Simulación eficiente de mecanismos flexibles mediante formulaciones topológicas

El objetivo de esta Tesis es presentar un método eficiente para la evaluación de sistemas multi-cuerpo con elementos flexibles con pequeñas deformaciones, basado en métodos topológicos para la simulación de sistemas tan complejos como los que se utilizan en la práctica y en tiempo real o próximo al real. Se ha puesto un especial énfasis en la resolución eficiente de aquellos aspectos que conllevan mayor coste computacional, tales como la evaluación de las ecuaciones dinámicas y el cálculo de los términos de inercia. Las ecuaciones dinámicas se establecen en función de las variables independientes del sistema, y la integración de las mismas se realiza mediante formulaciones implícitas de index-3. Esta Tesis se articula en seis Capítulos. En el Capítulo 1 se realiza una revisión bibliográfica de la simulación de sistemas flexibles y los métodos más relevantes de integración de las ecuaciones diferenciales del movimiento. Asimismo, se presentan los objetivos de esta Tesis. En el Capítulo 2 se presenta un método semi-recursivo para la evaluación de las ecuaciones de los sistemas multi-cuerpo con elementos flexibles basado en formulaciones topológicas y síntesis modal. Esta Tesis determina la posición de cada punto del cuerpo flexible en función de un sistema de referencia flotante que se mueve con dicho cuerpo y de las amplitudes de ciertos modos de deformación calculados a partir de un mallado obtenido mediante el Método de Elementos Finitos. Se presta especial atención en las condiciones de contorno que se han de tener en cuenta a la hora de establecer las variables que definen la deformación del cuerpo flexible. El Capítulo 3 se centra en la evaluación de los términos de inercia de los sistemas flexibles que generalmente conllevan un alto coste computacional. Se presenta un método que permite el cálculo de dichos términos basado en el uso de 24 matrices constantes que pueden ser calculadas previamente al proceso de integración. Estas matrices permiten evaluar la matriz de masas y el vector de fuerzas de inercia dependientes de la velocidad sin que sea necesario evaluar la posición deformada de todos los puntos del cuerpo flexible. Se realiza un análisis pormenorizado de dichas matrices con el objetivo de optimizar su cálculo estableciendo aproximaciones que permitan reducir el número de dichos términos y optimizar aún más su evaluación. Se analizan dos posibles simplificaciones: la primera utiliza una discretización no-consistente basada en elementos finitos en los que se definen únicamente los desplazamientos axiales de los nodos; en la segunda propuesta se hace uso de una matriz de masas concentradas (Lumped Mass). Basándose en la formulación presentada, el Capítulo 4 aborda la integración eficiente de las ecuaciones dinámicas. Se presenta un método iterativo para la integración con fórmulas de index-3 basado en la proyección de las ecuaciones dinámicas según las variables independientes del sistema multi-cuerpo. El cálculo del residuo del sistema de ecuaciones no lineales que se ha de resolver de modo iterativo se realiza mediante un proceso recursivo muy eficiente que aprovecha la estructura topológica del sistema. Se analizan tres formas de evaluar la matriz tangente del citado sistema no lineal: evaluación aproximada, numérica y recursiva. El método de integración presentado permite el uso de distintas fórmulas. En esta Tesis se analizan la Regla Trapezoidal, la fórmula BDF de segundo orden y un método híbrido TR-BDF2. Para este último caso se presenta un algoritmo de paso variable. En el Capítulo 5 plantea la implementación del método propuesto en un programa general de simulación de mecanismos que permita la resolución de cualquier sistema multi-cuerpo definiéndolo mediante un fichero de datos. La implementación de este programa se ha realizado tanto en C++ como en Java. Se muestran los resultados de las formulaciones presentadas en esta Tesis mediante la simulación de cuatro ejemplos de distinta complejidad. Mediante análisis concretos se comparan la formulación presentada con otras existentes. También se analiza el efecto del lenguaje de programación utilizado en la implementación y los efectos de las posibles simplificaciones planteadas. Por último, el Capítulo 6 resume las principales conclusiones alcanzadas en la Tesis y las futuras líneas de investigación que con ella se abren. ABSTRACT This Thesis presents an efficient method for solving the forward dynamics of a multi-body sys-tem formed by rigid and flexible bodies with small strains for real-time simulation of real-life models. It is based on topological formulations. The presented work focuses on the efficient solution of the most time-consuming tasks of the simulation process, such as the numerical integration of the motion differential equations and in particular the evaluation of the inertia terms corresponding to the flexible bodies. The dynamic equations are formulated in terms of independent variables of the muti-body system, and they are integrated by means of implicit index-3 formulae. The Thesis is arranged in six chapters. Chapter 1 presents a review of the most relevant and recent contributions related to the modelization of flexible multi-body systems and the integration of the corresponding dynamic equations. The main objectives of the Thesis are also presented in detail. Chapter 2 presents a semi-recursive method for solving the equations of a multi-body system with flexible bodies based on topological formulations and modal synthesis. This Thesis uses the floating frame approach and the modal amplitudes to define the position of any point at the flexible body. These modal deformed shapes are obtained by means of the Finite Element Method. Particular attention has been taken to the boundary conditions used to define the deformation of the flexible bodies. Chapter 3 focuses on the evaluation of the inertia terms, which is usually a very time-consuming task. A new method based on the use of 24 constant matrices is presented. These matrices are evaluated during the set-up step, before the integration process. They allow the calculation of the inertia terms in terms of the position and orientation of the local coordinate system and the deformation variables, and there is no need to evaluate the position and velocities of all the nodes of the FEM mesh. A deep analysis of the inertia terms is performed in order to optimize the evaluation process, reducing both the terms used and the number of arithmetic operations. Two possible simplifications are presented: the first one uses a non-consistent approach in order to define the inertia terms respect to the Cartesian coordinates of the FEM mesh, rejecting those corresponding to the angular rotations; the second approach makes use of lumped mass matrices. Based on the previously presented formulation, Chapter 4 is focused on the numerical integration of the motion differential equations. A new predictor-corrector method based on index-3 formulae and on the use of multi-body independent variables is presented. The evaluation of the dynamic equations in a new time step needs the solution of a set on nonlinear equations by a Newton-Raphson iterative process. The computation of the corresponding residual vector is performed efficiently by taking advantage of the system’s topological structure. Three methods to compute the tangent matrix are presented: an approximated evaluation that considers only the most relevant terms, a numerical approach based on finite differences and a recursive method that uses the topological structure. The method presented for integrating the dynamic equations can use a variety of integration formulae. This Thesis analyses the use of the trapezoidal rule, the 2nd order BDF formula and the hybrid TR-BDF2 method. A variable-time step strategy is presented for the last one. Chapter 5 describes the implementation of the proposed method in a general purpose pro-gram for solving any multibody defined by a data file. This program is implemented both in C++ and Java. Four examples are used to check the validity of the formulation and to compare this method with other methods commonly used to solve the dynamic equations of multi-body systems containing flexible bodies. The efficiency of the programming methodology used and the effect of the possible simplifications proposed are also analyzed. Chapter 6 summarizes the main Conclusions obtained in this Thesis and the new lines of research that have been opened.

Anomalous Consistency in Mild Cognitive Impairment: A Complex Networks Approach

Increased variability in performance has been associated with the emergence of several neurological and psychiatric pathologies. However, whether and how consistency of neuronal activity may also be indicative of an underlying pathology is still poorly understood. Here we propose a novel method for evaluating consistency from non-invasive brain recordings. We evaluate the consistency of the cortical activity recorded with magnetoencephalography in a group of subjects diagnosed with Mild Cognitive Impairment (MCI), a condition sometimes prodromal of dementia, during the execution of a memory task. We use metrics coming from nonlinear dynamics to evaluate the consistency of cortical regions. A representation known as parenclitic networks is constructed, where atypical features are endowed with a network structure, the topological properties of which can be studied at various scales. Pathological conditions correspond to strongly heterogeneous networks, whereas typical or normative conditions are characterized by sparsely connected networks with homogeneous nodes. The analysis of this kind of networks allows identifying the extent to which consistency is affected in the MCI group and the focal points where MCI is especially severe. To the best of our knowledge, these results represent the first attempt at evaluating the consistency of brain functional activity using complex networks theory.

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.

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ! 21 nM; Vmax 49 ! 21 pmol Ni2+ min"1 mg"1 bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(II) ions. This Ni2+ transport is not inhibited by similar cations such as Mn2+, Zn2+, or Co2+, but is blocked by Cu2+. Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein

Broken symmetry and strangeness of the semiconductor impurity band metal-insulator transition

The filamentary model of the metal-insulator transition in randomly doped semiconductor impurity bands is geometrically equivalent to similar models for continuous transitions in dilute antiferromagnets and even to the λ transition in liquid He, but the critical behaviors are different. The origin of these differences lies in two factors: quantum statistics and the presence of long range Coulomb forces on both sides of the transition in the electrical case. In the latter case, in addition to the main transition, there are two satellite transitions associated with disappearance of the filamentary structure in both insulating and metallic phases. These two satellite transitions were first identified by Fritzsche in 1958, and their physical origin is explained here in geometrical and topological terms that facilitate calculation of critical exponents.

Exploring the folding free energy surface of a three-helix bundle protein

The multidimensional free energy surface for a small fast folding helical protein is explored based on first-principle calculations. The model represents the 46-residue segment from fragment B of staphylococcal protein A. The relationship between collapse and tertiary structure formation, and the order of collapse and secondary structure formation, are investigated. We find that the initial collapse process gives rise to a transition state with about 30% of the native tertiary structure and 50–70% of the native helix content. We also observe two distinct distributions of native helix in this collapsed state (Rg ≈ 12 Å), one with about 20% of the native helical hydrogen bonds, the other with near 70%. The former corresponds to a local minimum. The barrier from this metastable state to the native state is about 2 kBT. In the latter case, folding is essentially a downhill process involving topological assembly. In addition, the order of formation of secondary structure among the three helices is examined. We observe cooperative formation of the secondary structure in helix I and helix II. Secondary structure in helix III starts to form following the formation of certain secondary structure in both helix I and helix II. Comparisons of our results with those from theory and experiment are made.

HOP-1, a Caenorhabditis elegans presenilin, appears to be functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 signaling

Mutant presenilins have been found to cause Alzheimer disease. Here, we describe the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro

Long-range promoter–enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken βA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein–protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.

HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

From residue matching patterns to protein folding topographies: General model and bovine pancreatic trypsin inhibitor

A coarse-grained model for protein-folding dynamics is introduced based on a discretized representation of torsional modes. The model, based on the Ramachandran map of the local torsional potential surface and the class (hydrophobic/polar/neutral) of each residue, recognizes patterns of both torsional conformations and hydrophobic-polar contacts, with tolerance for imperfect patterns. It incorporates empirical rates for formation of secondary and tertiary structure. The method yields a topological representation of the evolving local torsional configuration of the folding protein, modulo the basins of the Ramachandran map. The folding process is modeled as a sequence of transitions from one contact pattern to another, as the torsional patterns evolve. We test the model by applying it to the folding process of bovine pancreatic trypsin inhibitor, obtaining a kinetic description of the transitions between the contact patterns visited by the protein along the dominant folding pathway. The kinetics and detailed balance make it possible to invert the result to obtain a coarse topographic description of the potential energy surface along the dominant folding pathway, in effect to go backward or forward between a topological representation of the chain conformation and a topographical description of the potential energy surface governing the folding process. As a result, the strong structure-seeking character of bovine pancreatic trypsin inhibitor and the principal features of its folding pathway are reproduced in a reasonably quantitative way.

TnTIN and TnTAP: Mini-transposons for site-specific proteolysis in vivo

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.

Transport of torsional stress in DNA

It is well known that transcription can induce torsional stress in DNA, affecting the activity of nearby genes or even inducing structural transitions in the DNA duplex. It has long been assumed that the generation of significant torsional stress requires the DNA to be anchored, forming a limited topological domain, because otherwise it would spin almost freely about its axis. Previous estimates of the rotational drag have, however, neglected the role of small natural bends in the helix backbone. We show how these bends can increase the drag several thousandfold relative to prior estimates, allowing significant torsional stress even in linear unanchored DNA. The model helps explain several puzzling experimental results on structural transitions induced by transcription of DNA.

Positional enhancer-blocking activity of the chicken β-globin insulator in transiently transfected cells

It is thought that insulators demarcate transcriptionally and structurally independent chromatin domains. Insulators are detected by their ability to block enhancer–promoter interactions in a directional manner, and protect a transgene from position effects. Most studies are performed in stably transformed cells or organisms. Here we analyze the enhancer-blocking activity of the chicken β-globin insulator in transient transfection experiments in both erythroid and nonerythroid cell lines. We show that four tandem copies of a 90-bp fragment of this insulator were able to block an enhancer in these experiments. In circular plasmids, placement on either side of the enhancer reduced activity, but when the plasmid was linearized, the enhancer-blocking activity was observed only when the insulator was placed between the promoter and the enhancer. These observations are consistent with the position-dependent enhancer-blocking activity of the insulator observed in stable transformation experiments.

The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization

Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.