Design of a new neutron delivery system for high flux source

La construcci��n en la actualidad de nuevas fuentes para el uso de haces de neutrones as�� como los programas de renovaci��n en curso en algunas de las instalaciones experimentales existentes han evidenciado la necesidad urgente de desarrollar la tecnolog��a empleada para la construcci��n de gu��as de neutrones con objeto de hacerlas mas eficientes y duraderas. Esto viene motivado por el hecho de que varias instalaciones de experimentaci��n con haces de neutrones han reportado un n��mero de incidentes mec��nicos con tales gu��as, lo que hace urgente el progresar en nuestro conocimiento de los susbtratos v��treos sobre los cuales se depositan los espejos que permiten la reflexi��n total de los neutrones y como aquellos se degradan con la radiaci��n. La presente tesis se inscribe en un acuerdo de colaboraci��n establecido entre el Institut Max von Laue - Paul Langevin (ILL) de Grenoble y el Consorcio ESS-Bilbao con objeto de mejorar el rendimiento y sostenibilidad de los sistemas futuros de guiado de neutrones. El caso de la Fuente Europea de Espalaci��n en construcci��n en Lund sirve como ejemplo ya que se contempla la instalaci��n de gu��as de neutrones de m��s de 100 metros en algunos de los instrumentos. Por otro lado, instalaciones como el ILL prev��n tambi��n dentro del programa Endurance de rejuvenecimiento la reconstrucci��n de varias l��neas de transporte de haz. Para el presente estudio se seleccionaron cuatro tipos de vidrios borosilicatados que fueron el Borofloat, N-ZK7, N-BK7 y SBSL7. Los tres primeros son bien conocidos por los especialistas en instrumentaci��n neutr��nica ya que se han empleado en la construcci��n de varias instalaciones mientras que el ��ltimo es un candidato potencial en la fabricaci��n de substratos para espejos neutr��nicos en un futuro. Los cuatro vidrios tiene un contenido en ��xido de Boro muy similar, approximadamente un 10 mol.%. Tal hecho que obedece a las regulaciones para la fabricaci��n de estos dispositivos hace que tales substratos operen como protecci��n radiol��gica absorbiendo los neutrones transmitidos a trav��s del espejo de neutrones. Como contrapartida a tal beneficio, la reacci��n de captura 10B(n,_)7Li puede degradar el substrato v��treo debido a los 2.5 MeV de energ��a cin��tica depositados por la part��cula _ y los n��cleos en retroceso y de hecho la fragilidad de tales vidrios bajo radiaci��n ha sido atribuida desde hace ya tiempo a los efectos de esta reacci��n. La metodolog��a empleada en esta tesis se ha centrado en el estudio de la estructura de estos vidrios borosilicatados y como esta se comporta bajo condiciones de radiaci��n. Los materiales en cuesti��n presentan estructuras que dependen de su composici��n qu��mica y en particular del ratio entre formadores y modificadores de la red iono-covalente. Para ello se han empleado un conjunto de t��cnicas de caracterizaci��n tanto macro- como microsc��picas tales como estudios de dureza, TEM, Raman, SANS etc. que se han empleado tambi��n para determinar el comportamiento de estos materiales bajo radiaci��n. En particular, algunas propiedades macrosc��picas relacionadas con la resistencia de estos vidrios como elementos estructurales de las gu��as de neutrones han sido estudiadas as�� como tambi��n los cambios en la estructura v��trea consecuencia de la radiaci��n. Para este prop��sito se ha dise��ado y fabricado por el ILL un aparato para irradiaci��n de muestras con neutrones t��rmicos en el reactor del ILL que permite controlar la temperatura alcanzada por la muestra a menos de 100 ��C. Tal equipo en comparaci��n con otros ya existences permite en cuesti��n de dias acumular las dosis recibidas por una gu��a en operaci��n a lo largo de varios a��os. El uso conjunto de varias t��cnicas de caracterizaci��n ha llevado a revelar que los vidrios aqui estudiados son significativamente diferentes en cuanto a su estructura y que tales diferencias afectan a sus propiedades macrosc��picas asi como a su comportamiento bajo radiaci��n. Tal resultado ha sido sorprendente ya que, como se ha mencionado antes, algunos de estos vidrios eran bien conocidos por los fabricantes de gu��as de neutrones y hasta el momento eran considerados pr��cticamente similares debido a su contenido comparable en ��xido de Boro. Sin embargo, los materiales N-BK7 and S-BSL7 muetran gran homogeneidad a todas las escalas de longitud, y m��s espec��ficamente, a escalas nanom��tricas las subredes de S��lice y ��xido de Boro se mezclan dando logar a estructuras locales que recuerdan a la del cristal de Reedmergnerita. Por el contrario, N-ZK7 y Borofloat muestran dominios separados ricos en S��lice o Boro. Como era de esperar, las importantes diferencias arriba mencionadas se traducen en comportamientos dispares de estos materiales bajo un haz de neutrones t��rmicos. Los resultados muestran que el N-BK7 y el S-BSL7 son los m��s estables bajo radiaci��n, lo que macrosc��picamente hace que estos materiales muestren un comportamiento similar expandi��ndose lentamente en funci��n de la dosis recibida. Por el contario, los otros dos materiales muestran un comportamiento mucho m��s reactivo, que hace que inicialmente se compacten con la dosis recibida lo que hace que las redes de Silicio y Boro se mezclen resultando en un incremento en densidad hasta alcanzar un valor l��mite, seguido por un proceso de expansi��n lenta que resulta comparable al observado para N-BK7 y SBSL7. Estos resultados nos han permitido explicar el origen de las notorias diferencias observadas en cuanto a las dosis l��mite a partir de las cuales estos materiales desarrollan procesos de fragmentaci��n en superficie. ABSTRACT The building of new experimental neutron beam facilities as well as the renewal programmes under development at some of the already existing installations have pinpointed the urgent need to develop the neutron guide technology in order to make such neutron transport devices more efficient and durable. In fact, a number of mechanical failures of neutron guides have been reported by several research centres. It is therefore important to understand the behaviour of the glass substrates on top of which the neutron optics mirrors are deposited and how these materials degrade under radiation conditions. The case of the European Spallation Source (ESS) at present under construction at Lund is a good example. It previews the deployment of neutron guides having more than 100 metres of length for most of the instruments. Also, the future renovation programme of the ILL, called Endurance, foresees the refurbishment of several beam lines. This Ph.D. thesis was the result of a collaboration agreement between the ILL and ESS-Bilbao aiming to improve the performance and sustainability of future neutron delivery systems. Four different industrially produced alkali-borosilicate glasses were selected for this study: Borofloat, N-ZK7, N-BK7 and SBSL7. The first three are well known within the neutron instrumentation community as they have already been used in several installations whereas the last one is at present considered as a candidate for making future mirror substrates. All four glasses have a comparable content of boron oxide of about 10 mol.%. The presence of such a strong neutron absorption element is in fact a mandatory component for the manufacturing of neutron guides because it provides a radiological shielding for the environment. This benefit is however somewhat counterbalanced since the resulting 10B(n,_)7Li reactions degrade the glass due to the deposited energy of 2.5 MeV by the _ particle and the recoil nuclei. In fact, the brittleness of some of these materials has been ascribed to this reaction. The methodology employed by this study consisted in understanding the general structure of borosilicates and how they behave under irradiation. Such materials have a microscopic structure strongly dependent upon their chemical content and particularly on the ratios between network formers and modifiers. The materials have been characterized by a suite of macroscopic and structural techniques such as hardness, TEM, Raman, SANS, etc. and their behaviour under irradiation was analysed. Some macroscopic properties related to their resistance when used as guide structural elements were monitored. Also, changes in the vitreous structure due to radiation were observed by means of several experimental tools. For such a purpose, an irradiation apparatus has been designed and manufactured to enable irradiation with thermal neutrons within the ILL reactor while keeping the samples below 100 ��C. The main advantage of this equipment if compared to others previously available was that it allowed to reach in just some days an equivalent neutron dose to that accumulated by guides after several years of use. The concurrent use of complementary characterization techniques lead to the discovery that the studied glasses were deeply different in terms of their glass network. This had a strong impact on their macroscopic properties and their behaviour under irradiation. This result was a surprise since, as stated above, some of these materials were well known by the neutron guide manufacturers, and were considered to be almost equivalent because of their similar boron oxide content. The N-BK7 and S-BSL7 materials appear to be fairly homogeneous glasses at different length scales. More specifically, at nanometre scales, silicon and boron oxide units seem to mix and generate larger structures somewhat resembling crystalline Reedmergnerite. In contrast, N-ZK7 and Borofloat are characterized by either silicon or boron rich domains. As one could expect, these drastic differences lead to their behaviour under thermal neutron flux. The results show that N-BK7 and S-BSL7 are structurally the most stable under radiation. Macroscopically, such stability results in the fact that these two materials show very slow swelling as a function or radiation dose. In contrast, the two other glasses are much more reactive. The whole glass structure compacts upon radiation. Specifically, the silica network, and the boron units tend to blend leading to an increase in density up to some saturation, followed by a very slow expansion which comes to be of the same order than that shown by N-BK7 and S-BSL7. Such findings allowed us to explain the drastic differences in the radiation limits for macroscopic surface splintering for these materials when they are used in neutron guides.

Design of scour Protection Systems in Offshore Wind Farms

Scour compromises the operation of offshore facilities. This article describes the results of an investigation aimed at the analysis of different methods used in the scour protection systems design at offshore wind farms. The study is focused on transitional waters, where monopile foundations present medium or large diameters. Using the experience of offshore wind farms currently installed, a new design formula is proposed. All of this with the aim of improving a preliminary design of scour protection systems considering maritime parameters

VHDL-based system design of a cognitive sensorimotor loop (CSL) for haptic Human-Machine Interaction (HMI)

This document is a summary of the Bachelor thesis titled ���VHDL-Based System Design of a Cognitive Sensorimotor Loop (CSL) for Haptic Human-Machine Interaction (HMI)��� written by Pablo de Miguel Morales, Electronics Engineering student at the Universidad Polit��cnica de Madrid (UPM Madrid, Spain) during an Erasmus+ Exchange Program at the Beuth Hochschule f��r Technik (BHT Berlin, Germany). The tutor of this project is Dr. Prof. Hild. This project has been developed inside the Neurobotics Research Laboratory (NRL) in close collaboration with Benjamin Panreck, a member of the NRL, and another exchange student from the UPM Pablo Gabriel Lezcano. For a deeper comprehension of the content of the thesis, a deeper look in the document is needed as well as the viewing of the videos and the VHDL design. In the growing field of automation, a large amount of workforce is dedicated to improve, adapt and design motor controllers for a wide variety of applications. In the specific field of robotics or other machinery designed to interact with humans or their environment, new needs and technological solutions are often being discovered due to the existing, relatively unexplored new scenario it is. The project consisted of three main parts: Two VHDL-based systems and one short experiment on the haptic perception. Both VHDL systems are based on a Cognitive Sensorimotor Loop (CSL) which is a control loop designed by the NRL and mainly developed by Dr. Prof. Hild. The CSL is a control loop whose main characteristic is the fact that it does not use any external sensor to measure the speed or position of the motor but the motor itself. The motor always generates a voltage that is proportional to its angular speed so it does not need calibration. This method is energy efficient and simplifies control loops in complex systems. The first system, named CSL Stay In Touch (SIT), consists in a one DC motor system controller by a FPGA Board (Zynq ZYBO 7000) whose aim is to keep contact with any external object that touches its Sensing Platform in both directions. Apart from the main behavior, three features (Search Mode, Inertia Mode and Return Mode) have been designed to enhance the haptic interaction experience. Additionally, a VGA-Screen is also controlled by the FPGA Board for the monitoring of the whole system. This system has been completely developed, tested and improved; analyzing its timing and consumption properties. The second system, named CSL Fingerlike Mechanism (FM), consists in a fingerlike mechanical system controlled by two DC motors (Each controlling one part of the finger). The behavior is similar to the first system but in a more complex structure. This system was optional and not part of the original objectives of the thesis and it could not be properly finished and tested due to the lack of time. The haptic perception experiment was an experiment conducted to have an insight into the complexity of human haptic perception in order to implement this knowledge into technological applications. The experiment consisted in testing the capability of the subjects to recognize different objects and shapes while being blindfolded and with their ears covered. Two groups were done, one had full haptic perception while the other had to explore the environment with a plastic piece attached to their finger to create a haptic handicap. The conclusion of the thesis was that a haptic system based only on a CSL-based system is not enough to retrieve valuable information from the environment and that other sensors are needed (temperature, pressure, etc.) but that a CSL-based system is very useful to control the force applied by the system to interact with haptic sensible surfaces such as skin or tactile screens. RESUMEN. Este documento es un resumen del proyecto fin de grado titulado ���VHDL-Based System Design of a Cognitive Sensorimotor Loop (CSL) for Haptic Human-Machine Interaction (HMI)��� escrito por Pablo de Miguel, estudiante de Ingenier��a Electr��nica de Comunicaciones en la Universidad Polit��cnica de Madrid (UPM Madrid, Espa��a) durante un programa de intercambio Erasmus+ en la Beuth Hochschule f��r Technik (BHT Berlin, Alemania). El tutor de este proyecto ha sido Dr. Prof. Hild. Este proyecto se ha desarrollado dentro del Neurorobotics Research Laboratory (NRL) en estrecha colaboraci��n con Benjamin Panreck (un miembro del NRL) y con Pablo Lezcano (Otro estudiante de intercambio de la UPM). Para una comprensi��n completa del trabajo es necesaria una lectura detenida de todo el documento y el visionado de los videos y an��lisis del dise��o VHDL incluidos en el CD adjunto. En el creciente sector de la automatizaci��n, una gran cantidad de esfuerzo est�� dedicada a mejorar, adaptar y dise��ar controladores de motor para un gran rango de aplicaciones. En el campo espec��fico de la rob��tica u otra maquinaria dise��ada para interactuar con los humanos o con su entorno, nuevas necesidades y soluciones tecnol��gicas se siguen desarrollado debido al relativamente inexplorado y nuevo escenario que supone. El proyecto consta de tres partes principales: Dos sistemas basados en VHDL y un peque��o experimento sobre la percepci��n h��ptica. Ambos sistemas VHDL est��n basados en el Cognitive Sesnorimotor Loop (CSL) que es un lazo de control creado por el NRL y cuyo desarrollador principal ha sido Dr. Prof. Hild. El CSL es un lazo de control cuya principal caracter��stica es la ausencia de sensores externos para medir la velocidad o la posici��n del motor, usando el propio motor como sensor. El motor siempre genera un voltaje proporcional a su velocidad angular de modo que no es necesaria calibraci��n. Este m��todo es eficiente en t��rminos energ��ticos y simplifica los lazos de control en sistemas complejos. El primer sistema, llamado CSL Stay In Touch (SIT), consiste en un sistema formado por un motor DC controlado por una FPGA Board (Zynq ZYBO 7000) cuyo objetivo es mantener contacto con cualquier objeto externo que toque su plataforma sensible en ambas direcciones. Aparte del funcionamiento b��sico, tres modos (Search Mode, Inertia Mode y Return Mode) han sido dise��ados para mejorar la interacci��n. Adicionalmente, se ha dise��ado el control a trav��s de la FPGA Board de una pantalla VGA para la monitorizaci��n de todo el sistema. El sistema ha sido totalmente desarrollado, testeado y mejorado; analizando su propiedades de timing y consumo energ��tico. El segundo sistema, llamado CSL Fingerlike Mechanism (FM), consiste en un mecanismo similar a un dedo controlado por dos motores DC (Cada uno controlando una falange). Su comportamiento es similar al del primer sistema pero con una estructura m��s compleja. Este sistema no formaba parte de los objetivos iniciales del proyecto y por lo tanto era opcional. No pudo ser plenamente desarrollado debido a la falta de tiempo. El experimento de percepci��n h��ptica fue dise��ado para profundizar en la percepci��n h��ptica humana con el objetivo de aplicar este conocimiento en aplicaciones tecnol��gicas. El experimento consist��a en testear la capacidad de los sujetos para reconocer diferentes objetos, formas y texturas en condiciones de privaci��n del sentido del o��do y la vista. Se crearon dos grupos, en uno los sujetos ten��an plena percepci��n h��ptica mientras que en el otro deb��an interactuar con los objetos a trav��s de una pieza de pl��stico para generar un h��ndicap h��ptico. La conclusi��n del proyecto fue que un sistema h��ptico basado solo en sistemas CSL no es suficiente para recopilar informaci��n valiosa del entorno y que debe hacer uso de otros sensores (temperatura, presi��n, etc.). En cambio, un sistema basado en CSL es id��neo para el control de la fuerza aplicada por el sistema durante la interacci��n con superficies h��pticas sensibles tales como la piel o pantallas t��ctiles.

Design of a generalized tool for the performance assessment under sail based on analytical, numerical and empirical results : a global sailing yacht meta-model

La evaluaci��n de las prestaciones de las embarcaciones a vela ha constituido un objetivo para ingenieros navales y marinos desde los principios de la historia de la navegaci��n. El conocimiento acerca de estas prestaciones, ha crecido desde la identificaci��n de los factores clave relacionados con ellas(eslora, estabilidad, desplazamiento y superficie v��lica), a una comprensi��n m��s completa de las complejas fuerzas y acoplamientos involucrados en el equilibrio. Junto con este conocimiento, la aparici��n de los ordenadores ha hecho posible llevar a cabo estas tareas de una forma sistem��tica. Esto incluye el c��lculo detallado de fuerzas, pero tambi��n, el uso de estas fuerzas junto con la descripci��n de una embarcaci��n a vela para la predicci��n de su comportamiento y, finalmente, sus prestaciones. Esta investigaci��n tiene como objetivo proporcionar una definici��n global y abierta de un conjunto de modelos y reglas para describir y analizar este comportamiento. Esto se lleva a cabo sin aplicar restricciones en cuanto al tipo de barco o c��lculo, sino de una forma generalizada, de modo que sea posible resolver cualquier situaci��n, tanto estacionaria como en el dominio del tiempo. Para ello se comienza con una definici��n b��sica de los factores que condicionan el comportamiento de una embarcaci��n a vela. A continuaci��n se proporciona una metodolog��a para gestionar el uso de datos de diferentes or��genes para el c��lculo de fuerzas, siempre con el la soluci��n del problema como objetivo. Esta ��ltima parte se plasma en un programa de ordenador, PASim, cuyo prop��sito es evaluar las prestaciones de diferentes ti pos de embarcaciones a vela en un amplio rango de condiciones. Varios ejemplos presentan diferentes usos de PASim con el objetivo de ilustrar algunos de los aspectos discutidos a lo largo de la definici��n del problema y su soluci��n . Finalmente, se presenta una estructura global de cara a proporcionar una representaci��n virtual de la embarcaci��n real, en la cual, no solo e l comportamiento sino tambi��n su manejo, son cercanos a la experiencia de los navegantes en el mundo real. Esta estructura global se propone como el n��cleo (un motor de software) de un simulador f��sico para el que se proporciona una especificaci��n b��sica. ABSTRACT The assessment of the performance of sailing yachts, and ships in general, has been an objective for naval architects and sailors since the beginning of the history of navigation. The knowledge has grown from identifying the key factors that influence performance(length, stability, displacement and sail area), to a much more complete understanding of the complex forces and couplings involved in the equilibrium. Along with this knowledge, the advent of computers has made it possible to perform the associated tasks in a systematic way. This includes the detailed calculation of forces, but also the use of those forces, along with the description of a sailing yacht, to predict its behavior, and ultimately, its performance. The aim of this investigation is to provide a global and open definition of a set of models and rules to describe and analyze the behavior of a sailing yacht. This is done without applying any restriction to the type of yacht or calculation, but rather in a generalized way, capable of solving any possible situation, whether it is in a steady state or in the time domain. First, the basic definition of the factors that condition the behavior of a sailing yacht is given. Then, a methodology is provided to assist with the use of data from different origins for the calculation of forces, always aiming towards the solution of the problem. This last part is implemented as a computational tool, PASim, intended to assess the performance of different types of sailing yachts in a wide range of conditions. Several examples then present different uses of PASim, as a way to illustrate some of the aspects discussed throughout the definition of the problem and its solution. Finally, a global structure is presented to provide a general virtual representation of the real yacht, in which not only the behavior, but also its handling is close to the experience of the sailors in the real world. This global structure is proposed as the core (a software engine) of a physical yacht simulator, for which a basic specification is provided.

Design of broadband transmitarray unit cells with comparative study of different numbers of layers

A generalized methodology to design low-profile transmitarray (TA) antennas made of several stacked layers with nonresonant printed phasing elements is presented. A study of the unit cell bandwidth, phase-shift range and tolerances has been conducted considering different numbers of layers. A structure with three metalized layers with capacitive and inductive elements enabling a phase range of nearly 360�� and low insertion loss is introduced. A study of the four-layer structure shows improvement in the performance of the unit cells in terms of bandwidth from 2% to more than 20% and a complete phase coverage. Implementations on a flexible substrate of TAs with progressive phase shift operating at 19 GHz are used for validation.

Optimal design of a beam subjected to compression forces in the framework of different structural models

The optimal design of a vertical cantilever beam is presented in this paper. The beam is assumed immersed in an elastic Winkler soil and subjected to several loads: a point force at the tip section, its self weight and a uniform distributed load along its length. lbe optimal design problem is to find the beam of a given length and minimum volume, such that the resultant compressive stresses are admisible. This prohlem is analyzed according to linear elasticity theory and within different alternative structural models: column, Navier-Bernoulli beam-column, Timoshenko beamcolumn (i.e. with shear strain) under conservative loads, typically, constant direction loads. Results obtained in each case are compared, in order to evaluate the sensitivity of model on the numerical results. The beam optimal design is described by the section distribution layout (area, second moment, shear area etc.) along the beam span and the corresponding beam total volume. Other situations, some of them very interesting from a theoretical point of view, with follower loads (Beck and Leipholz problems) are also discussed, leaving for future work numerical details and results.

Exploring the origins of topological frustration: Design of a minimally frustrated model of fragment B of protein A

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from Staphylococcus aureus was investigated. This G��-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht �� values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate ��, whereas highly frustrated systems display a bimodal distribution peaked at low and high �� values. The distribution of �� values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at �� = 0.3 with tails extending to low and high �� values, indicating the presence of a small amount of topological frustration. The contacts with high �� values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of �� values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration.

Design of compounds that increase the absorption of polar���molecules

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the c2j gene substituted for the wild-type tyrosinase allele: Utilization in design of syngeneic immunotherapy���models

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase���PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the ��1b and ��1d mRNA splice variants. The spontaneous c2j albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In c2j/c2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably ��1b) present in C/C were obligatorily absent, others (��3 and ��1d) were elevated. In c2j/c2j melanomas, the percentage of total tyrosinase transcripts attributable to ��3 reached approximately 2-fold the incidence in c2j/c2j or C/C skin melanocytes. The percentage attributable to ��1d rose to approximately 2-fold the incidence in c2j/c2j skin, and to 10-fold that in C/C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C/C or c2j/c2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability

We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to ���1.49 (more stable N239Y) kcal mol���1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol���1 and Tm raised by 5.6��C is of potential interest for trials in vivo.

Design of potent and selective human cathepsin K inhibitors that span the active���site

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Design of fast enzymes by optimizing interaction potential in active���site

The diffusional encounter between substrate and enzyme, and hence catalytic efficiency, can be enhanced by mutating charged residues on the surface of the enzyme. In this paper we present a simple method for screening such mutations. This is based on our earlier result that electrostatic enhancement of the enzyme-substrate binding rate constant can be accounted for just by the interaction potential within the active site. Assuming that catalytic and structural integrity is maintained, the catalytic efficiency can be optimized by surface charge mutations which lead to stronger interaction potential within the active site. Application of the screening method on superoxide dismutase shows that only charge mutations close to the active site will have practical effect on the catalytic efficiency. This rationalizes a large number of findings obtained in previous simulation and experimental studies.

Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC

Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure���activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the ��-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

Design of three-dimensional domain-swapped dimers and fibrous oligomers

Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 ��� resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.