ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

Do the interactions between glucocorticoids and sex hormones regulate the development of the metabolic syndrome?

The metabolic syndrome is basically a maturity-onset disease. Typically, its manifestations begin to flourish years after the initial dietary or environmental aggression began. Since most hormonal, metabolic, or defense responses are practically immediate, the procrastinated response do not seem justified. Only in childhood, the damages of the metabolic syndrome appear with minimal delay. Sex affects the incidence of the metabolic syndrome, but this is more an effect of timing than absolute gender differences, females holding better than males up to menopause, when the differences between sexes tend to disappear. The metabolic syndrome is related to an immune response, countered by a permanent increase in glucocorticoids, which keep the immune system at bay but also induce insulin resistance, alter the lipid metabolism, favor fat deposition, mobilize protein, and decrease androgen synthesis. Androgens limit the operation of glucocorticoids, which is also partly blocked by estrogens, since they decrease inflammation (which enhances glucocorticoid release). These facts suggest that the appearance of the metabolic syndrome symptoms depends on the strength (i.e., levels) of androgens and estrogens. The predominance of glucocorticoids and the full manifestation of the syndrome in men are favored by decreased androgen activity. Low androgens can be found in infancy, maturity, advanced age, or because of their inhibition by glucocorticoids (inflammation, stress, medical treatment). Estrogens decrease inflammation and reduce the glucocorticoid response. Low estrogen (infancy, menopause) again allow the predominance of glucocorticoids and the manifestation of the metabolic syndrome. It is postulated that the equilibrium between sex hormones and glucocorticoids may be a critical element in the timing of the manifestation of metabolic syndrome-related pathologies.

Confirmatory factor analysis of an inventory of perception of insecurity and fear of crime

This paper focuses on the study of the factorial structure of an inventory to estimate the subjective perception of insecurity and fear of crime. Made from the review of the literature on the subject and the results obtained in previous works, this factor structure shows that this attitude towards insecurity and fear of crime is identified through a number of latent factors which are schematically summarized in (a) personal safety, (b) the perception of personal and social control, (c) the presence of threatening people or situations, (d) the processes of identity and space appropriation, (e) satisfaction with the environment, and (f) the environmental and the use of space. Such factors are relevant dimensions to analyze the phenomenon. Method: A sample of 571 participants in a neighborhood of Barcelona was evaluated with the proposed inventory, which yielded data from the distributions of all the items provided. The administration was conducted by researchers specially trained for it and the results were analyzed by using standard procedures in the confirmatory factor analysis (CFA) from the hypothesized theoretical structure. The analysis was performed by decatypes according to the different response scales prepared in the inventory and their ordinal nature, and by estimating the polychoric correlation coefficients. The results show an acceptable fit of the proposed model, an appropriate behavior of the residuals and statistically significant estimates of the factor loadings. This would indicate the goodness of the proposed factor structure.

Role of microRNAs in the age-associated decline of pancreatic beta cell function in rat islets.

AIMS/HYPOTHESIS: Ageing can lead to reduced insulin sensitivity and loss of pancreatic beta cell function, predisposing individuals to the development of diabetes. The aim of this study was to assess the contribution of microRNAs (miRNAs) to age-associated beta cell dysfunction. METHODS: The global mRNA and miRNA profiles of 3- and 12-month-old rat islets were collected by microarray. The functional impact of age-associated differences in miRNA expression was investigated by mimicking the observed changes in primary beta cells from young animals. RESULTS: Beta cells from 12-month-old rats retained normal insulin content and secretion, but failed to proliferate in response to mitotic stimuli. The islets of these animals displayed modifications at the level of several miRNAs, including upregulation of miR-34a, miR-124a and miR-383, and downregulation of miR-130b and miR-181a. Computational analysis of the transcriptomic modifications observed in the islets of 12-month-old rats revealed that the differentially expressed genes were enriched for miR-34a and miR-181a targets. Indeed, the induction of miR-34a and reduction of miR-181a in the islets of young animals mimicked the impaired beta cell proliferation observed in old animals. mRNA coding for alpha-type platelet-derived growth factor receptor, which is critical for compensatory beta cell mass expansion, is directly inhibited by miR34a and is likely to be at least partly responsible for the effects of this miRNA. CONCLUSIONS/INTERPRETATION: Changes in the level of specific miRNAs that occur during ageing affect the proliferative capacity of beta cells. This might reduce their ability to expand under conditions of increased insulin demand, favouring the development of type 2 diabetes.

Growth factor- and cytokine-driven pathways governing liver stemness and differentiation.

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/progenitor cell expansion and differentiation, and the relevance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to proliferation, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the transforming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-β mediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expansion of liver stem cells. Hedgehog family ligands are necessary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell factor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.

AMPc - Sinalização intracelular: um software educacional

Chemicals binding to membrane receptors may induce events within the cell changing its behavior. Since these events are simultaneous and hard to be understood by students, we developed a computational model to dynamically and visually explore the cAMP signaling system to facilitate its understanding. The animation is shown in parts, from the hormone-receptor binding to the cellular response. There are some questions to be answered after using the model. The software was field-tested and an evaluation questionnaire (concerning usability, animations, models, and the software as an educational tool) was answered by the students, showing the software to be a valuable aid for content comprehension.

Efecto de los usos del suelo en la emisión de dióxido de carbono del suelo a la atmósfera en un agroecosistema semiárido del Valle del Ebro

Los cambios en los usos del suelo han contribuido de manera importante al incremento de gases de efecto invernadero en la atmósfera, especialmente de dióxido de carbono, aumentando sus emisiones desde 1970 en un 80%. Estos cambios causan la alteración de los suelos provocando un impacto sobre el ciclo del carbono, aumentando las tasas de descomposición de la fracción orgánica creando así un flujo de CO2 a la atmosfera. Entre las recomendaciones del Panel Intergubernamental de expertos sobre el Cambio Climático (IPCC, en inglés), y contemplado en el Protocolo de Kyoto, se encuentra el proceso de secuestro de carbono en suelos, que implica la eliminación del CO2 atmosférico por parte de las plantas y su almacenamiento como materia orgánica del suelo. Para poder favorecer dicho proceso, en un determinado tipo de ecosistema, es fundamental conocer cuáles son los factores que gobiernan la respiración del suelo y el impacto que tienen los diferentes usos en la emisión de CO2. En el presente trabajo se han estudiado 4 usos del suelo representativos del secano aragonés: un monocultivo de cebada en siembra directa (NT), un suelo abandonado labrado (AC), un suelo abandonado no alterado (AU) y un suelo forestal (FR) con el objetivo de conocer sus tasas de respiración, la influencia de diferentes parámetros edáficos en ellas, y proponer cambios en el uso del suelo que ayuden a mitigar estas emisiones. Además, se ha dedicado un apartado para conocer cómo influyen diferentes técnicas de fertilización nitrogenada (mineral y orgánica) en la respiración de un monocultivo de cebada en siembra directa. En cuanto a los usos, los resultados obtenidos tanto in situ como en laboratorio muestran una mayor respiración en AC, siendo los valores más bajos los de NT y FR. Una de las principales conclusiones es que la supresión del laboreo y del periodo de barbecho largo, así como la conversión de tierras abandonadas y marginales a cultivos y zonas forestales se presentan en este tipo de ecosistemas como prácticas de secuestro de carbono. En el estudio de aplicación de fertilizantes, no se observó ningún cambio en la respiración del suelo después de la aplicación de nitrógeno mineral. En cambio, el suelo fertilizado con purín sí que mostró picos de emisión durante las siguientes horas a la incorporación de éste, debido fundamentalmente a su alto contenido de carbono lábil.

Experimento didático sobre cromatografia gasosa: uma abordagem analítica e ambiental

This paper describes an experiment to teach the principles of gas chromatography exploring the boiling points and polarities to explain the elution order of a series of alcohols, benzene and n-propanone, as well as to teach the response factor concept and the internal standard addition method. Retention times and response factors are used for qualitative identification and quantitative analysis of a hypothetical contamination source in a simulated water sample. The internal standard n-propanol is further used for quantification of benzene and n-butanol in the water sample. This experiment has been taught in the instrumental analysis course offered to chemistry and oceanography students.

Resposta antioxidante, formação de fitoquelatinas e composição de pigmentos fotoprotetores em Brachiaria decumbens Stapf submetida à contaminação com Cd e Zn

In order to evaluate the response of Brachiaria decumbens Stapf to Cd and Zn, plants were hydroponically exposed to 50 and 100 mmol L-1 of Cd and 500 and 2000 mmol L-1 of Zn. Metal content of shoots and roots was determined, as well as alterations in photosynthetic and photoprotective pigments, antioxidant metabolites and phytochelatin synthesis. Plants concentrated elevated levels of Cd and Zn, especially in roots. Zinc exposure negatively affected chlorophyll and β-carotene content, whereas the highest dose of Cd reduced VAZ cycle pigments and tocopherol levels in plant shoots. Cadmium was the maximum inducer of the phytochelatin synthesis pathway.

Aumento da resposta analítica por meio da otimização do sistema de injeção sem divisão de fluxo em cromatografia gasosa empregando a lei dos gases ideais

This paper reports the development of an easy, fast and effective procedure for the verification of the ideal gas law in splitless injection systems in order to improve the response. Results of a group of pesticides were used to demonstrate the suitability of the approach. The procedure helps establish experimental parameters through theoretical aspects. The improved instrumental response allowed extraction with lower sample volumes, the minimization of time and costs and the simplification of sample preparation.

Experimento didático de quimiometria para planejamento de experimentos: avaliação das condições experimentais na determinação espectrofotométrica de ferro II com o-fenantrolina. Um tutorial, parte III

The objective of this manuscript is to describe a practical experiment that can be employed for teaching concepts related to design of experiments using Matlab or Octave computing environment to beginners, undergraduate and graduate students. The classical experiment for determination of Fe (II) using o-phenanthroline was selected because it is easy to understand, and all the required materials are readily available in most analytical laboratories. The approach used in this tutorial is divided in two steps: first, the students are introduced to the concept of multivariate effects, how to calculate and interpret them, and the construction and evaluation of a linear model to describe the experimental domain by using a 2³ factorial design. Second, an extension of the factorial design by adding axial points is described, thereby, providing a central composite design. The quadratic model is then introduced and used to build the response surface.

Influence of plasma modification on surface properties and offset printability of coated paper

The properties of the paper surface play a crucial role in ensuring suitable quality and runnability in various converting and finishing operations, such as printing. Plasma surface modification makes it possible to modify the surface chemistry of paper without altering the bulk material properties. This also makes it possible to investigate the role of the surface chemistry alone on printability without influencing the porous structure of the pigment-coated paper. Since the porous structure of a pigment coating controls both ink setting and optical properties, surface chemical changes created by a plasma modification have a potential to decouple these two effects and to permit a better optimization of them both. The aim of this work was to understand the effects of plasma surface modification on paper properties, and how it influences printability in the sheet-fed offset process. The objective was to broaden the fundamental understanding of the role of surface chemistry on offset printing. The effects of changing the hydrophilicity/ hydrophobicity and the surface chemical composition by plasma activation and plasma coatings on the properties of coated paper and on ink-paper interactions as well as on sheet-fed offset print quality were investigated. In addition, the durability of the plasma surface modification was studied. Nowadays, a typical sheet-fed offset press also contains units for surface finishing, for example UVvarnishing. The role of the surface chemistry on the UV-varnish absorption into highly permeable and porous pigment-coated paper was also investigated. With plasma activation it was possible to increase the surface energy and hydrophilicity of paper. Both polar and dispersion interactions were found to increase, although the change was greater in the polar interactions due to induced oxygen molecular groups. The results indicated that plasma activation takes place particularly in high molecular weight components such as the dispersion chemicals used to stabilize the pigment and latex particles. Surface composition, such as pigment and binder type, was found to influence the response to the plasma activation. The general trend was that pilot-scale treatment modified the surface chemistry without altering the physical coating structure, whereas excessive laboratory-scale treatment increased the surface roughness and reduced the surface strength, which led to micro-picking in printing. It was shown that pilot-scale plasma activation in combination with appropriate ink oils makes it possible to adjust the ink-setting rate. The ink-setting rate decreased with linseed-oil-based inks, probably due to increased acid-base interactions between the polar groups in the oil and the plasma-treated paper surface. With mineral-oil-based inks, the ink setting accelerated due to plasma activation. Hydrophobic plasma coatings were able to reduce or even prevent the absorption of dampening water into pigmentcoated paper, even when the dampening water was applied under the influence of nip pressure. A uniform hydrophobic plasma coating with sufficient chemical affinity with ink gave an improved print quality in terms of higher print density and lower print mottle. It was also shown that a fluorocarbon plasma coating reduced the free wetting of the UV-varnish into the highly permeable and porous pigment coating. However, when the UV-varnish was applied under the influence of nip pressure, which leads to forced wetting, the role of the surface chemical composition seems to be much less. A decay in surface energy and wettability occurred during the first weeks of storage after plasma activation, after which it leveled off. However, the oxygen/carbon elemental ratio did not decrease as a function of time, indicating that ageing could be caused by a re-orientation of polar groups or by a contamination of the surface. The plasma coatings appeared to be more stable when the hydrophobicity was higher, probably due to fewer interactions with oxygen and water vapor in the air.

A multibody dynamic model of the cross-country ski-skating technique

The objective of this thesis is the development of a multibody dynamic model matching the observed movements of the lower limb of a skier performing the skating technique in cross-country style. During the construction of this model, the formulation of the equation of motion was made using the Euler - Lagrange approach with multipliers applied to a multibody system in three dimensions. The description of the lower limb of the skate skier and the ski was completed by employing three bodies, one representing the ski, and two representing the natural movements of the leg of the skier. The resultant system has 13 joint constraints due to the interconnection of the bodies, and four prescribed kinematic constraints to account for the movements of the leg, leaving the amount of degrees of freedom equal to one. The push-off force exerted by the skate skier was taken directly from measurements made on-site in the ski tunnel at the Vuokatti facilities (Finland) and was input into the model as a continuous function. Then, the resultant velocities and movement of the ski, center of mass of the skier, and variation of the skating angle were studied to understand the response of the model to the variation of important parameters of the skate technique. This allowed a comparison of the model results with the real movement of the skier. Further developments can be made to this model to better approximate the results to the real movement of the leg. One can achieve this by changing the constraints to include the behavior of the real leg joints and muscle actuation. As mentioned in the introduction of this thesis, a multibody dynamic model can be used to provide relevant information to ski designers and to obtain optimized results of the given variables, which athletes can use to improve their performance.

Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia

Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days), receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies) and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs.

Research of Klebsiella pneumoniae in dairy herds

Klebsiella pneumoniae is a common environmental agent of clinical and subclinical mastitis affecting dairy herds, and may be present in the final product decreasing its quality. Mastitis caused by K. pneumoniae is even more severe due to its poor response to antibiotic therapy, rapid evolution to toxic shock and death of the animal. This paper aimed to study the prevalence of this pathogen among dairy herds in ten farms located in different municipalities of São Paulo State based on size and use of milking technology. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20mL) was collected from all CMT-positive quarters and bulk tanks, whereas swab samples were collected from feces, hind limbs of the animals, bedding and milking parlor. Identification of K. pneumoniae was performed using conventional microbiology culture, biochemical assay and Polimerase Chain Reaction. The primers were designed and tested at the Laboratory of Molecular Biology applied to Zoonoses (FMVZ, Unesp-Botucatu) targeting the 16S rRNA gene. This study included 1067 animals. Six cases of intramammary infection by K. pneumoniae were detected in six different cows in two farms. Moreover, K. pneumoniae was isolated in 77 swabs (34 from bedding in 9 farms, 7 from waiting rooms in 5 farms, 6 from milking parlors in 4 farms, 11 from rectums in six farms, and 19 from hindlimbs in 7 farms. Molecular analysis confirmed the agent was K. pneumoniae. At least one strain of the agent was identified in a certain site in all farms, showing the need of maintaining the hygiene in dairy farms.