Role of ATH5 in the specification of retinal ganglion cells and expression and cell compartmentalization of EFEMP1, a protein associated with Malattia Leventinese

ABSTRACT : The development of the retina is a very complex process, occurring through the progressive restriction of cell fates, from pluripotent cell populations to complex tissues and organs. In all vertebrate species analyzed so far, retinal differentiation starts with the generation of retinal ganglion cells (RGC)s. One of the documented key essential events in the specification of RGCs is the expression of ATHS, an atonal homolog encoding a bHLH transcription factor. Despite the putative role of master regulator of RGC differentiation, the mechanism of integrating its functions into a coherent program underlying the production of this subclass of retinal neurons has not yet been elucidated. By using chromatin immunoprecipitation combined with microarray (ChIP-on-chip) we have screened for ATH5 direct targets in the developing chick retina at two consecutive periods: E3.5 (stage HH22) and E6 (stage HH30), covering the stages of progenitor proliferation, neuroepithelium patterning, RGC specification, cell cycle exit and early neuronal differentiation. In parallel, complementary analysis with Affymetrix expression microarrays was conducted. We compared RGCs versus retina to see if the targets correspond to genes preferentially expressed in RGCs. We also precociously overexpressed ATH5 in the retina of individual embryo, and contralateral retina vas used as a control. Our integrated approach allowed us to establish a compendium of ATH5-targets and enabled us to position ATH5 in the transcription network underlying neurogenesis in the retina. Malattia Leventinese (ML) is an autosomal, dominant retinal dystrophy characterized by extracellular, amorphous deposits known as drusen, between the retinal pigment epithelium (RPE) and Bruch's membrane. On the genetic level, it has been associated with a single missense mutation (R345W) in a widely expressed gene with unknown function called EFEMP1. We determined expression patterns of the EFEMP1 gene in normal and ML human retinas. Our data shown that the upregulation of EFEMP1 is not specific to ML eye, except for the region of the ciliary body. We also analyzed the cell compartmentalization of different versions of the protein (both wild type and mutant). Our studies indicate that both abnormal expression of the EFEMP1 gene and mutation and accumulation of EFEMP 1 protein (inside or outside the cells) might contribute to the ML pathology. Résumé : 1er partie : L'ontogenèse de la rétine est un processus complexe au cours duquel des cellules progénitrices sont engagée, par vagues successives, dans des lignées où elles vont d'abord être déterminées puis vont se différencier pour finalement construire un tissu rétinien composé de cinq classes de neurones (les photorécepteurs, les cellules horizontales, bipolaires, amacrines et ganglionnaires) et d'une seule de cellules gliales (les cellules de Muller). Chez tous les vertébrés, la neurogenèse rétinienne est d'abord marquée par la production des cellules ganglionnaires (RGCs). La production de cette classe de neurone est liée à l'expression du gène ATH5 qui est un homologue du gène atonal chez la Drosophile et qui code pour un facteur de transcription de la famille des protéines basic Helix-Loop-Helix (bHLH). Malgré le rôle central que joue ATH5 dans la production des RGCs, le mécanisme qui intègre la fonction de cette protéine dans le programme de détermination neuronale et ceci en relation avec le développement de la rétine n'est pas encore élucidé. Grâce à une technologie qui permet de combiner la sélection de fragments de chromatine liant ATH5 et la recherche de séquences grâce à des puces d'ADN non-codants (ChIP-on-chip), nous avons recherché des cibles potentielles de la protéine ATH5 dans la rétine en développement. Nous avons conduit cette recherche à deux stades de développement de manière à englober la phase de prolifération cellulaire, la détermination des RGCs, la sortie du cycle cellulaire ainsi que les premières étapes de la différentiation de ces neurones. Des expériences complémentaires nous ont permis de définir les patrons d'expression des gènes sélectionnés ainsi que l'activité promotrice des éléments de régulation identifiés lors de notre criblage. Ces approches expérimentales diverses et complémentaires nous ont permis de répertorier des gènes cibles de la protéine ATH5 et d'établir ainsi des liens fonctionnels entre des voies métaboliques dont nous ne soupçonnions pas jusqu'alors qu'elles puissent être associées à la production d'une classe de neurones centraux. 2ème partie : Malattia Leventinese (ML) est une maladie génétique qui engendre une dystrophie de la rétine. Elle se caractérise par l'accumulation de dépôt amorphe entre l'épithélium pigmentaire et la membrane de Bruch et connu sous le nom de drusen. Cette maladie est liée à une simple mutation non-sens (R345W) dans un gène dénommé EFEMP1 qui est exprimé dans de nombreux tissus mais dont la fonction reste mal définie. Une étude détaillée de l'expression de ce gène dans des rétines humaines a révélé une expression à un niveau élevé du gène EFEMP1 dans divers tissus de l'oeil ML mais également dans des yeux contrôles. Alors que l'accumulation d'ARN messager EFEMP1 dans les cellules de l'épithélium pigmentaire n'est pas spécifique à ML, l'expression de ce gène dans le corps cilié n'a été observée que dans l'oeil ML. Nous avons également comparé la sécrétion de la protéine sauvage avec celle porteuse de la mutation. En résumé, notre étude révèle que le niveau élevé d'expression du gène EFEMP1 ainsi que l'accumulation de la protéine dans certains compartiments cellulaires pourraient contribuer au développement de pathologies rétiniennes liées à ML.

FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

Background: During early steps of embryonic development the hindbrain undergoes a regionalization process along the anterior-posterior (AP) axis that leads to a metameric organization in a series of rhombomeres (r). Refinement of the AP identities within the hindbrain requires the establishment of local signaling centers, which emit signals that pattern territories in their vicinity. Previous results demonstrated that the transcription factor vHnf1 confers caudal identity to the hindbrain inducing Krox20 in r5 and MafB/Kreisler in r5 and r6, through FGF signaling [1].Results: We show that in the chick hindbrain, Fgf3 is transcriptionally activated as early as 30 min after mvHnf1 electroporation, suggesting that it is a direct target of this transcription factor. We also analyzed the expression profiles of FGF activity readouts, such as MKP3 and Pea3, and showed that both are expressed within the hindbrain at early stages of embryonic development. In addition, MKP3 is induced upon overexpression of mFgf3 or mvHnf1 in the hindbrain, confirming vHnf1 is upstream FGF signaling. Finally, we addressed the question of which of the FGF-responding intracellular pathways were active and involved in the regulation of Krox20 and MafB in the hindbrain. While Ras-ERK1/2 activity is necessary for MKP3, Krox20 and MafB induction, PI3K-Akt is not involved in that process.Conclusion: Based on these observations we propose that vHnf1 acts directly through FGF3, and promotes caudal hindbrain identity by activating MafB and Krox20 via the Ras-ERK1/2 intracellular pathway.

Panel index VAR models: Specification, Estimation, Testing and Leading Indicators

This paper proposes a method to conduct inference in panel VAR models with cross unit interdependencies and time variations in the coefficients. The approach can be used to obtain multi-unit forecasts and leading indicators and to conduct policy analysis in a multiunit setups. The framework of analysis is Bayesian and MCMC methods are used to estimate the posterior distribution of the features of interest. The model is reparametrized to resemble an observable index model and specification searches are discussed. As an example, we construct leading indicators for inflation and GDP growth in the Euro area using G-7 information.

Female labour supply in Spain: The importance of behavioural assumptions and unobserved heterogeneity specification

We estimate four models of female labour supply using a Spanish sampleof married women from 1994, taking into account the complete form of theindividual s budget set. The models differ in the hypotheses relating tothe presence of optimisation errors and/or the way non-workers contributeto the likelihood function. According to the results, the effects of wagesand non-labour income on the labour supply of Spanish married women dependon the specification used. The model which has both preference andoptimisation errors and allows for both voluntarily and involuntarilyunemployed females desiring to participate seems to better fit the evidencefor Spanish married women.

Strategy of specification of management areas: rice grain yield as related to soil fertility

It is well-known nowadays that soil variability can influence crop yields. Therefore, to determine specific areas of soil management, we studied the Pearson and spatial correlations of rice grain yield with organic matter content and pH of an Oxisol (Typic Acrustox) under no- tillage, in the 2009/10 growing season, in Selvíria, State of Mato Grosso do Sul, in the Brazilian Cerrado (longitude 51º24' 21'' W, latitude 20º20' 56'' S). The upland rice cultivar IAC 202 was used as test plant. A geostatistical grid was installed for soil and plant data collection, with 120 sampling points in an area of 3.0 ha with a homogeneous slope of 0.055 m m-1. The properties rice grain yield and organic matter content, pH and potential acidity and aluminum content were analyzed in the 0-0.10 and 0.10-0.20 m soil layers. Spatially, two specific areas of agricultural land management were discriminated, differing in the value of organic matter and rice grain yield, respectively with fertilization at variable rates in the second zone, a substantial increase in agricultural productivity can be obtained. The organic matter content was confirmed as a good indicator of soil quality, when spatially correlated with rice grain yield.

Guide Specification for Highway Construction Texturing Concrete Pavement for Reduced Tire/Pavement Noise using Diamond Grinding, March 1, 2011

This document provides language that can be used by an Owner-Agency to develop materials and construction specifications with the objective of reducing tire/pavement noise. While the practices described herein are largely prescriptive, they have been demonstrated to increase the likelihood of constructing a durable, quieter concrete surface.

Guide Specification for Highway Construction Texturing Concrete Pavement for Reduced Tire/Pavement Noise using Artificial Turf Drag, March 1, 2011

This document provides language that can be used by an Owner-Agency to develop materials and construction specifications with the objective of reducing tire/pavement noise. While the practices described herein are largely prescriptive, they have been demonstrated to increase the likelihood of constructing a durable, quieter concrete surface. Guidance is provided herein for texturing the concrete surface since texture geometry has a paramount effect on tire/pavement noise. Guidance for curing is also provided to improve strength and durability of the surface mortar, and thus to improve texture durability.

Guide Specification for Highway Construction Texturing Concrete Pavement for Reduced Tire/Pavement Noise using Longitudinal Tining, March 1, 2011

This document provides language that can be used by an Owner-Agency to develop materials and construction specifications with the objective of reducing tire/pavement noise. While the practices described herein are largely prescriptive, they have been demonstrated to increase the likelihood of constructing a durable, quieter concrete surface. Guidance is provided herein for texturing the concrete surface since texture geometry has a paramount effect on tire/pavement noise. Guidance for curing is also provided to improve strength and durability of the surface mortar, and thus to improve texture durability.

Concrete Pavement Mixture Design and Analysis (MDA) Guide Specification for Highway Concrete Pavements: Commentary, TPF-5(205), 2012

This guide specification and commentary for concrete pavements presents current state-of-the art thinking with respect to materials and mixture selection, proportioning, and acceptance. This document takes into account the different environments, practices, and materials in use across the United States and allows optional inputs for local application. The following concrete pavement types are considered: jointed plain concrete pavement, the most commonly used pavement type and may be doweled or non-doweled at transverse joints; and continuously reinforced concrete pavement, typically constructed without any transverse joints, typically used for locations with high truck traffic loads and/or poor support conditions.

Concrete Pavement Mixture Design and Analysis (MDA) Guide Specification for Highway Concrete Pavements, TPF-5(205), 2012

A guide specification and commentary have been prepared that lay out current state-of-the art thinking with respect to materials and mixture selection, proportioning, and acceptance. These documents take into account the different environments, practices, and materials in use across the US and allow optional inputs for local application.

Report and Specification for Transportation Program Management System, HR-394, 1997

TPMS is proposed as a distributed, PC-based system for automating two processes required for road improvements in Iowa: a) the annual preparation, submission, and approval of road improvement programs. b) the ongoing process of developing plans and obtaining approval for projects to be let for bids.

Evaluation of PCC Specification Changes Impact on Durability: 1992 - 1997 Core Study, MLR-98-06, 2005

In 1990, early distress had shown up on US 20 in Hamilton/Webster counties, three years after paving. Since that time, over a dozen more projects, constructed between 1984 and 1994, have been found to exhibit similar early distress. Several changes to the concrete and Portland cement specifications occurred in 1994 and 1996. This study was undertaken to investigate in place concrete pavements before and after specification changes were implemented. The objective of this research is to evaluate the impact of Portland cement and concrete specification changes made in 1994 and 1996 on PCC durability. Cores were obtained in 1998 and 2003 from projects constructed in 1992, before specification changes, and 1997 after specification changes. The following is a brief summary of the conclusions: 1. The pavements in the study constructed under the new specifications are performing much better after 5 years of service than the pavements constructed under the old specifications. 2. According to ISU, micro-cracking is evident in all concrete that has been in service, due to thermal stresses and loading stresses. Also, the low vacuum SEM will desiccate the concrete enough to cause micro-cracking. The SEM should not be used as a tool to indicate micro-cracking. 3. Use of Type II cement (C3A <8%) and a 3.0% SO3 limit does not completely eliminate ettringite infilling in air voids, as indicated in the bottom of the 1997 cores. 4. In areas of high moisture (bottom of the core), infilling is present in most of the 1997 cores. 5. Low air content and high spacing factor in the top of 1992 cores apparently causes F/T cycling cracking and then increased moisture paths from cracking causes infilling. 6. Use of ground granulated blast furnace slag (GGBFS) and fly ash reduces ettringite infilling either by diluting the aluminate (C3A) or lowering permeability, which slows ingress of moisture. 7. The specification changes that made the biggest impact on pavement durability are the limits on vibration and increase in air content in September 1994. 8. Investigations of cores from pavements placed in 2002 and 2003 indicate improved air contents and spacing factors. In-place air content and spacing factors should be monitored to determine if appropriate air void parameters are being met.

Alternative tests for correct specification of conditional predictive densities

We propose new methods for evaluating predictive densities that focus on the models' actual predictive ability in finite samples. The tests offer a simple way of evaluatingthe correct specification of predictive densities, either parametric or non-parametric.The results indicate that our tests are well sized and have good power in detecting mis-specification in predictive densities. An empirical application to the Survey ofProfessional Forecasters and a baseline Dynamic Stochastic General Equilibrium modelshows the usefulness of our methodology.

Java Specification Request development on a mobile device

Nykyaikaiset Java-teknologiaa sisältävät matkapuhelimet kehittyvät vauhdikkaasti prosessoritehon, muistin määrän sekä uusien käyttöjärjestelmäversioiden tarjoamien ominaisuuksien myötä. Laitteiden näyttöjen koko tulee pysymään pienenä,mutta silti moninaista multimediasisältöä äänen, videon ja kuvan osilta voidaanhuomattavasti parantaa JSR 234:n eli kehittyneen multimedialaajennuksen avulla.Erityisesti edistyneet ääniominaisuudet ovat tervetullut lisä, sillä viime aikojen kehitys matkapuhelimissa on saanut aikaan niiden muuntumisen myös kannettavaksi musiikkisoittimiksi. Diplomityössä JSR 234 -spesifikaation tietty osa kehitettiin ympäristössä, joka koostui Series 60 -ohjelmistoalustankolmannesta versiosta sekä Symbian OS v9.1 käyttöjärjestelmästä. Tuloksena syntynyt Java-rajapinta tarjoaa sovelluskehittäjille yksinkertaisemman lähestymistavan Symbianin efektirajapintaan piilottaen samalla alla olevan käyttöjärjestelmänmonimutkaisuuden. Toteutuksen täytyy olla läpikotaisin testattu, jotta voidaan varmentua sen noudattavan tarkkaan JSR 234 -spesifikaatiota. Työssä on esitelty useita eri testausmenetelmiä tarkoituksena saavuttaa projektissa paras mahdollinen laatu.