Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

O pátio jesuítico no Palácio Anchieta: narrativas tipomorfológicas e paisagísticas na cidade de Vitória (ES)

A hipótese defendida nesta pesquisa se baseia na possibilidade de a arquitetura jesuítica implantada em terras brasileiras (século XVI) dialogar e agenciar, num mesmo corpo edificado, e de modo inter-relacionado, aspectos relativos à morfologia urbana, tipologia e paisagem. Lama explica que, como disciplina, a morfologia urbana agrega para si não somente o ambiente construído, mas os meios pelos quais este foi construído em sua interação com a forma urbana, ou seja, os “fenômenos sociais, econômicos e outros motores da urbanização” (LAMAS, 1992). Entender a forma urbana é entender seus elementos constituintes, “quer em ordem à leitura ou análise do espaço, quer em ordem à sua concepção ou produção” (LAMAS, 1992). Estudar a forma urbana significa compreender o lugar onde se insere a cidade e seus elementos constituintes, seus espaços e a inter-relação entre eles e seu contexto, em um espectro abrangente do que se denomina cidade, e urbano. A tipologia arquitetônica e a morfologia urbana estão interligadas no cerne de suas análises, considerando que ambas, segundo Pereira, estudam “duas ordens de fatos homogêneos” (PEREIRA, 2012); estudam elementos constituintes da cidade – arquitetônicos e espaciais – que se sobrepõem ou se complementam de acordo com a escala de análise utilizada. A arquitetura jesuítica do Brasil colonial modela de modo determinante a construção de distintos núcleos urbanos originários na costa brasileira no século XVI. Isso, por meio da implantação de tipologia edilícia que acompanha a doutrina jesuítica de localização e escolha do sítio para suas construções, preconizando segurança, visibilidade do entorno e facilidade de acesso por rios ou pelo mar. Essas construções, realizadas em áreas elevadas, marcaram, por conseguinte, no tempo e no espaço, a paisagem dos primeiros núcleos urbanos brasileiros. A pesquisa analisou um dos exemplares históricos da arquitetura jesuítica no Estado do Espírito Santo, especificamente na cidade de Vitória, capital e núcleo urbano original da colonização portuguesa neste Estado. A instalação dos jesuítas na antiga Vila da Vitória, no séc. XVI, através de sua igreja dedicada a São Tiago e de seu colégio anexo, marca a presença tipológica de uma arquitetura religiosa que influencia a própria morfologia da cidade – caracterizando esta arquitetura como um tipomorfológico - e, por reflexo, participa da construção de sua paisagem urbana secular. Entende-se que o antigo complexo jesuítico de São Tiago e atual Palácio Anchieta, sede governamental e prédio cultural capixaba, é uma arquitetura que permeia estas três grandes narrativas arquitetônicas e urbanas: a tipologia, a morfologia e a paisagem.

Ação mediada em aulas de Biologia : um enfoque a partir dos conceitos de fotossíntese e respiração celular

Esta pesquisa teve como objetivo investigar o papel da ação mediada na configuração dos sentidos que perpassam um processo de interação discursiva em uma sala de aula de Biologia, da 1ª série do ensino médio, de uma escola pública estadual do município de Vitória-ES, durante aulas que visavam abordar os conceitos de fotossíntese e respiração celular. Para isso, apoiamo-nos na tese de que a ação mediada qualificada como organizada, dialógica, compreensiva e interativa favorece a formação desses conceitos científicos por alunos de ensino médio. Assim, partimos de uma concepção teórico-metodológica ancorada na matriz histórico-cultural. A pesquisa-ação crítico-colaborativa foi utilizada como aporte metodológico e os instrumentos de coleta de dados se basearam em observações do cotidiano escolar e da sala de aula com registros em diários de campo, filmagem das aulas em vídeo, questionários, provas escritas dos alunos e entrevistas reflexivas. As análises dos dados se basearam na análise microgenética proposta por Vigotski, complementada com uma análise compreensiva ancorada nas ideias de Bakhtin. Dois grandes eixos de análise foram delimitados: 1 - a ação mediada no trabalho com os alunos; 2 - os enunciados e sentidos produzidos durante o processo da pesquisa na perspectiva da professora. Os resultados revelam evidências de que a ação mediada qualificada como intencional, organizada, dialógica, compreensiva e interativa favorece a formação dos conceitos científicos de fotossíntese e respiração celular por alunos do ensino médio, promovendo um ensino fecundo, na concepção defendida por Vigotski. Os resultados indicam também que o processo de pesquisa-ação crítico- colaborativa apresentou resultados positivos no que concerne à formação continuada da professora de Biologia. Concluímos que se fazem necessários investimentos em programas de formação de professores que procuram articular escola e universidade, integrando formação inicial e continuada de professores. Para isso, defendemos que esse processo de formação seja feito dentro de uma perspectiva crítica e colaborativa, baseada em uma ação mediada intencional e dialógica que favoreça o desenvolvimento de um ensino fecundo

Towards Emotional Interaction: Using Movies to Automatically Learn Users' Emotional States

The HCI community is actively seeking novel methodologies to gain insight into the user’s experience during interaction with both the application and the content. We propose an emotional recognition engine capable of automatically recognizing a set of human emotional states using psychophysiological measures of the autonomous nervous system, including galvanic skin response, respiration, and heart rate. A novel pattern recognition system, based on discriminant analysis and support vector machine classifiers is trained using movies’ scenes selected to induce emotions ranging from the positive to the negative valence dimension, including happiness, anger, disgust, sadness, and fear. In this paper we introduce an emotion recognition system and evaluate its accuracy by presenting the results of an experiment conducted with three physiologic sensors.

The host-tourist interaction in a world heritage site: the case of Guimarães

Guimarães, in the northwest of Portugal, is a city of strong symbolic and cultural significance and its nomination by UNESCO as world heritage, in 2001, enlarged its tourism potential. In this paper we present a few results of a survey that envisaged capturing the Guimarães residents’ perceptions of tourism impacts and their attitudes towards tourists. Specifically, one analyzes the type of relationship that exists between some socio-demographic groups and the perceived tourism impacts, as well as their socio-characteristics and the existing level of interaction between residents and tourists. The survey was implemented between January and March 2010 to a convenience sample of 540 inhabitants of the municipality of Guimarães resulting in 400 questionnaires with complete data. For this, we made use of various statistical techniques. Using a factorial analysis, we can conclude that the three factors used explain 52.3% of the variance contained in the original variables obtained from the survey. By another side, using a logit model in the analysis and taking as the dependent variable the frequent or very frequent contact with tourists, we found that only the variables referred to perceived positive impacts of tourism, education and the place of residence in urban areas have shown to be statistically significant. We are aware of the multiple ways the issue of residents’ perceptions and attitudes towards tourism can be approached and of the difficulties to get useful policy-oriented insights. This paper is a step in that trail.

Access to information: assessment of the use of automated interaction technologies in call centers

With the purpose of at lowering costs and reendering the demanded information available to users with no access to the internet, service companies have adopted automated interaction technologies in their call centers, which may or may not meet the expectations of users. Based on different areas of knowledge (man-machine interaction, consumer behavior and use of IT) 13 propositions are raised and a research is carried out in three parts: focus group, field study with users and interviews with experts. Eleven automated service characteristics which support the explanation for user satisfaction are listed, a preferences model is proposed and evidence in favor or against each of the 13 propositions is brought in. With balance scorecard concepts, a managerial assessment model is proposed for the use of automated call center technology. In future works, the propositions may become verifiable hypotheses through conclusive empirical research.

Interaction between information systems and work in the Brazilian banking sector

This article presents the results of a research to understand the conditions of interaction between work and three specific information systems (ISs) used in the Brazilian banking sector. We sought to understand how systems are redesigned in work practices, and how work is modified by the insertion of new systems. Data gathering included 46 semi-structured interviews, together with an analysis of system-related documents. We tried to identify what is behind the practices that modify the ISs and work. The data analysis revealed an operating structure: a combination of different practices ensuring that the interaction between agents and systems will take place. We discovered a structure of reciprocal conversion caused by the increased technical skills of the agent and the humanization of the systems. It is through ongoing adjustment between work and ISs that technology is tailored to the context and people become more prepared to handle with technology.

The institutional configuration of sport policy in Brazil: organization, evolution and dilemmas

Brazil has become the center of the spotlight of the whole world recently, amongst many other reasons, one of them was because it was chosen to host a series of mega sporting events - Pan American Games in 2007, Confederations Football Cup in 2013, Fifa Football World Cup 2014 Games and 2016 Olympic and Paralympic Games in 2016. However, little is known about the country's administrative governmental structure focused on sport policy. The available studies focus their analysis on the sport policies content, but not on the arrangement of its structural decision-making. The main aim of this article is indeed to describe, based on official documentation, the evolution and the current arrangements of the government responsible for the administrative structure for the planning and implementation of sports policies in Brazil. Thus, we tried to list the main problems arising from the organization of the Brazilian sports' management. These problems are: (1) inappropriate institutional structure in terms of human resources and obstacles to participation by other social actors beyond the officials (parliament and members of the Ministry of Sports) in the sports policy; (2) disarticulation between public institutions generating redundancies and conflicts of jurisdiction due to the poor division of labor between bureaucracy agencies; and (3) inadequate planning proved by the lack of organization of some institutions, and by the lack of assessment and continuity of public policies over time. Therefore, we must emphasize those problems from above, and due to these administrative arrangements, Brazilian sports' policy has big challenges in the sport development in this country, which includes the creation of a national "system" for sports and a priority investment in sport education.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 3 - On the interaction of vector species and parasite strain in the reaction of bugs to infection by Trypanosoma cruzi

The reaction of nine vector species of Chagas' disease to infection by seven different Trypanosoma cruzi strains; Berenice, Y, FL, CL, S. Felipe, Colombiana and Gávea, are examined and compared. On the basis of the insects' ability to establish and maintain the infection, vector species could be divided into two distinct groups which differ in their reaction to an acute infection by T. cruzi. While the proportion of positive bugs was found to be low in Triatoma infestans and Triatoma dimidiata it was high, ranging from 96.9% to 100% in the group of wild (Rhodnius neglectus, Triatoma rubrovaria)and essentially sylvatic vectors in process of adaptation to human dwellings, maintained under control following successful insecticidal elimination of Triatoma infestans (Panstrongylus megistus, Triatoma sordida and Triatoma pseudomaculata). An intermediate position is held by Triatoma brasiliensis and Rhodnius prolixus. This latter has been found to interchange between domestic and sylvatic environments. The most important finding is the strikingly good reaction between each species of the sylvatic bugs and practically all T. cruzi strains herein studied, thus indicating that the factors responsible for the excellent reaction of P.megistus to infection by Y strain, as previously reported also come into operation in the reaction of the same vector species to acute infections by five of the remaining T.cruzi strains. Comparison or data reported by other investigators with those herein described form the basis of the discussion of Dipetalogaster maximus as regards its superiority as a xenodiagnostic agent.

Equilibrium self-assembly of colloids with distinct interaction sites: Thermodynamics, percolation, and cluster distribution functions

We calculate the equilibrium thermodynamic properties, percolation threshold, and cluster distribution functions for a model of associating colloids, which consists of hard spherical particles having on their surfaces three short-ranged attractive sites (sticky spots) of two different types, A and B. The thermodynamic properties are calculated using Wertheim's perturbation theory of associating fluids. This also allows us to find the onset of self-assembly, which can be quantified by the maxima of the specific heat at constant volume. The percolation threshold is derived, under the no-loop assumption, for the correlated bond model: In all cases it is two percolated phases that become identical at a critical point, when one exists. Finally, the cluster size distributions are calculated by mapping the model onto an effective model, characterized by a-state-dependent-functionality (f) over bar and unique bonding probability (p) over bar. The mapping is based on the asymptotic limit of the cluster distributions functions of the generic model and the effective parameters are defined through the requirement that the equilibrium cluster distributions of the true and effective models have the same number-averaged and weight-averaged sizes at all densities and temperatures. We also study the model numerically in the case where BB interactions are missing. In this limit, AB bonds either provide branching between A-chains (Y-junctions) if epsilon(AB)/epsilon(AA) is small, or drive the formation of a hyperbranched polymer if epsilon(AB)/epsilon(AA) is large. We find that the theoretical predictions describe quite accurately the numerical data, especially in the region where Y-junctions are present. There is fairly good agreement between theoretical and numerical results both for the thermodynamic (number of bonds and phase coexistence) and the connectivity properties of the model (cluster size distributions and percolation locus).

Interaction of formaldehyde and tobacco smoking in the frequency of micronucleus and the XRCC3 Thr241Met polymorphism

Formaldehyde is classified by IARC as carcinogenic to humans (nasopharyngeal cancer). Tobacco smoke has been epidemiologically associated to a higher risk of development of cancer, especially in the oral cavity, larynx and lungs, as these are places of direct contact with many carcinogenic tobacco’s compounds. XRCC3 is involved in homologous recombination repair of cross-links and chromosomal double-strand breaks (Thr241Met polymorphism). The aim of the study is to determine whether there is an in vivo association between genetic polymorphism of the gene XRCC3 and the frequency of genotoxicity biomarkers in subjects exposed or not to formaldehyde and with or without tobacco consumption.

Percolation of colloids with distinct interaction sites

We generalize the Flory-Stockmayer theory of percolation to a model of associating (patchy) colloids, which consists of hard spherical particles, having on their surfaces f short-ranged-attractive sites of m different types. These sites can form bonds between particles and thus promote self-assembly. It is shown that the percolation threshold is given in terms of the eigenvalues of a m x m matrix, which describes the recursive relations for the number of bonded particles on the ith level of a cluster with no loops; percolation occurs when the largest of these eigenvalues equals unity. Expressions for the probability that a particle is not bonded to the giant cluster, for the average cluster size and the average size of a cluster to which a randomly chosen particle belongs, are also derived. Explicit results for these quantities are computed for the case f = 3 and m = 2. We show how these structural properties are related to the thermodynamics of the associating system by regarding bond formation as a (equilibrium) chemical reaction. This solution of the percolation problem, combined with Wertheim's thermodynamic first-order perturbation theory, allows the investigation of the interplay between phase behavior and cluster formation for general models of patchy colloids.

Effects of the interaction of tobacco smoke and alcohol consumption on buccal micronucleus in workers exposed occupationally to formaldehyde

Occupational exposure to formaldehyde (FA) has been shown to induce nasopharyngeal cancer and has been classified as carcinogenic to humans (group 1) on the basis of sufficient evidence in humans. Tobacco smoke has been associated to a higher risk of development of cancer, especially in the oral cavity, larynx and lungs, as these are places of direct contact with many carcinogenic tobacco’s compounds. Alcohol is a recognized agent that influence cells in a genotoxic form, been citied as a strong agent with potential in the development of carcinogenic lesions. Epidemiological evidence points to a strong synergistic effect between cigarette smoking and alcohol consumption in the induction of cancers in the oral cavity. Approximately 90% of human cancers originate from epithelial cells. Therefore, it could be argued that oral epithelial cells represent a preferred target site for early genotoxic events induced by carcinogenic agents entering the body via inhalation and ingestion. The MN assay in buccal cells was also used to study cancerous and precancerous lesions and to monitor the effects of a number of chemopreventive agents.

A dramatic effect of double bond configuration in N-oxy-3-aza Cope rearrangements: a simple synthesis of functionalised allenes

The first examples of low temperature N-oxy-3-aza Cope rearrangements, leading to functionalised allenes are described, where the Z-configuration of the enaminic double bond in the rearranging system proves critical.

Automatic assignment of absolute configuration from 1D NMR data

Opposite enantiomers exhibit different NMR properties in the presence of an external common chiral element, and a chiral molecule exhibits different NMR properties in the presence of external enantiomeric chiral elements. Automatic prediction of such differences, and comparison with experimental values, leads to the assignment of the absolute configuration. Here two cases are reported, one using a dataset of 80 chiral secondary alcohols esterified with (R)-MTPA and the corresponding 1H NMR chemical shifts and the other with 94 13C NMR chemical shifts of chiral secondary alcohols in two enantiomeric chiral solvents. For the first application, counterpropagation neural networks were trained to predict the sign of the difference between chemical shifts of opposite stereoisomers. The neural networks were trained to process the chirality code of the alcohol as the input, and to give the NMR property as the output. In the second application, similar neural networks were employed, but the property to predict was the difference of chemical shifts in the two enantiomeric solvents. For independent test sets of 20 objects, 100% correct predictions were obtained in both applications concerning the sign of the chemical shifts differences. Additionally, with the second dataset, the difference of chemical shifts in the two enantiomeric solvents was quantitatively predicted, yielding r2 0.936 for the test set between the predicted and experimental values.