993 resultados para Ars Analytica


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Epigenetic silencing of foreign genes introduced into plants poses an unsolved problem for transgenic technology. Here we have used the simple multicellular green alga Volvox carteri as a model to analyse the relation of DNA methylation to transgenic silencing. Volvox DNA contains on average 1.1% 5-methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated 32P-labelled nucleotides. In two nuclear transformants of V.carteri, produced in 1993 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500–1000 generations. Each transformant clone contains multiple intact copies of C-ars, most of them integrated into the genome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that the inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-level (7%) CpG methylation. These are average values from 40 PCR clones sequenced from each DNA strand in the two portions of C-ars. The observed correlation of CpG methylation and transgene inactivation in a green alga will be discussed in the light of transcriptional silencing.

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The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ∼2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ∼60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

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Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5′ end of the LEU2 gene at one end was transformed into a strain in which the 5′ end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2+. Ade1+ Leu2+ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

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At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

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Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.

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The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

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Com o escopo de fornecer subsídios para compreender como o processo de colaboração científica ocorre e se desenvolve em uma instituição de pesquisas, particularmente o IPEN, o trabalho utilizou duas abordagens metodológicas. A primeira utilizou a técnica de análise de redes sociais (ARS) para mapear as redes de colaboração científica em P&D do IPEN. Os dados utilizados na ARS foram extraídos da base de dados digitais de publicações técnico-científicas do IPEN, com o auxílio de um programa computacional, e basearam-se em coautoria compreendendo o período de 2001 a 2010. Esses dados foram agrupados em intervalos consecutivos de dois anos gerando cinco redes bienais. Essa primeira abordagem revelou várias características estruturais relacionadas às redes de colaboração, destacando-se os autores mais proeminentes, distribuição dos componentes, densidade, boundary spanners e aspectos relacionados à distância e agrupamento para definir um estado de redes mundo pequeno (small world). A segunda utilizou o método dos mínimos quadrados parciais, uma variante da técnica de modelagem por equações estruturais, para avaliar e testar um modelo conceitual, apoiado em fatores pessoais, sociais, culturais e circunstanciais, para identificar aqueles que melhor explicam a propensão de um autor do IPEN em estabelecer vínculos de colaboração em ambientes de P&D. A partir do modelo consolidado, avaliou-se o quanto ele explica a posição estrutural que um autor ocupa na rede com base em indicadores de ARS. Nesta segunda parte, os dados foram coletados por meio de uma pesquisa de levantamento com a utilização de um questionário. Os resultados mostraram que o modelo explica aproximadamente 41% da propensão de um autor do IPEN em colaborar com outros autores e em relação à posição estrutural de um autor na rede o poder de explicação variou entre 3% e 3,6%. Outros resultados mostraram que a colaboração entre autores do IPEN tem uma correlação positiva com intensidade moderada com a produtividade, da mesma forma que, os autores mais centrais na rede tendem a ampliar a sua visibilidade. Por fim, vários outros indicadores estatísticos bibliométricos referentes à rede de colaboração em P&D do IPEN foram determinados e revelados, como, a média de autores por publicação, média de publicações por autores do IPEN, total de publicações, total de autores e não autores do IPEN, entre outros. Com isso, esse trabalho fornece uma contribuição teórica e empírica aos estudos relacionados à colaboração científica e ao processo de transferência e preservação de conhecimento, assim como, vários subsídios que contribuem para o contexto de tomada de decisão em ambientes de P&D.

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Esta Tesis tiene dos partes. La Primera Parte es Teórica y Metodológica y trata de la actual crisis de paradigma en las Ciencias Sociales, y de cómo se puede remontar con la Teoría del Pensamiento Complejo, siempre que sus propuestas se centren en modelos empíricos de Análisis de Redes Sociales debidamente matematizados y estadísticamente refrendados. La propuesta del tesista propone enriquecer el actual homo economicus, incorporando la importancia de las relaciones con el grupo (coactivas, coercitivas o motivacionales), a través de un nuevo objeto de estudio: los Proyectos. Es mediante los Proyectos, donde los individuos y los grupos en los que interactúan, transan y organizan sus esfuerzos. El problema reside en que, no existe hasta la fecha, una sistematización y modelización de los Proyectos como objeto de estudio en las Ciencias Sociales. Sin embargo, hay una amplia experiencia de análisis y sistematización de Proyectos tanto en la Economía de la Empresa (Management, Business Administration), como en la Economía Pública. En esta Tesis se estudia todo lo publicado recientemente sobre los Proyectos de Inversión Pública (PIPs) y su eficiencia en Latinoamérica. En la Segunda Parte, centrada en un Trabajo Empírico y su modelización, el tesista crea una Base de Datos (BdD) primaria, a partir del Banco de Proyectos (BdP) del Ministerio de Economía y Finanzas (MEF) del Perú (2001-2014), que recoge todos los Proyectos de Inversión Pública (PIP), cerca de 400.000 PIPs Iniciales, los tabula en 48 categorías y posteriormente, “deja hablar a los datos” jugando a relacionar, correlacionar, inducir hipótesis y verificarlas mediante un sistema que se centra en la operativa tipo “Big Data”. A esto le denomina “triangular” porque mezcla en el esfuerzo, herramientas de Estadística Descriptiva, Estadística Inferencial y Econometría para poder refrendar el conocimiento inducido, que siempre en ciencia, es una mera certeza probabilística. El tesista concluye que en el caso del Sistema Nacional de Inversión Pública del Perú (SNIP) y más específicamente, de los procesos administrativos que emplea -denominados “Ciclo PIP”-, queda claro que se está trabajando con “fenómenos emergentes” cuyo comportamiento no se adapta a una Distribución Normal. Y que dicho comportamiento errático se debe a que la Inversión Pública es cíclica (Ecuación Evolutiva de Price) y a que el “Ciclo PIP” opera a todo nivel (GN, GR, GL) en función de las relaciones entre los miembros que componen su red. Ergo, es un tema a Analizar con Social Network Analysis (Análisis Social de Redes, ARS). El tesista concluye que las redes de “Ciclo PIP” en el Perú fallan principalmente por problemas de escasez de personal técnico multisectorial debidamente cualificado. A manera de conclusión, propone la creación de una Plataforma Web 3.0 (metadatos), que utilice un Sistema de Razonamiento Basado en Casos (SRBC) para aprovechar el conocimiento que dimana de los éxitos y fracasos de los propios PIPs, con el fin de facilitar las gestiones de los miembros de la red que formulan, evalúan y ejecutan los PIPs en el Perú, tanto a nivel Municipal (GP) como Regional (GR) y Nacional (GN).

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A novel approach is presented to determine mercury in urine samples, employing vortex-assisted ionic liquid dispersive liquid–liquid microextraction and microvolume back-extraction to prepare samples, and screen-printed electrodes modified with gold nanoparticles for voltammetric analysis. Mercury was extracted directly from non-digested urine samples in a water-immiscible ionic liquid, being back-extracted into an acidic aqueous solution. Subsequently, it was determined using gold nanoparticle-modified screen-printed electrodes. Under optimized microextraction conditions, standard addition calibration was applied to urine samples containing 5, 10 and 15 μg L−1 of mercury. Standard addition calibration curves using standards between 0 and 20 μg L−1 gave a high level of linearity with correlation coefficients ranging from 0.990 to 0.999 (N = 5). The limit of detection was empirical and statistically evaluated, obtaining values that ranged from 0.5 to 1.5 μg L−1, and from 1.1 to 1.3 μg L−1, respectively, which are significantly lower than the threshold level established by the World Health Organization for normal mercury content in urine (i.e., 10–20 μg L−1). A certified reference material (REC-8848/Level II) was analyzed to assess method accuracy finding 87% and 3 μg L−1 as the recovery (trueness) and standard deviation values, respectively. Finally, the method was used to analyze spiked urine samples, obtaining good agreement between spiked and found concentrations (recovery ranged from 97 to 100%).

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Title from f. 1r.

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Fragment from an early ʻAbbāsid Qurʼān on parchment dyed orange-red (compare cat.11, p.58 in Déroche, The Abbasid tradition, Nasser D. Khalili collection of Islamic art, v.1 and Metropolitan Museum of Art accession nos. 40.164.1a and 40.164.1b) carrying Sūrat Hūd (11) verses 88 through 103 (11:88-11:103).