Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

Mineralogy and structural specification of ODP Leg 125 peridotite samples

Abundant serpentinite seamounts are found along the outer high of the Mariana forearc at the top of the inner slope of the trench. One of them, Conical Seamount, was drilled at Sites 778, 779, and 780 during Leg 125. The rocks recovered at Holes 779A and 780C, respectively, on the flanks and at the summit of the seamount, include moderately serpentinized depleted harzburgites and some dunites. These rocks exhibit evidence of resorption of the orthopyroxene, when present, and the local presence of very calcic-rich diopside in veins oblique to the main high-temperature foliation of the rock. The peridotites, initially well-foliated with locally poikiloblastic textures, show overprints of a two-stage deformation history: (1) a high-temperature (>1000°C), low-stress (0.02 GPa), homogeneous deformation that has led to the present Porphyroclastic textures displayed by the rocks and (2) heterogeneous ductile shearing at a much higher stress (0.05 GPa). This heterogeneous shearing probably describes a single tectonic event because it began at high temperatures, producing dynamic recrystallization of olivine in the shear zone, and ended at low temperatures in the stability field of chlorite and serpentine. In a few samples, olivine shows evidence of quasi-hydrostatic recrystallization at a very high temperature. Here, we propose that this recrystallization was related to fluid/magma percolation, a process that can also account for the resorption of the orthopyroxene and for the late crystallization of diopside veins in the rock. The impregnation by fluid or magma, development of the main high-temperature, low-stress deformation, and subsequent migration recrystallization of olivine probably occurred in a mantle fragment involved in the arc formation. In addition, this mantle has preserved structures that may have formed earlier in the oceanic lithosphere upon which the arc formed. Heterogeneous ductile shear zones in the peridotites may have developed during uplift. The "cold" deformation may have taken place during diapiric rise of hot mantle that underwent subsequent serpentinization or gliding along normal faults associated with the extension of the eastern margin of the forearc.

Simulation analysis of the EU ELV/RoHS directives based on an applied general equilibrium model with Melitz-type trade specification

This paper explores the potential usefulness of an AGE model with the Melitz-type trade specification to assess economic effects of technical regulations, taking the case of the EU ELV/RoHS directives as an example. Simulation experiments reveal that: (1) raising the fixed exporting cost to make sales in the EU market brings results that exports of the targeted commodities (motor vehicles and parts for ELV and electronic equipment for RoHS) to the EU from outside regions/countries expand while the domestic trade in the EU shrinks when the importer's preference for variety (PfV) is not strong; (2) if the PfV is not strong, policy changes that may bring reduction in the number of firms enable survived producers with high productivity to expand production to be large-scale mass producers fully enjoying the fruit of economies of scale; and (3) When the strength of the importer's PfV is changed from zero to unity, there is the value that totally changes simulation results and their interpretations.

On the specification and testing of inverters for stand-alone PV systems

Inverter features are reviewed from a PV systems perspective, with a view to contributing to possible codes, procurement specifications and testing procedures, in order to assure the technical quality of these systems. A laboratory testing campaign has been carried out on a representative set of sixteen currently available inverters and a set of the most common AC appliances. The results of the tests are discussed with the aim of divulging the particular features of operating AC appliances in PV systems and the provisions to be taken into account in PV system design. The development of testing procedures has followed the motto ?keep it as simple as possible?, in order to make their application easier in conventional laboratories in developing countries.

Experiences on the specification of algorithms for fire and smoke control in road tunnels

The main objective of ventilation systems in case of fire is the reduction of the possible consequences by achieving the best possible conditions for the evacuation of the users and the intervention of the emergency services. The required immediate transition, from normal to emergency functioning of the ventilation equipments, is being strengthened by the use of automatic and semi-automatic control systems, what reduces the response times through the help to the operators, and the use of pre-defined strategies. A further step consists on the use of closed-loop algorithms, which takes into account not only the initial conditions but their development (air velocity, traffic situation, etc.), optimizing smoke control capacity.

Ontology Requirements Specification

The goal of the ontology requirements specification activity is to state why the ontology is being built, what its intended uses are, who the end users are, and which requirements the ontology should fulfill. This chapter presents detailed methodological guidelines for specifying ontology requirements efficiently. These guidelines will help ontology engineers to capture ontology requirements and produce the ontology requirements specification document (ORSD). The ORSD will play a key role during the ontology development process because it facilitates, among other activities, (1) the search and reuse of existing knowledge resources with the aim of reengineering them into ontologies, (2) the search and reuse of ontological resources (ontologies, ontology modules, ontology statements as well as ontology design patterns), and (3) the verification of the ontology along the ontology development.

LED bulb lamps technical specification and testing procedure for solar home systems.

With the consolidation of the new solid state lighting LEOs devices, te5t1n9 the compliance 01 lamps based on this technology lor Solar Home Systems (SHS) have been analyzed. The definition of the laboratory procedures to be used with final products 15 a necessary step in arder to be able to assure the quality of the lamps prior to be installed [1]. As well as with CFL technology. particular attention has been given to simplicity and technical affordability in arder to facilitate the implementation of the test with basie and simple laboratory too15 even on the same SHS electrification program locations. The block of test procedures has been applied to a set of 14 low-cost lamps. They apply to lamp resistance, reliability and performance under normal, extreme and abnormal operating conditions as a simple but complete quality meter tool 01 any LEO bulb.

An ITS architecture specification - The FOTsis project experience

The recent continuous development of Cooperative ITS has resulted in several initiatives which focus on different parts of the Cooperative environment landscape. The FOTsis project focuses on the infrastructure side of the Cooperative environment and will deploy and test 7 services designed to maximise the benefits of the integration of the road operator and infrastructure-based information providers into the ITS environment. This integration can take place in any of the stages of data collection, processing and actuations of the services, but also support and trigger external tasks such as operations of the emergency response entities, etc. This paper describes the current status of the project and focuses on the specification of the supporting architecture to the services tested: references, a brief outline of the requirements’ definition, and the FOTsis architecture proposal, with some conclusions about the architecture tests conducted. The outlook on the project’s next steps is given in the last section of the paper.

LED bulbs technical specification and testing procedure for solar home systems

The definition of technical specifications and the corresponding laboratory procedures are necessary steps in order to assure the quality of the devices prior to be installed in Solar Home Systems (SHS). To clarify and unify criteria a European project supported the development of the Universal Technical Standard for Solar Home Systems (UTSfSHS). Its principles were to generate simple and affordable technical requirements to be optimized in order to facilitate the implementation of tests with basic and simple laboratory tools even on the same SHS electrification program countries. These requirements cover the main aspects of this type of installations and its lighting chapter was developed based on the most used technology at that time: fluorescent tubes and CFLs. However, with the consolidation of the new LED solid state lighting devices, particular attention is being given to this matter and new procedures are required. In this work we develop a complete set of technical specifications and test procedures that have been designed within the frame of the UTSfSHS, based on an intense review of the scientific and technical publications related to LED lighting and their practical application. They apply to lamp reliability, performance and safety under normal, extreme and abnormal operating conditions as a simple but complete quality meter tool for any LED bulb.

An ITS architecture specification - The FOTsis project experience

The recent continuous development of Cooperative ITS has resulted in several initiatives which focus on different parts of the Cooperative environment landscape. The FOTsis project focuses on the infrastructure side of the Cooperative environment and will deploy and test 7 services designed to maximise the benefits of the integration of the road operator and infrastructure-based information providers into the ITS environment. This paper describes the current status of the project and focuses on the road safety approach within the project: safety services and safety impact assessment. The outlook on the project's next steps is given in the last section of the paper.

Anteroposterior neural tissue specification by activin-induced mesoderm

The transforming growth factor β superfamily member, activin, is able to induce mesodermal tissues in animal cap explants from Xenopus laevis blastula stage embryos. Activin can act like a morphogen of the dorsoventral axis in that lower doses induce more ventral, and higher doses more dorsal, tissue types. Activin has also previously been reported to induce neural tissues in animal caps. From cell mixing experiments it was inferred that this might be an indirect effect of induced mesoderm signaling to uninduced ectoderm. Here we demonstrate directly that neural tissues do indeed arise by the action of induced mesoderm on uninduced ectoderm. Dorsal mesoderm is itself subdivided into posterior and anterior domains in vivo, but this had not been demonstrated for induced mesoderm. We therefore tested whether different concentrations of activin recreate these different anteroposterior properties as well. We show that the anteroposterior positional value of induced mesoderm, including its neuroinductive properties, depends on the dose of activin applied to the mesoderm, with lower doses inducing more posterior and higher doses giving more anterior markers. We discuss the implications of these results for patterning signals and the relationship between anteroposterior and dorsoventral axes.

Proximo–distal specification in the wing disc of Drosophila by the nubbin gene

Mutations in the nubbin (nub) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior–posterior nor the dorsal–ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of nub el and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of nub in the wing’s proximo–distal axis and in the formation of compartment boundaries.

A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete–scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.