Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of I-125-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with I-125-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the I-125 methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than I-125-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Régimen jurídico de las lenguas y reconocimiento de la diversidad lingüística en el Tratado por el que se establece una Constitución para Europa

The territory of the European Union is made up of a rich and wide-ranging universe of languages, which is not circumscribed to the «State languages». The existence of multilingualism is one of Europe’s defining characteristics and it should remain so in the constantly evolving model of Europe’s political structure. Nonetheless, until now, the official use of languages has been limited to the «State languages» and has been based on a concept of state monolingualism that has led to a first level of hierarchization among the languages of Europe. This has affected the very concept of European language diversity. The draft of the treaty establishing a European Constitution contains various language-related references that can be grouped in two major categories: on the one hand, those references having to do the constitutional status of languages, and on the other, those regarding the recognition of European language diversity. Both issues are dealt with in this article. In analyzing the legal regime governing languages set forth in the draft of the constitutional treaty, we note that the draft is not based on the concept of the official status of languages. The language regulation contained in the draft of the constitutional treaty is limited in character. The constitutional language regime is based on the concept of Constitutional languages but the official status of languages is not governed by this rule. The European Constitution merely enunciates rights governing language use for European citizens vis-à-vis the languages of the Constitution and refers the regulation of the official status of languages to the Council, which is empowered to set and modify that status by unanimous decision. Because of its broad scope, this constitutes a regulatory reservation. In the final phase of the negotiation process a second level of constitutional recognition of languages would be introduced, linked to those that are official languages in the member states (Catalan, Basque, Galician, etc.). These languages, however, would be excluded from the right to petition; they would constitute a tertium genus, an intermediate category between the lan guages benefiting from the language rights recognized under the Constitution and those other languages for which no status is recognized in the European institutional context. The legal functionality of this second, intermediate category will depend on the development of standards, i.e., it will depend on the entrée provided such languages in future reforms of the institutional language regime. In a later section, the article reflects on European Union language policy with regard to regional or minority languages, concluding that the Union has not acted in accordance with defined language policy guidelines when it has been confronted, in the exercise of its powers, with regional or minority languages (or domestic legislation having to do with language demands). The Court of Justice has endeavoured to resolve on a case by case basis the conflicts raised between community freedoms and the normative measures that protect languages. Thus, using case law, the Court has set certain language boundaries for community freedoms. The article concludes by reflecting on the legal scope of the recognition of European language diversity referred to in Article II-82 of the European Constitution and the possible measures to implement the precept that might constitute the definition of a true European language policy on regional or minority languages. Such a policy has yet to be defined.

"I'm on it 24/7 at the moment" : A qualitative examination of multi-screen viewing behaviours among UK 10-11 year olds

Background: Screen-viewing has been associated with increased body mass, increased risk of metabolic syndrome and lower psychological well-being among children and adolescents. There is a shortage of information about the nature of contemporary screen-viewing amongst children especially given the rapid advances in screen-viewing equipment technology and their widespread availability. Anecdotal evidence suggests that large numbers of children embrace the multi-functionality of current devices to engage in multiple forms of screen-viewing at the same time. In this paper we used qualitative methods to assess the nature and extent of multiple forms of screen-viewing in UK children. Methods: Focus groups were conducted with 10-11 year old children (n = 63) who were recruited from five primary schools in Bristol, UK. Topics included the types of screen-viewing in which the participants engaged; whether the participants ever engaged in more than one form of screen-viewing at any time and if so the nature of this multiple viewing; reasons for engaging in multi-screen-viewing; the room within the house where multi-screen-viewing took place and the reasons for selecting that room. All focus groups were transcribed verbatim, anonymised and thematically analysed. Results: Multi-screen viewing was a common behaviour. Although multi-screen viewing often involved watching TV, TV viewing was often the background behaviour with attention focussed towards a laptop, handheld device or smart-phone. There were three main reasons for engaging in multi-screen viewing: 1) tempering impatience that was associated with a programme loading; 2) multi-screen facilitated filtering out unwanted content such as advertisements; and 3) multi-screen viewing was perceived to be enjoyable. Multi-screen viewing occurred either in the child's bedroom or in the main living area of the home. There was considerable variability in the level and timing of viewing and this appeared to be a function of whether the participants attended after-school clubs. Conclusions: UK children regularly engage in two or more forms of screen-viewing at the same time. There are currently no means of assessing multi-screen viewing nor any interventions that specifically focus on reducing multi-screen viewing. To reduce children's overall screen-viewing we need to understand and then develop approaches to reduce multi-screen viewing among children.

The Metabolic Core and Catalytic Switches Are Fundamental Elements in the Self-Regulation of the Systemic Metabolic Structure of Cells

19 p.

La funcionalidad de las creencias

[ES]Este trabajo muestra de forma comparativa la simbología existente con relación a la muerte en dos comunidades gallegas. Aquí presento unas breves reflexiones sobre los estereotipos, todo ello articulado en los silencios que se producen en los saltos generacionales. Finalizando con la delimitación de los espacios físicos correspondientes a las diferencias simbólicas en la construcción de los “unos” y los “otros”.

Communities in complex networks

The study of complex networks has attracted the attention of the scientific community for many obvious reasons. A vast number of systems, from the brain to ecosystems, power grid, and the Internet, can be represented as large complex networks, i.e, assemblies of many interacting components with nontrivial topological properties. The link between these components can describe a global behaviour such as the Internet traffic, electricity supply service, market trend, etc. One of the most relevant topological feature of graphs representing these complex systems is community structure which aims to identify the modules and, possibly, their hierarchical organization, by only using the information encoded in the graph topology. Deciphering network community structure is not only important in order to characterize the graph topologically, but gives some information both on the formation of the network and on its functionality.

A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

Background: The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. Methods: Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. Results: When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. Conclusions: We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.

Evaluación de la política de Espacios Naturales Protegidos: una propuesta metodológica para la Comunidad Autónoma del País Vasco

395 p.

Kalman Filtering in R

Support in R for state space estimation via Kalman filtering was limited to one package, until fairly recently. In the last five years, the situation has changed with no less than four additional packages offering general implementations of the Kalman filter, including in some cases smoothing, simulation smoothing and other functionality. This paper reviews some of the offerings in R to help the prospective user to make an informed choice.

Global Self-Organization of the Cellular Metabolic Structure

Background: Over many years, it has been assumed that enzymes work either in an isolated way, or organized in small catalytic groups. Several studies performed using "metabolic networks models'' are helping to understand the degree of functional complexity that characterizes enzymatic dynamic systems. In a previous work, we used "dissipative metabolic networks'' (DMNs) to show that enzymes can present a self-organized global functional structure, in which several sets of enzymes are always in an active state, whereas the rest of molecular catalytic sets exhibit dynamics of on-off changing states. We suggested that this kind of global metabolic dynamics might be a genuine and universal functional configuration of the cellular metabolic structure, common to all living cells. Later, a different group has shown experimentally that this kind of functional structure does, indeed, exist in several microorganisms. Methodology/Principal Findings: Here we have analyzed around 2.500.000 different DMNs in order to investigate the underlying mechanism of this dynamic global configuration. The numerical analyses that we have performed show that this global configuration is an emergent property inherent to the cellular metabolic dynamics. Concretely, we have found that the existence of a high number of enzymatic subsystems belonging to the DMNs is the fundamental element for the spontaneous emergence of a functional reactive structure characterized by a metabolic core formed by several sets of enzymes always in an active state. Conclusions/Significance: This self-organized dynamic structure seems to be an intrinsic characteristic of metabolism, common to all living cellular organisms. To better understand cellular functionality, it will be crucial to structurally characterize these enzymatic self-organized global structures.

Lehiaketa motor baten direkzio piparen diseinua

[EU]Hurrengo orrialdeetan aurkezten den proiektu honetan, lehiaketa motor baten direkzio piparen diseinua proposatzen da zeinak Moto Engineering Foundation fundazioak sustatutako nazioarteko Motostudent txapelketan parte hartzeko motorrera egokituta egon beharko den. Honako helburu honekin beraz, direkzio piparen diseinu egoki bat egin ahal izateko piezak sufrituko dituen karga egoerak aurreikusi beharko ditugu hauen arabera jasan beharreko tentsio egoerak aztertu ahal izateko. Piezaren tentsioen analisia aurrera eraman beharko da beraz, diseinaturiko piezaren funtzionamendu egokia ziurtatu ahal izateko. Historian zehar existitutako diseinu modelo ezberdinak eta baita gaur egunean gehien erabilitakoak ere aurkeztu eta gero, gure kasura hobekien moldatuko zaion adibidetik ekingo zaio diseinuari, aldez aurretik eskuratutako datu eta errodamenduetatik abiatuz. Horrenbestez, oinarritzat hartutako direkzio sistema modeloa eta bestelako elementuen neurriak datutzat hartuta, tija zein norabide ardatzarena esate baterako, direkzio piparen hasierako geometria definituko da. Eskuragarri ditugun analisi metodo ezberdinak aztertu eta gure kasura hobekien moldatzen dena aukeratu eta eraikitako piezari aplikatuko zaio. Lorturiko emaitzen arabera hasiera batean planteaturiko diseinua onartu edo baztertzea erabaki beharko dugu. Beraz, gure diseinua optimizatuz joateko aukera izango dugu, baldintzak eta betebeharrak egiztatuko dituen amaierako pieza diseinua lortu arte. Bukatzeko, proiektuari itxiera emango dion atala aurkeztuko da, hala nola, proiektuaren garapenean zehar lorturiko emaitzak eta ondorioak biltzen dituen atala non lorturiko direkzio piparen funtzionamendu egokia bermatzen dituzten parametroak aurkeztuko diren. Gainera, direkzio sistemaren funtsezko atala izango diren errodamenduen kalkulu eta aukeraketa bideratuko da baita ere.

Análisis comparativo de alternativas o soluciones de sistemas estructurales en acero y madera para marquesinas de aparcamiento

[ES]En este trabajo se hace un análisis y una comparación de las diferentes formas estructurales que puede adoptar una marquesina de aparcamiento, principalmente estructuras hechas en madera y acero. Para la realización del citado análisis se han valorado las ventajas y desventajas de los materiales, funcionalidad, mantenimiento, estética, dificultad de ejecución y economicidad, realizándose asimismo un diseño de marquesina utilizando uno u otro de los materiales citados. Para la ejecución de la marquesina, se formula un diseño que cumplimente los condicionantes de funcionalidad del elemento. A su vez, se analiza, parte por parte, todo el sistema estructural, empezando por determinar las acciones que van a aparecer y deberán ser transmitidas al terreno. Una vez obtenidos los esfuerzos que se han de transmitir, se formula una hipótesis de calidad del terreno de cimentación, con el fin de determinar las características de la cimentación. Previamente se realiza un predimensionado del sistema estructural, para cuyo cálculo se ha utilizado el programa informático Tricalc. Obtenidas las dimensiones exactas y los detalles constructivos para cada una de las opciones de materiales a analizar (madera y acero), se evalúa económicamente cada diseño obtenido, para determinar la solución más económica y rentable desde el punto de vista, por ejemplo del montaje y desmontaje, así como por el mantenimiento. Además de las funciones estructurales, también se analiza la adaptación al entorno de la madera y el acero.

Diseño de sistema de suspensión progresiva Pro-Link

[ES]Mediante este trabajo se va a diseñar un mecanismo para crear un sistema de suspensión progresiva para la suspensión trasera de una motocicleta a partir de un muelle lineal. El muelle introducido en la suspensión será lineal, por lo tanto, tendrá una sola rigidez, constante y la cual delimita el muelle, pero gracias a un mecanismo diseñado especialmente para la suspensión trasera de una moto, se va conseguir que la rigidez varíe. El mecanismo utilizado para este trabajo se llama Pro-Link y la empresa que lo emplea es Honda. Para ello, se creará un esquema del mecanismo en sí y tras un análisis cinemático mediante el programa Creo Parametric, y haciendo varios cambios en el sistema para ajustarlo a la mejor progresividad posible, se obtendrá la gráfica en la que se observará la curva que marca la rigidez progresiva. Esto es, se verá la evolución de la nueva rigidez dependiendo de la oscilación vertical de la rueda. Por otra parte, gracias a un análisis dinámico del mismo programa se conseguirán las reacciones del sistema y se calcularán los elementos auxiliares de éste para el buen funcionamiento del mismo. Finalmente, se creará el diseño del triángulo y un diseño preliminar del basculante mediante el método de elementos finitos del programa.

Design and evaluation of an optical switching node

[EN]For a good development of elastic optical networks, the design of flexible optical switching nodes is required. This work analyses the previously proposed flexible architectures and, based on the most appropriate, which is the Architecture on Demand (AoD), proposes a specific configuration of the node that includes spatial and spectral switching and the wavelength conversion functionality with a low blocking probability and the minimum amount of modules; the characteristics of the traffic that the designed node is able to cope with are specified in the last chapter. An evaluation of the designed node is also done, and, compared to the other architectures, it is shown that the Architecture on Demand gives better results than others and that it has a higher potential for future developments.