Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes. (C) 2003 Wiley-Liss, Inc.

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes

Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 It recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Invertebrate D2 type dopamine receptor exhibits age-based plasticity of expression in the mushroom bodies of the honeybee brain

We have isolated a cDNA clone from the honeybee brain encoding a dopamine receptor, AmDop2, which is positively coupled to adenylyl cyclase. The transmembrane domains of this receptor are 88% identical to the orthologous Drosophila D2 dopamine receptor, DmDop2, though phylogenetic analysis and sequence homology both indicate that invertebrate and vertebrate D2 receptors are quite distinct. In situ hybridization to mRNA in whole-mount preparations of honeybee brains reveals gene expression in the mushroom bodies, a primary site of associative learning. Furthermore, two anatomically distinct cell types in the mushroom bodies exhibit differential regulation of AmDop2 expression. In all nonreproductive females (worker caste) and reproductive males (drones) the receptor gene is strongly and constitutively expressed in all mushroom body interneurons with small cell bodies. In contrast, the large cell-bodied interneurons exhibit dramatic plasticity of AmDop2 gene expression. In newly emerged worker bees (cell-cleaning specialists) and newly emerged drones, no AmDop2 transcript is observed in the large interneurons whereas this transcript is abundant in these cells in the oldest worker bees (resource foragers) and older drones. Differentiation of the mushroom body interneurons into two distinct classes (i.e., plastic or nonplastic with respect to AmDop2 gene expression) indicates that this receptor contributes to the differential regulation of distinct neural circuits. Moreover, the plasticity of expression observed in the large cells implicates this receptor in the behavioral maturation of the bee.

Autosomal dominant hypocalcemia: A novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A, GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, co-segregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca2+ on both intracellular Ca2+ mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca2+ concentrations.

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the traps-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of Hela cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the traps-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

A Generic Library for GUI Reasoning and Testing

Graphical user interfaces (GUIs) make software easy to use by providing the user with visual controls. Therefore, correctness of GUI's code is essential to the correct execution of the overall software. Models can help in the evaluation of interactive applications by allowing designers to concentrate on its more important aspects. This paper presents a generic model for language-independent reverse engineering of graphical user interface based applications, and we explore the integration of model-based testing techniques in our approach, thus allowing us to perform fault detection. A prototype tool has been constructed, which is already capable of deriving and testing a user interface behavioral model of applications written in Java/Swing.

Development of a library for clients of ONVIF video cameras: challenges and solutions

The interoperability of IP video equipment is a critical problem for surveillance systems and other video application developers. ONVIF is one of the two specifications addressing the standardization of networked devices interface, and it is based on SOAP. This paper addresses the development of an ONVIF library to develop clients of video cameras. We address the choice of a web services toolkit, and how to use the selected toolkit to develop a basic library. From that, we discuss the implementation of features that ...

XAdES4J: a java library for XAdES signature services

As comunicações electrónicas são cada vez mais o meio de eleição para negócios entre entidades e para as relações entre os cidadãos e o Estado (e-government). Esta diversidade de transacções envolve, muitas vezes, informação sensível e com possível valor legal. Neste contexto, as assinaturas electrónicas são uma importante base de confiança, fornecendo garantias de integridade e autenticação entre os intervenientes. A produção de uma assinatura digital resulta não só no valor da assinatura propriamente dita, mas também num conjunto de informação adicional acerca da mesma, como o algoritmo de assinatura, o certificado de validação ou a hora e local de produção. Num cenário heterogéneo como o descrito anteriormente, torna-se necessária uma forma flexível e interoperável de descrever esse tipo de informação. A linguagem XML é uma forma adequada de representar uma assinatura neste contexto, não só pela sua natureza estruturada, mas principalmente por ser baseada em texto e ter suporte generalizado. A recomendação XML Signature Syntax and Processing (ou apenas XML Signature) foi o primeiro passo na representação de assinaturas em XML. Nela são definidas sintaxe e regras de processamento para criar, representar e validar assinaturas digitais. As assinaturas XML podem ser aplicadas a qualquer tipo de conteúdos digitais identificáveis por um URI, tanto no mesmo documento XML que a assinatura, como noutra qualquer localização. Além disso, a mesma assinatura XML pode englobar vários recursos, mesmo de tipos diferentes (texto livre, imagens, XML, etc.). À medida que as assinaturas electrónicas foram ganhando relevância tornou-se evidente que a especificação XML Signature não era suficiente, nomeadamente por não dar garantias de validade a longo prazo nem de não repudiação. Esta situação foi agravada pelo facto da especificação não cumprir os requisitos da directiva 1999/93/EC da União Europeia, onde é estabelecido um quadro legal para as assinaturas electrónicas a nível comunitário. No seguimento desta directiva da União Europeia foi desenvolvida a especificação XML Advanced Electronic Signatures que define formatos XML e regras de processamento para assinaturas electrónicas não repudiáveis e com validade verificável durante períodos de tempo extensos, em conformidade com a directiva. Esta especificação estende a recomendação XML Signature, definindo novos elementos que contêm informação adicional acerca da assinatura e dos recursos assinados (propriedades qualificadoras). A plataforma Java inclui, desde a versão 1.6, uma API de alto nível para serviços de assinaturas digitais em XML, de acordo com a recomendação XML Signature. Contudo, não existe suporte para assinaturas avançadas. Com este projecto pretende-se desenvolver uma biblioteca Java para a criação e validação de assinaturas XAdES, preenchendo assim a lacuna existente na plataforma. A biblioteca desenvolvida disponibiliza uma interface com alto nível de abstracção, não tendo o programador que lidar directamente com a estrutura XML da assinatura nem com os detalhes do conteúdo das propriedades qualificadoras. São definidos tipos que representam os principais conceitos da assinatura, nomeadamente as propriedades qualificadoras e os recursos assinados, sendo os aspectos estruturais resolvidos internamente. Neste trabalho, a informação que compõe uma assinatura XAdES é dividia em dois grupos: o primeiro é formado por características do signatário e da assinatura, tais como a chave e as propriedades qualificadoras da assinatura. O segundo grupo é composto pelos recursos assinados e as correspondentes propriedades qualificadoras. Quando um signatário produz várias assinaturas em determinado contexto, o primeiro grupo de características será semelhante entre elas. Definiu-se o conjunto invariante de características da assinatura e do signatário como perfil de assinatura. O conceito é estendido à verificação de assinaturas englobando, neste caso, a informação a usar nesse processo, como por exemplo os certificados raiz em que o verificador confia. Numa outra perspectiva, um perfil constitui uma configuração do serviço de assinatura correspondente. O desenho e implementação da biblioteca estão também baseados no conceito de fornecedor de serviços. Um fornecedor de serviços é uma entidade que disponibiliza determinada informação ou serviço necessários à produção e verificação de assinaturas, nomeadamente: selecção de chave/certificado de assinatura, validação de certificados, interacção com servidores de time-stamp e geração de XML. Em vez de depender directamente da informação em causa, um perfil — e, consequentemente, a operação correspondente — é configurado com fornecedores de serviços que são invocados quando necessário. Para cada tipo de fornecedor de serviços é definida um interface, podendo as correspondentes implementações ser configuradas de forma independente. A biblioteca inclui implementações de todos os fornecedores de serviços, sendo algumas delas usadas for omissão na produção e verificação de assinaturas. Uma vez que o foco do projecto é a especificação XAdES, o processamento e estrutura relativos ao formato básico são delegados internamente na biblioteca Apache XML Security, que disponibiliza uma implementação da recomendação XML Signature. Para validar o funcionamento da biblioteca, nomeadamente em termos de interoperabilidade, procede-se, entre outros, à verificação de um conjunto de assinaturas produzidas por Estados Membros da União Europeia, bem como por outra implementação da especificação XAdES.

OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors

High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice.

Exploração da Point Cloud Library aplicada à percepção em sistemas autónomos

A navegação e a interpretação do meio envolvente por veículos autónomos em ambientes não estruturados continua a ser um grande desafio na actualidade. Sebastian Thrun, descreve em [Thr02], que o problema do mapeamento em sistemas robóticos é o da aquisição de um modelo espacial do meio envolvente do robô. Neste contexto, a integração de sistemas sensoriais em plataformas robóticas, que permitam a construção de mapas do mundo que as rodeia é de extrema importância. A informação recolhida desses dados pode ser interpretada, tendo aplicabilidade em tarefas de localização, navegação e manipulação de objectos. Até à bem pouco tempo, a generalidade dos sistemas robóticos que realizavam tarefas de mapeamento ou Simultaneous Localization And Mapping (SLAM), utilizavam dispositivos do tipo laser rangefinders e câmaras stereo. Estes equipamentos, para além de serem dispendiosos, fornecem apenas informação bidimensional, recolhidas através de cortes transversais 2D, no caso dos rangefinders. O paradigma deste tipo de tecnologia mudou consideravelmente, com o lançamento no mercado de câmaras RGB-D, como a desenvolvida pela PrimeSense TM e o subsequente lançamento da Kinect, pela Microsoft R para a Xbox 360 no final de 2010. A qualidade do sensor de profundidade, dada a natureza de baixo custo e a sua capacidade de aquisição de dados em tempo real, é incontornável, fazendo com que o sensor se tornasse instantaneamente popular entre pesquisadores e entusiastas. Este avanço tecnológico deu origem a várias ferramentas de desenvolvimento e interacção humana com este tipo de sensor, como por exemplo a Point Cloud Library [RC11] (PCL). Esta ferramenta tem como objectivo fornecer suporte para todos os blocos de construção comuns que uma aplicação 3D necessita, dando especial ênfase ao processamento de nuvens de pontos de n dimensões adquiridas a partir de câmaras RGB-D, bem como scanners laser, câmaras Time-of-Flight ou câmaras stereo. Neste contexto, é realizada nesta dissertação, a avaliação e comparação de alguns dos módulos e métodos constituintes da biblioteca PCL, para a resolução de problemas inerentes à construção e interpretação de mapas, em ambientes indoor não estruturados, utilizando os dados provenientes da Kinect. A partir desta avaliação, é proposta uma arquitectura de sistema que sistematiza o registo de nuvens de pontos, correspondentes a vistas parciais do mundo, num modelo global consistente. Os resultados da avaliação realizada à biblioteca PCL atestam a sua viabilidade, para a resolução dos problemas propostos. Prova da sua viabilidade, são os resultados práticos obtidos, da implementação da arquitectura de sistema proposta, que apresenta resultados de desempenho interessantes, como também boas perspectivas de integração deste tipo de conceitos e tecnologia em plataformas robóticas desenvolvidas no âmbito de projectos do Laboratório de Sistemas Autónomos (LSA).

Novel transcription factors regulating the expression of the rice gene OsDREB1B

Dissertation presented to obtain the Ph.D degree in Biology

A private library in the Mateus Palace (Vila Real). The D. Luís António de Sousa Botelho Mourão (1722-1798) Books Collection

Fundação para a Ciência e Tecnologia no âmbito de Bolsa de Doutoramento (SFRH/BD/86280/2012)