented. The compiled topologies are compared using a set benchmark to provide the necessary information to assess whether non-isolated topologies are suitable for AC-module application. The main contributions of this thesis are: • The application of the boundary mode control with constant off-time to a forward converter, to obtain a simple and low-cost single-stage forward micro-inverter. • A modification of the forward micro-inverter with primary-parallel secondary-series connected transformers to reduce the current stress and improve the voltage gain with highly coupled transformers. •The application of the interleaved configuration with different phase shedding strategies to the proposed forward micro-inverter. • An analysis and comparison of the influence in size and efficiency of increasing the number of transformers in the parallel-series and interleaved multiphase configurations. • Elimination of the current loop and current measurements in the multiphase topologies by adopting DCM operation and a current sensorless MPPT. • A compilation and comparison with the same specifications of suitable non-isolated step-up inverters. This thesis is organized in six chapters. In Chapter 1 the background of single-phase PV-connected systems is discussed and the scope of the thesis is defined. Chapter 2 compiles the existing solutions for isolated micro-inverters and transformerless step-up inverters suitable for AC-module application. In addition, the most convenient non-isolated inverters are compared using a defined benchmark. Chapter 3 focuses on the originally proposed single-stage forward micro-inverter. The application of multiphase techniques is addressed in Chapter 4 and Chapter 5, and the impact in different parameters of increasing the number of phases is analyzed. In Chapter 4 an original primary-parallel secondary-series variation of the forward micro-inverter is presented, while Chapter 5 focuses on the application of the interleaved configuration. Finally, Chapter 6 discusses the contributions of the thesis and the future work.

A Modified Positive Velocity and Position Feedback scheme with delay compensation for improved nanopositioning performance

Acknowledgments This paper was sponsored by the Spanish FPU12/00984 Program (Ministerio de Educacion, Cultura y Deporte). It was also sponsored by the Spanish Government Research Program with the Project DPI2012-37062-CO2-01 (Ministerio de Economia y Competitividad) and by the European Social Fund.

Parasite-mediated nuclear factor κB regulation in lymphoproliferation caused by Theileria parva infection

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor κB (NFκB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFκB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IκB molecules which normally sequester NFκB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IκBα. However, IκBα reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFκB-mediated positive feedback loop which restores cytoplasmic IκBα. In contrast, T. parva mediated continuous degradation of IκBβ resulting in persistently low cytoplasmic IκBβ levels. Normal IκBβ levels were only restored following T. parva killing, indicating that viable parasites are required for IκBβ degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IκB degradation and consequent enhanced expression of NFκB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IκB levels or NFκB activation, indicating that the parasite subverts the normal IκB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.

The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

RhoA GTPase and Serum Response Factor Control Selectively the Expression of MyoD without Affecting Myf5 in Mouse Myoblasts

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2 could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

A modifier screen in the eye reveals control genes for Krüppel activity in the Drosophila embryo

Irregular facets (If) is a dominant mutation of Drosophila that results in small eyes with fused ommatidia. Previous results showed that the gene Krüppel (Kr), which is best known for its early segmentation function, is expressed ectopically in If mutant eye discs. However, it was not known whether ectopic Kr activity is either the cause or the result of the If mutation. Here, we show that If is a gain-of-function allele of Kr. We then used the If mutation in a genetic screen to identify dominant enhancers and suppressors of Kr activity on the third chromosome. Of 30 identified Kr-interacting loci, two were cloned, and we examined whether they also represent components of a natural Kr-dependent developmental pathway of the embryo. We show that the two genes, eyelid (eld) and extramacrochaetae (emc), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique Kr-expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the Kr gain-of-function mutation If provides a tool to identify genes that are active during eye development and that a number of them function also in the control of Kr-dependent developmental processes.

Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of “mitotic chromatin.” Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop.

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.

Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a receptor-mediated mechanism.

Pulmonary neuroendocrine cells are localized predominantly at airway branchpoints. Previous work showed that gastrin-releasing peptide (GRP), a major pulmonary bombesin-like peptide, occurred in neuroendocrine cells exclusively in branching human fetal airways. We now demonstrate that GRP and GRP receptor genes are expressed in fetal mouse lung as early as embryonic day 12 (E12), when lung buds are beginning to branch. By in situ hybridization, GRP receptor transcripts were at highest levels in mesenchymal cells at cleft regions of branching airways and blood vessels. To explore the possibility that bombesin-like peptides might play a role in branching morphogenesis, E12 lung buds were cultured for 48 hr in serum-free medium. In the presence of 0.10-10 microM bombesin, branching was significantly augmented as compared with control cultures, with a peak of 94% above control values at 1 microM (P < 0.005). The bombesin receptor antagonist [Leu13- psi(CH2NH)Leu14]bombesin alone (100 nM) had no effect on baseline branching but completely abolished bombesin-induced branching. A bombesin-related peptide, [Leu8]phyllolitorin also increased branching (65% above control values at 10 nM, P < 0.005). [Leu8]Phyllolitorin also significantly augmented thymidine incorporation in cultured lung buds. Fibronectin, which is abundant at branchpoints, induces GRP gene expression in undifferentiated cell lines. These observations suggest that BLPs secreted by pulmonary neuroendocrine cells may contribute to lung branching morphogenesis. Furthermore, components of branchpoints may induce pulmonary neuroendocrine cell differentiation as part of a positive feedback loop, which could account in part for the high prevalence of these cells at branchpoints.

Controle da mistura ar/combustível em um motor a combustão interna: sistema em malha fechada.

O controle da mistura ar/combustível é muito importante para o correto funcionamento dos motores à combustão interna ciclo Otto. A relação entre o ar e o combustível influencia diretamente no funcionamento do motor, na emissão de poluentes e no consumo de combustível. Este trabalho apresenta o desenvolvimento de um controle da mistura ar/combustível a partir do estudo de modelos de malha fechada deste sistema. Esse controle tem por objetivo manter a mistura o mais próxima possível do ponto estequiométrico, a fim de otimizar a taxa de conversão de gases poluentes pelo catalisador, e utiliza um sensor de oxigênio, conhecido como sonda lambda, para realizar a realimentação do sistema, indicando se a mistura está no ponto estequiométrico. Este trabalho também apresenta o desenvolvimento de um compensador em malha fechada para controlar a mistura a/c (ar/combustível) em outros pontos, além do estequiométrico, através do uso de uma sonda lambda de banda larga.