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Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling.

The in vitro Cell Transformation Assay (CTA) is a powerful tool for mechanistic studies of carcinogenesis. The endpoint is the classification of transformed colonies (foci) by means of standard morphological features. To increase throughput and reliability of CTAs, one of the suggested follow-up activities is to exploit the comprehension of the mechanisms underlying cell transformation. To this end, we have performed CTAs testing CdCl2, a widespread environmental contaminant classified as a human carcinogen with the underlying mechanisms of action not completely understood. We have isolated and re-seeded the cells at the end (6 weeks) of in vitro CTAs to further identify the biochemical pathways underlying the transformed phenotype of foci. Morphological evaluations and proliferative assays confirmed the loss of contact-inhibition and the higher proliferative rate of transformed clones. The biochemical analysis of EGFR pathway revealed that, despite the same initial carcinogenic stimulus (1 μM CdCl2 for 24 h), transformed clones are characterized by the activation of two different molecular pathways: proliferation (Erk activation) or survival (Akt activation). Our preliminary results on molecular characterization of cell clones from different foci could be exploited for CTAs improvement, supporting the comprehension of the in vivo process and complementing the morphological evaluation of foci.

Cadmium is a widely distributed industrial and environmental pollutant. Principle target organs are soft tissues such as the liver, where cadmium accumulates with a biological half-life of approximately 20-30 years causing a variety of toxic responses. In HepG2, CdCl(2) exposure for short periods (from 1 to 24h) induces differential expression of stress proteins, including MT and hsp70. However, less is known about the stress response during a prolonged exposure to this metal. MTT assay showed a low cytotoxicity of CdCl(2) (0.1, 0.5, 1, 2, 5, 10microM), over a period of 72h. Cadmium uptake by ICP-AES technique and the corresponding expression of stress proteins (MT, hsp70) during the same prolonged time were also analysed. Results show that Cd was continuously and increasingly accumulated, at the highest of the concentrations tested. Metallothionein expression was up-regulated with a saturation curve at 48 as well as 72h after CdCl(2) exposure. High levels of MT probably confer an acquired tolerance to the stress and protection against cell injury as demonstrated by low cytotoxicity values. On the contrary, the unchanged pattern of hsp70 expression suggests that this protective mechanism, unlike other members of the family, is less involved during CdCl(2) prolonged exposure.

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.

This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro -to- in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5–400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.

On May 19, 2015, a meeting was held on the use of alternative methods for the evaluation of hepatotoxicity. It was chaired by Prof. Francesca Caloni and Prof. Giovanna Mazzoleni, and organized by CELLTOX, the Italian Association of in vitro Toxicology, in partnership with the Italian Reference Center for Alternative Methods, Welfare and Care of Laboratory Animal and MISTRAL (Integrated Models for Prevention and Protection in Environmental and Occupational Health) Research Center, University of Brescia, Italy. The meeting was hosted by the Istituto Zooprofilattico Sperimentale della Lombardia e Emilia Romagna, Brescia. The conference was introduced by Dr Maura Ferrari, who reported on the activity of the Italian Reference Center for Alternative Methods, Welfare and Care of Laboratory Animals, indicating the need for alternative liver models.

With complexity comes errors, and unexpected errors lead to unexpected unhappiness. Join us and learn how to add contextual data to errors, design error hierarchies, take charge of control flow, create re-usable error handlers, and integrate with error reporting solutions. We'll talk about recoverable versus irrecoverable errors and discuss how and how not to use exceptions. From internationalization to background jobs, we'll cover the gamut. Regardless of your Rail proficiency, you'll learn why expecting the unexpected makes for happier developers, happier businesses and happier users.

¿Cómo hacer partícipe al habitante de una ciudad determinada de la tradición escrita que le pertenece? El objetivo principal del proyecto de investigación Cartografía literaria de Ciudad Juárez, como su nombre lo indica, es ubicar referencias literarias en mapas urbanos y transitables. De esta forma tanto el desarrollo histórico de esta peculiar metrópolis fronteriza como su estado actual podrán ser conocidos a través de su representación en obras narrativas, poéticas y dramáticas que la han elegido como un espacio protagónico en donde interactúan sus personajes. La salvaguarda del patrimonio intangible que yace en un rico corpus literario adquiere materialidad al hacer el traslado, tanto simbólico como físico, del espacio ficticio al lugar concreto en donde se desarrolla la trama de una obra. A través del diseño de rutas literarias el lector-caminante puede transitar libremente entre hojas y sitios que de a poco se irá apropiando., How to make the inhabitant of a given city part of the written tradition that belongs to her? The main objective of the research project is to locateliterary references in urban and accessible maps. In this way, both the historical development of this peculiar border town and its present state can be known through its representation in narrative, poetic and dramatic works that have chosen it as a protagonist space where their characters interact. Preservation of the intangible heritage that lies in a rich literary corpus acquires materiality by transferring, both symbolic and physical, the fictional space to the specific place where the plot of a work develops. Through the design of literary routes the reader-walker can move freely between pages and sites that will gradually be appropriated., Universidad Pablo de Olavide. Área de Historia del Arte, Versión del editor

Joint physiologically-based toxicokinetic and toxicodynamic (PBTK/TD) modelling was applied to simulate concentration–time profiles of nicotine, a well-known stimulant, in the human body following single and repeated dosing. Both kinetic and dynamic models were first calibrated by using in vivo literature data for the Caucasian population. The models were then used to estimate the blood and liver concentrations of nicotine in terms of the Area Under Curve (AUC) and the peak concentration (Cmax) for selected exposure scenarios based on inhalation (cigarette smoking), oral intake (nicotine lozenges) and dermal absorption (nicotine patches). The model simulations indicated that whereas frequent cigarette smoking gives rise to high AUC and Cmax in blood, the use of nicotine-rich dermal patches leads to high AUC and Cmax in the liver. Venous blood concentrations were used to estimate one of the most common acute effects, mean heart rate, both at rest and during exercise. These estimations showed that cigarette smoking causes a high peak heart rate, whereas dermal absorption causes a high mean heart rate over 48h. This study illustrates the potential of using PBTK/TD modelling in the safety assessment of nicotine-containing products.

For Emilio Carballido, theatre was a pedagogical tool that could reach young people. Not only did he put several plays that dialogue with Spanish classical theater on stage, but also went on further and recast Golden Age dramas. That is what he did with «Sainetes y mojigangas», published in Teatro para adolescentes, sharing authorship with Calderon de la Barca: La tía, El desafío de Juan Rana, La rabia, and La muerte (Editores Mexicanos Unidos, 1985). The fictitious co-authorship was not only meant to circulate classical drama among Mexican people, but also brought young readers/actors close to apparently remote authors. This article studies the function and the adaptation of Calderonian interludes on the hands of Emilio Carballido through an analysis of their composition techniques, recasting, and scenic resources. The double authorship transplants the plays in time and space to make them intelligible to an audience that the Spanish author did not address originally; thus, defending the validity and importance of classical theatre today.&nbsp

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Economia, Administração e Contabilidade, Departamento de Economia, Programa de Pós-Graduação em Economia, 2016. Neste trabalho é analisada a interação entre o ambiente macroeconômico e as decisões individuais das firmas desde a livre flutuação do câmbio em $1999$. Num primeiro momento, estima-se a influência do ambiente macroeconômico na estrutura da dívida das firmas. É possível ver que o fator de maior importância para que firmas possam obter endividamento de longo prazo e endividamento em moeda estrangeira é o tamanho das mesmas. Isto é, empresas maiores têm mais acesso ao mercado de crédito de longo prazo e ao mercado de crédito estrangeiro. Em seguida, estima-se os efeitos patrimoniais das variações cambiais, com base no investimento realizado pelas empresas. Os efeitos não são significantes, mas tornam-se negativos e altamente significantes ao se inserir um controle para exportação e importação a nível setorial. Ao controlar para imperfeições nos mercados de capitais, usando a medida Q de Tobin, os resultados voltam a ser insignificantes, apontando que outros fatores podem ser mais importantes nas decisões das firmas nacionais. In this work we focus on the interaction between the macroeconomic environment and firms individual decisions since Brazil started a free-float regime in 1999. In a first moment, we estimate the influence of the macroeconomic environment inf firms debt structure. It is possible to observe that the major factor influencing that firms obtain long terms financing or financing in foreign currency is the size of the firms. In other words, larger firms have more access to long term and external credit than smaller ones. The following step was to estimate exchange rate balance sheet effects observing firms investment. The results are inconclusive in a first estimation, but became negative and highly significant when controlled for exportation and importation. However, those same results turned to be inconclusive again when controlled for capital market imperfections using the Tobin’s Q, witch indicates that there are other factors playing a more important role in firms decisions.

The Codex Espangliensis, a collective work dealing with political and social matters concerning Chicana/o culture, presents a unique interpretation of the pre-Columbian world, the conquest, the cul...

La interpretacion del teatro breve de la segunda mitad del siglo XVI ha exigido, tanto a su publico contemporaneo, como al lector actual, completar el texto por medio de la reconstruccion del contexto. La extension de la obra requiere el empleo de la informacion del mundo del espectador, fenomeno contrario al de una comedia que crea y expone sus propias reglas, convenciones y lenguaje. En 1570 el librero valenciano Juan Timoneda publico el Registro de representantes, una coleccion de "los passos mas modernos y graciosos." De los siete entremeses que conforman el libro, tres son apocrifos y pertenecen a la pluma del comediante Lope de Rueda, de quien se usa fama y nombre para asegurar la venta. En la construccion de los personajes-tipo, anclados en modelos estables y comportamientos previamente delineados, hay cierta novedad que los guia hacia la diferencia y que genera tension entre la denuncia y la promocion, entre la critica y el escarnio, entre el retrato de costumbres de una determinada localidad y la produccion dramatica que se exporta a los dominios del imperio hispanico. El Registro de representantes esta sujeto a un proceso de actualizacion simultaneo donde, por un lado, la tradicion teatral incorpora ideas locales, mientras que, por otro, las piezas se incorporan y adaptan a nuevas condiciones sociales que funcionan como signos de una modernidad incipiente. Mi trabajo contempla al entremes como un canal de informacion que representa una porcion de la realidad en un momento donde la publicacion de antologias, diccionarios, poeticas, cartografias y calendarios esta vinculada de forma directa con la estandarizacion de sistemas que almacenan, transmiten y organizan informacion.

This paper describes the use of two in vitro systems (stage 35 Xenopus laevis embryos and the human hepatoblastoma cell line, Hep G2) to study effects of some environmental contaminants (benzo[a]pyrene, copper and zinc), which are representative of chemicals with different cell targets and mechanisms of action. The ability to activate benzo[a]pyrene and to metabolise it with the cytochrome P4501A isozyme were demonstrated in both in vitro systems by assessing the formation of water-soluble and protein-bound benzo[a]pyrene metabolites and by immunochemical analysis. In X. laevis embryos, the formation of DNA adducts demonstrated the ability to produce benzo[a]pyrene reactive metabolites. Moreover, in Hep G2 cells, the cytoskeletal protein, tubulin, and the reduced form of glutathione proved to be the cellular targets of copper and zinc toxicity. In response to metal-induced stress in Hep G2 cells, there was a cytoplasmic reorganisation of heat shock protein, Hsp 70. In conclusion, the in vitro systems provide for a rapid evaluation of heterogeneous compounds such as benzo[a]pyrene and heavy metals that differ in toxic potency and mechanisms of action. They could also be used to study the mechanisms of toxic action and to identify specific cellular and molecular targets.

The application of physiologically based toxicokinetic (PBTK) modelling in route-to-route (RtR) extrapolation of three cosmetic ingredients: coumarin, hydroquinone and caffeine is shown in this study. In particular, the oral no-observed-adverse-effect-level (NOAEL) doses of these chemicals are extrapolated to their corresponding dermal values by comparing the internal concentrations resulting from oral and dermal exposure scenarios. The PBTK model structure has been constructed to give a good simulation performance of biochemical processes within the human body. The model parameters are calibrated based on oral and dermal experimental data for the Caucasian population available in the literature. Particular attention is given to modelling the absorption stage (skin and gastrointestinal tract) in the form of several sub-compartments. This gives better model prediction results when compared to those of a PBTK model with a simpler structure of the absorption barrier. In addition, the role of quantitative structure–property relationships (QSPRs) in predicting skin penetration is evaluated for the three substances with a view to incorporating QSPR-predicted penetration parameters in the PBTK model when experimental values are lacking. Finally, PBTK modelling is used, first to extrapolate oral NOAEL doses derived from rat studies to humans, and then to simulate internal systemic/liver concentrations – Area Under Curve (AUC) and peak concentration – resulting from specified dermal and oral exposure conditions. Based on these simulations, AUC-based dermal thresholds for the three case study compounds are derived and compared with the experimentally obtained oral threshold (NOAEL) values.

El tema de este trabajo es la simbolización del mensaje contenido en la ¿Canción de la danza del arquero flechador¿ de Los cantares de Dzitbalché, que instruye y prepara a un guerrero maya para el sacrificio y ofrenda de una vida humana. El enfoque comunicativo centra su atención en los participantes, en la secuencia del ritual y en el efecto impuesto a su auditorio, quien actualiza los procedimientos conductores de la información, convirtiéndose a sí mismo ¿y a lo largo del tiempo¿ en el canal transmisor del mensaje. El propósito comunicativo de los actores debe ser considerado vis à vis con su contexto social, la fijación oral y escrita que reitera el ritual, así como los poderes a los cuales va dirigido el sacrificio., The theme of this work is the symbolization of the message contained in ¿Canción de la danza del arquero flechador¿, from Los cantares de Dzitbalché, which directs and prepares a Mayan warrior to sacrifice and oblate a human life. A communicative approach focuses on participants, on the sequence of the ritual, and on the effect imposed to its audience. These spectators bring up today procedures that transfer information, turning into themselves ¿and over time¿ in the channel/means to transmit the message. The actors¿ communicative purpose should be considered visà-vis their social context, the written and oral tradition that repeats the ritual, as well as the powers to which the sacrifice is offered., Universidad Pablo de Olavide. Área de Historia del Arte, Versión del editor

Emilio Carballido tenía la convicción de que el teatro es una herramienta pedagógica capaz de formar a los adolescentes. No se detuvo con poner en escena varias obras que dialogan con el teatro clásico español, sino que realizó refundiciones de piezas de los Siglos de Oro. Así hizo con los «Sainetes y mojigangas» que aparecen en Teatro para adolescentes bajo su autoría y la de Calderón de la Barca: La tía, El desafío de Juan Rana, La rabia y La muerte (Editores Mexicanos Unidos, 1985). La ficticia coautoría intentaba no solo difundir la dramaturgia clásica entre el pueblo mexicano, sino también acercar a los jóvenes lectores/actores a autores en apariencia lejanos. Este trabajo estudia la función y adaptación del teatro breve calderoniano en manos de Emilio Carballido a través del análisis de sus técnicas de composición, refundición y recursos escénicos. La doble autoría trasplanta de tiempo y espacio dichas piezas para hacerlas inteligibles a un público al que no se dirigía el autor español originalmente, defendiendo así la vigencia e importancia del teatro clásico en nuestros días. For Emilio Carballido, theatre was a pedagogical tool that could reach young people. Not only did he put several plays that dialogue with Spanish classical theater on stage, but also went on further and recast Golden Age dramas. That is what he did with «Sainetes y mojigangas», published in Teatro para adolescentes, sharing authorship with Calderon de la Barca: La tía, El desafío de Juan Rana, La rabia, and La muerte (Editores Mexicanos Unidos, 1985). The fictitious co-authorship was not only meant to circulate classical drama among Mexican people, but also brought young readers/actors close to apparently remote authors. This article studies the function and the adaptation of Calderonian interludes on the hands of Emilio Carballido through an analysis of their composition techniques, recasting, and scenic resources. The double authorship transplants the plays in time and space to make them intelligible to an audience that the Spanish author did not address originally; thus, defending the validity and importance of classical theatre today.

They bridge your application and your database. They're object-relational mappers, and no two are alike. Join us as we compare ActiveRecord from Rails with Ecto from Phoenix, a web framework for Elixir. Comparing the same app implemented in both, we'll see why even with two different web frameworks in two different programming languages, it's the differing ORM designs that most affect the result. This tale of compromises and tradeoffs, where no abstraction is perfect, will teach you how to pick the right ORM for your next project, and how to make the best of the one you already use.

Recently the use of in vitro systems has gained acceptance for toxicological research, since they offer several advantages. We have used primary and immortalized cell cultures to evaluate the effect of the fungicide Methyl 1-(butylcarbamoyl)benzimidazole-2-carbamate (Benomyl), the insecticide O-2-diethylamino-6-methylpyrimidine-4-yl-0,0-dimethyl phosphorothioate (Pirimiphos-methyl), the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Benomyl resulted responsible for microtubular disorganization in time-dose dependent manner, and for glutathione depletion. Pirimiphos-methyl, alone or combined with Benomyl, had no effect on microtubule organization, but reinforced glutathione depletion. MCPA exerted its primary effect on mitochondria, increasing the mitochondrial membrane potential and inhibiting the ATP-synthesizing ATPase. Flow cytometric analysis suggests that MCPA affects C3H cell cycle and induces the formation of type II foci in standard transformation assay. The results presented demonstrate that cultured cells represent a rapid, controlled and useful method to test pesticides both individually and in combination.