Scour prediction and scour protections in offshore wind farms

This paper is the result of research whose main objective is to analyse different methods used for the prediction of maximum scour depth and scour extension, and for the design of scour protections in offshore wind farms located in shallow water, using medium and large diameter monopile foundations. Physical agents such as waves, currents and wind play a major role in the design of structures like offshore farms. As a result, the study has highlighted the need for introducing experience backed climate monomials such as the dimensionless wave height parameter (H0) and proposes the use of formulations that can express the extent of scour protections as a function of waves in transitional waters.

Survey on student motivation and approval of educational environment at Universidad Politécnica de Madrid within the European Higher Education area implementation

A recent study elaborated by Vicerrectorado de Ordenación Académica y Planificación Estratégica of Technical University of Madrid (UPM) defines the satisfaction of the university student body as "the response that the University offers to the expectations and demands of service of the students, considered in a general way ". Besides an indicator of academic and institutional insertion of the student, the assessment of student engagement allows us to adapt the academic offer and the extension services of the University to the real needs of the students. The process of convergence towards the European Higher Education Area (EHEA) raises the need to form in competitions, that is to say, of developing in our students capacities and knowledge beyond the purely theoretical-practical thing. Therefore, the perception and experience of the educational process and environment by the students is an important issue to be addressed to accomplish their expectations and achieve a curriculum accordingly to EHEA expectations. The present study aims to explore the student motivation and approval of the educational environment at the UPM. To this end a total of 97 students enrolled in the undergraduate program of Civil Engineering, Computer Engineering and Agronomic Engineering at UPM were surveyed. The survey consisted of 40 questions divided in three blocks. The first one of 20 questions of personal character in that they were gathering, besides the sex and the age, the degree of fulfilment, implication and dedication with the institution and the academic tasks. In the second block we identify 10 questions related to the perception of the student on the teaching quality, and finally a block of 10 questions regarding the Bologna Process. The students personal motivation was moderately high, with a score of 3.6 (all scores are provided on a 5-point scale), being the most valuable items obtaining a university degree (4,3) and the friendship between students (4,2). Any significant difference was shown between sexes (P=0.23) since the averages for this block of questions were of 3.7±0.3 and 3.5±0.4 for women and men respectively. The students are moderately satisfied with their graduate studies with an average score of 3,2, being the questions that reflect a minor satisfaction the research profile of the teachers (2,8) and the organization of the Schools (2,9). The best valued questions are related to the usefulness and quality of the degrees, with 3,5 and 3,4 respectively, and to the interest of the courses within the degree (3,4). For sexes, the results of this block of questions are similar (3.1±0.3 and 3.2±0.3 for men and women respectively=0.79). Also, there were no differences (P=0.39) between the students who arrange work and studies or do not work (3.1±0.2 and 3.2±0.3 respectively). In conclusion, students at UPM present an acceptable degree of motivation and satisfaction with regard to the studies and services that offer their respective Schools. Both characteristics receive the same value both for men and for women and so much for students who arrange work and studies as for those who devote themselves only to studying. In a significant way, students who are more engaged and are in-class attendants present the major degree of satisfaction.Overall, there is a great lack of information regarding the Bologna Process. In fact to the majority, they would like to know more on what it is, what it means and what changes will involve its implementation.

Uso de una red inalámbrica de sensores para estimar riesgo de incendio

Una red inalámbrica de sensores (Wireless Sensor Network, WSN) constituye un sistema de comunicación de datos flexible utilizado como alternativa a las redes cableadas o como extensión de éstas y está compuesta por elementos de cómputo, medición y comunicación, que permiten al administrador instrumentar, observar y reaccionar a eventos y fenómenos en un ambiente específico. Una de las aplicaciones de estas redes es su uso en sistemas de predicción y prevención de incendios en áreas naturales. Su implementación se basa en el despliegue de sensores inalámbricos, realizado en una zona de riesgo de incendio para que puedan recolectar información sobre parámetros ambientales como temperatura, humedad, luz o presión, entre otros. Desde una estación base (o nodo "sumidero"), se suministra la información de los sensores a un centro de monitorización y control de forma estructurada. En este centro la información recibida puede ser analizada, procesada y visualizada en tiempo real. Desde este centro de control se puede controlar también la red WSN modificando el comportamiento de los sensores según el nivel de riesgo de incendio detectado. Este proyecto se basa en el diseño, implementación y despliegue de una red inalámbrica de sensores en un entorno simulado para observar su comportamiento en diferentes situaciones y mostrar su eficacia ante un posible caso de incendio. La implementación de este sistema denominado Sistema de Estimación de Riesgo de Incendio Utilizando una WSN (SERIUW) , junto con el desarrollado, en paralelo, de otro proyecto denominado Sistema de Control y Visualización de Información sobre Riesgo de Incendio (SCVIRI) que implementa las funciones de los centros de monitorización y control, conforman un Sistema de Anticipación y Seguimiento de Fuegos (SASF). Se han realizado pruebas de funcionalidad y eficacia, incluidas en la presente memoria del sistema unitario de en conjunto (ambos proyectos), en un entorno controlado simulado. Este sistema es una solución para la lucha contra los incendios forestales ya que predice y previene, de forma temprana, posibles incendios en las áreas naturales bajo supervisión. Ante un evento de incendio declarado este sistema es un poderoso instrumento de apoyo permitiendo, por un lado, generar alertas automáticas (con localización y gravedad de fuegos detectados) y por el otro, hacer un seguimiento del incendio con mapas en tiempo real (con su consecuente apoyo para la protección e información con las brigadas de bomberos en las zonas activas). ABSTRACT. A wireless sensor network (WSN) is a flexible data communication system used as an alternative to wired networks or as an extension of them and consists of nodes that perform calculation, measurement and communication activities. This allows the administrator to observe and react to events and phenomena in a specific environment. One application of these networks is fire prediction and prevention in natural areas. Its implementation is based on a deployment of wireless sensors, in a fire risk area, capable of collecting information such as temperature, humidity, luminance and pressure. A base station (or "sink") sends the collected information to a monitoring and control center following a structured format. At this center, the information received can be analyzed, processed and displayed in real time with monitoring systems. From this control center the WSN can also be controlled by changing the sensors behavior according to the level of fire risk detection. This project is based on the design, implementation and deployment of a Wireless Sensor Network (WSN) in a simulated environment in order to observe its behavior in different situations and show its effectiveness against a possible fire environment. The implementation of this system called SERIUW, has been done in parallel with other system, called SCVIRI, which has been developed in another project that implements the functions of monitoring and control center. Together, these two systems, make up a general system of anticipation and monitoring of fires. Functionality and performance tests have been performed on the overall system, in a controlled and simulated environment. The results of these tests are included in this document. The global system is a solution to fight the forest fires because it makes it easier to predict and prevent, early, possible fires in natural areas under supervision. This sytem can be a powerful tool since, before a fire event is declared, it generates automatic alerts (including location and severity information) and allows the real-time motorization of fire evolution integrated with maps. This could be also very useful for the support protection and information of fire brigades in zones in which a fire is already active.

Desarrollo de frameworks con .NET

La expansión experimentada por la informática, las nuevas tecnologías e internet en los últimos años, no solo viene dada por la evolución del hardware subyacente, sino por la evolución del desarrollo de software y del crecimiento del número de desarrolladores. Este incremento ha hecho evolucionar el software de unos sistemas de gestión basados en ficheros, prácticamente sin interfaz gráfico y de unos pocos miles de líneas a grandes sistemas distribuidos multiplataforma. El desarrollo de estos grandes sistemas, requiere gran cantidad de personas involucradas en el desarrollo, y que las herramientas de desarrollo hayan crecido también para facilitar su análisis, diseño, codificación, pruebas, implantación y mantenimiento. La base de estas herramientas software las proveen las propias plataformas de desarrollo, pero la experiencia de los desarrolladores puede aportar un sinfín de utilidades y de técnicas que agilicen los desarrollos y cumplan los requisitos del software en base a la reutilización de soluciones lo suficientemente probadas y optimizadas. Dichas herramientas se agrupan ordenadamente, creando así frameworks personalizados, con herramientas de todo tipo, clases, controles, interfaces, patrones de diseño, de tal manera que se dan soluciones personalizadas a un amplio número de problemas para emplearlas cuantas veces se quiera, bien marcando directrices de desarrollo mediante el uso de patrones, bien con la encapsulación de complejidades de tal modo que los desarrolladores ya dispongan de componentes que asuman cierta lógica o cierta complejidad aliviando así la fase de construcción. En este trabajo se abordan temas sobre las tecnologías base y plataformas de desarrollo para poder acometer la creación de un framework personalizado, necesidades a evaluar antes de acometerlo, y técnicas a emplear para la consecución del mismo, orientadas a la documentación, mantenimiento y extensión del framework. La exposición teórica consiste en mostrar y evaluar los requisitos para crear un framework, requisitos de la plataforma de desarrollo, y explicar cómo funcionan las grandes plataformas de desarrollo actuales, que elementos los componen y su funcionamiento, así como marcar ciertas pautas de estructuración y nomenclatura que el desarrollo de un framework debe contemplar para su mantenimiento y extensión. En la parte metodológica se ha usado un subconjunto de Métrica V3, ya que para el desarrollo de controles no aplica dicha metodología en su totalidad, pero contempla el catálogo de requisitos, los casos de uso, diagramas de clase, diagramas de secuencia, etc… Aparte de los conceptos teóricos, se presenta un caso práctico con fines didácticos de cómo parametrizar y configurar el desarrollo bajo la plataforma .NET. Dicho caso práctico consiste en la extensión de un control de usuario genérico de la plataforma .NET, de tal modo que se aplican conceptos más allá del hecho de crear funciones como las funcionalidades que puede brindar un API. Conceptos sobre como extender y modificar controles ya existentes, que interactúan por medio de eventos con otros controles, con vistas a que ese nuevo control forme parte de una biblioteca de controles de usuario personalizados ampliamente divulgada. Los controles de usuario son algo que no solo tienen una parte funcional, sino que también tienen una parte visual, y definiciones funcionales distintas de las típicas del software de gestión, puesto que han de controlar eventos, visualizaciones mientras se dan estos eventos y requisitos no funcionales de optimización de rendimiento, etc… Para el caso práctico se toma como herramienta la plataforma de desarrollo .Net Framework, en todas sus versiones, ya que el control a extender es el control ListView y hacerlo editable. Este control está presente en todas las versiones de .NET framework y con un alto grado de reutilización. Esta extensión muestra además como se puede migrar fácilmente este tipo de extensiones sobre todos los frameworks. Los entornos de desarrollo usados son varias versiones de Visual Studio para el mostrar dicha compatibilidad, aunque el desarrollo que acompaña este documento esté realizado sobre Visual Studio 2013. ABSTRACT The expansion in computer science, new technologies and the Internet in recent years, not only is given by the evolution of the underlying hardware, but for the evolution of software development and the growing number of developers. This increase has evolved software from management systems based on files almost without graphical interface and a few thousand of code lines, to large multiplatform distributed systems. The development of these large systems, require lots of people involved in development, and development tools have also grown to facilitate analysis, design, coding, testing, deployment and maintenance. The basis of these software tools are providing by their own development platforms, but the experience of the developers can bring a lot of utilities and techniques to speed up developments and meet the requirements of software reuse based on sufficiently proven solutions and optimized. These tools are grouped neatly, creating in this way custom frameworks, with tools of all types, classes, controls, interfaces, design patterns,… in such a way that they provide customized solutions to a wide range of problems to use them many times as you want to occur, either by dialing development guidelines by using patterns or along with the encapsulation of complexities, so that developers already have components that take some logic or some complexity relieving the construction phase. This paper cover matters based on technologies and development platforms to undertake the creation of a custom framework, needs to evaluate before rush it and techniques to use in order to achieve it, a part from techniques oriented to documentation, maintenance and framework extension. The theoretical explanation consists in to demonstrate and to evaluate the requirements for creating a framework, development platform requirements, and explain how large current development platforms work, which elements compose them and their operation work, as well as mark certain patterns of structure and nomenclature that the development of a framework should include for its maintenance and extension. In the methodological part, a subset of Métrica V3 has been used, because of, for the development of custom controls this methodology does not apply in its entirety, but provides a catalogue of requirements, use cases, class diagrams, sequence diagrams, etc ... Apart from the theoretical concepts, a study case for teaching purposes about how to parameterize and configure the development under the .NET platform is presented. This study case involves the extension of a generic user control of the .NET platform, so that concepts apply beyond the fact of creating functions as the functionalities that can provide an API. Concepts on how to extend and modify existing controls that interact through events with other controls, overlooking that new control as a part of a custom user controls library widely publicized. User controls are something that not only have a functional part, but also have a visual part, and various functional definitions of typical management software, since that they have to control events, visualizations while these events are given and not functional of performance optimization requirements, etc ... For the study case the development platform .Net Framework is taken as tool, in all its versions, considering that control to extend is the ListView control and make it editable. This control is present in all versions of .NET framework and with a high degree of reuse. This extension also shows how you can easily migrate these extensions on all frameworks. The used development environments are several versions of Visual Studio to show that compatibility, although the development that accompanies this document is done on Visual Studio 2013.

Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ0 and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4+CD8– cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage

hMSH2⋅hMSH6 heterodimer (hMutSα) and hMLH1⋅hPMS2 complex (hMutLα) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce O6-methylguanine (O6MeG) adducts. This study demonstrates that O6MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the SN1 DNA methylators N-methyl-N-nitrosourea or N-methyl-N′-nitro-N-nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSα and hMutLα. Coupled with the previous demonstration that O6MeG⋅T and O6MeG⋅C pairs are recognized by hMutSα, these results implicate action of the mismatch repair system in the initial step of a damage-signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage.

Protein tyrosine phosphatase PTP1B suppresses p210 bcr-abl-induced transformation of Rat-1 fibroblasts and promotes differentiation of K562 cells

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.

Stretched and overwound DNA forms a Pauling-like structure with exposed bases

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from σ ≈ −0.015 down to σ = −1. Surprisingly, for positively supercoiled DNA (σ > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has ≈2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling’s early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84–97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671–674].

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Differential Roles of Syntaxin 7 and Syntaxin 8 in Endosomal Trafficking

To understand molecular mechanisms that regulate the intricate and dynamic organization of the endosomal compartment, it is important to establish the morphology, molecular composition, and functions of the different organelles involved in endosomal trafficking. Syntaxins and vesicle-associated membrane protein (VAMP) families, also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), have been implicated in mediating membrane fusion and may play a role in determining the specificity of vesicular trafficking. Although several SNAREs, including VAMP3/cellubrevin, VAMP8/endobrevin, syntaxin 13, and syntaxin 7, have been localized to the endosomal membranes, their precise localization, biochemical interactions, and function remain unclear. Furthermore, little is known about SNAREs involved in lysosomal trafficking. So far, only one SNARE, VAMP7, has been localized to late endosomes (LEs), where it is proposed to mediate trafficking of epidermal growth factor receptor to LEs and lysosomes. Here we characterize the localization and function of two additional endosomal syntaxins, syntaxins 7 and 8, and propose that they mediate distinct steps of endosomal protein trafficking. Both syntaxins are found in SNARE complexes that are dissociated by α-soluble NSF attachment protein and NSF. Syntaxin 7 is mainly localized to vacuolar early endosomes (EEs) and may be involved in protein trafficking from the plasma membrane to the EE as well as in homotypic fusion of endocytic organelles. In contrast, syntaxin 8 is likely to function in clathrin-independent vesicular transport and membrane fusion events necessary for protein transport from EEs to LEs.

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.

Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: A critical role for nuclear targeting

Parathyroid hormone-related protein (PTHrP) is a prohormone that is posttranslationally processed to a family of mature secretory forms, each of which has its own cognate receptor(s) on the cell surface that mediate the actions of PTHrP. In addition to being secreted via the classical secretory pathway and interacting with cell surface receptors in a paracrine/autocrine fashion, PTHrP appears to be able to enter the nucleus directly following translation and influence cellular events in an “intracrine” fashion. In this report, we demonstrate that PTHrP can be targeted to the nucleus in vascular smooth muscle cells, that this nuclear targeting is associated with a striking increase in mitogenesis, that this nuclear effect on proliferation is the diametric opposite of the effects of PTHrP resulting from interaction with cell surface receptors on vascular smooth muscle cells, and that the regions of the PTHrP sequence responsible for this nuclear targeting represent a classical bipartite nuclear localization signal. This report describes the activation of the cell cycle in association with nuclear localization of PTHrP in any cell type. These findings have important implications for the normal physiology of PTHrP in the many tissues which produce it, and suggest that gene delivery of PTHrP or modified variants may be useful in the management of atherosclerotic vascular disease.

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

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

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A1 and A2 are inhibited and delayed at site A0. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.