Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Produções independentes em arte e comunicação como ferramentas para a construção de realidades sociais: estudos de caso no Brasil

Dissertação de mestrado em Comunicação, Arte e Cultura

Kun pelko astuu lavalle : eli kuinka opin elämään esiintymisjännityksen kanssa

This thesis focuses on stage fright; what it is and how to find the ideal solution in connection with stage fright. To find the way toward free expression; to find the way from lack of control to con-trol. It also describes how to learn to live with stage fright. Different experiences of stage fright have been used as source material, along with relevant theoretical information. The thesis highlights the importance of handling stage fright and studies stage fright in-depth, with examples. It presents studies and collected theories according to Päivi Arjas. It discusses presence and contact in the work of an actor with reference to the Gestalt method. It also presents methods of reducing stage fright. Ideal presence on stage is addressed, and how to turn stage fright into a positive thing. Finally, it considers the good aspects of stage fright in more deepth and goes through learning to live with stage fright. The thesis concludes that it is possible to heal stage fright. Gestalt methodology was of assistance with the healing process, with becoming conscious of symptoms, and thus in finding one's own way of turning anxious suspense into a positive way forward.

Untersuchung zum Phosphorylierungsstatus der katalytischen Untereinheit der PKA mittels proteomischer Techniken

ZUSAMMENFASSUNG: Das Phosphorylierungsmuster eines Proteins ist kein statischer Zustand, sondern vielmehr ein dynamischer Status, den es in der modernen funktionellen (Phospho-) Proteomik und Analytik abzubilden gilt. Klassischerweise erfolgt der Nachweis der Proteinphosphorylierung auf Peptid-Ebene mittels MS/MS Sequenzierung. Diese Standardmethode der shotgun Phosphoproteomanalytik vernachlässigt jedoch wegen den in LC MS/MS Analysen oftmals schwer detektierbaren Phosphopeptiden gerade den variablen und oftmals nur geringen Phosphorylierungsgrad vieler Phosphorylierungsstellen (P-Stellen). Mittels phosphospezifischer Anreicherungsstrategien und MS/MS Sequenzierung konnten an der Modellkinase PKA-Cα nach rekombinanter Expression in E. coli insgesamt acht P-Stellen identifiziert werden. Der Phosphorylierungsgrad wurde in Kooperation mit Dr. J. Seidler über quantitative Signalintensitätsmessungen bestimmt und zeigte eine nahezu vollständige Phosphorylierung von pS10, pS139, pT197 und pS338, während der Phosphorylierungsgrad für pS34, pS53, pS65 und pS259 zwischen <5 und 45 % variierte. Neben der Quantifizierung der P-Stellen wurde auch das Auftreten und die Verteilung definierter Phosphoformen der PKA-Cα untersucht und deren Abhängigkeit von der primären Aminosäureabfolge, dem Auftreten von zusätzlichen Modifikationen sowie den gewählten Expressions- und Reinigungsbedingungen aufgezeigt. Endogene, aus Säugergewebe isolierte PKA-Cα wies nur eine einzige Phosphoform mit den P-Stellen pT197 und pS338 auf. Auch in vitro autophosphorylierte rekombinante PKA-Cα, die zuvor dephosphoryliert worden war, wies eine zweifach modifizierte Phosphoform auf. Im Vergleich zum endogenen Protein ließ sich dieses Protein an S10 und S338 exzessiv phosphorylieren, wohingegen an T197 keine Autophosphorylierung nachzuweisen war. Das Ausbleiben weiterer Phosphorylierungen stellt in Frage, ob die Hyperphosphorylierung in E. coli ausschließlich auf Autophosphorylierungsprozessen beruht, was anhand einer nicht phosphorylierten, katalytisch inaktiven Variante von PKA-Cα (PKA-Cα K72H) vermutet wurde. Im Hinblick auf die funktionellen P-Stellen pT197 und pS338 erfordert diese Entdeckung sowie der unabhängige Nachweis, dass zellfrei exprimierte PKA-Cα nur an S338 phosphoryliert ist, eine Modifizierung des sequenziellen Vorhersagemodells, wonach die Phosphorylierung an T197 eine zwingende Voraussetzung für die nachfolgende Phosphorylierung an S338 ist. Ferner konnte über phosphomimetische Mutagenese die Funktionalität der Phosphorylierung an S53 innerhalb der glycinreichen Schleife der PKA-Cα und somit ein potenzieller Weg zur Regulation der enzymatischen Aktivität gezeigt werden. Ein weiterer möglicher upstream Regulator von PKA-Cα ist die Proteinphosphatase 5, die in der Lage war, die bislang als phosphatasestabil beschriebene P Stelle pT197 in vitro zu dephosphorylieren. Die vorliegende Arbeit zeigt, dass der Phosphorylierungszustand eines Proteins von zahlreichen internen und externen Faktoren abhängt – eine Tatsache, die gerade für rekombinante Proteine, insbesondere enzymatisch aktive Kinasen, oft vernachlässigt wurde. Daher müssen auch in der shotgun Phosphoproteomanalytik P-Stellen nicht mehr nur identifiziert und quantifiziert werden, sondern die resultierenden Proteinphosphoformen differenziert auch in ihrem physiologischen Kontext beschrieben werden.

Push button parliament–why India needs a non-partisan, recorded vote system

Decisions of national importance are made by Parliamentary voting. Yet Indian Members of Parliament (MPs) vote with a remarkable lack of freedom and accountability. The introduction of the Tenth Schedule in the Constitution has crippled free expression, since it provides that MPs voting against ‘any direction’ of their Party are liable to disqualification from the legislature  In addition, except for Constitutional amendments, Indian Parliamentary Procedure Rules do not require votes of MPs to be recorded unless the Speaker’s decision is contested in the House. The result is that voting in the House has become mechanical, controlled by Party politics and devoid of responsibility. This paper comments on a general theory of democratic accountability through the lens of Parliamentary voting. It suggests that the voting system adopted in the Parliament is an effective indicator to measure the level of accountability of its Members. In the context of India, this paper argues that the level of accountability will increase to a desirable extent only when there is adoption of a recorded system for every important House vote. Upon examination of India’s record thus far (through the sample of the 14th Lok Sabha) it becomes evident that the level of divisions (recorded votes) is substantially lower than other countries. This leads the paper to probe, as to why that might be the case. Part II of the paper answers that question by examining the Tenth Schedule of the Constitution. The paper scrutinizes the disproportionate influence of the Party in decision making in the Parliament. Apart from dealing with the inherent problem of the Tenth Schedule, this paper suggests two procedural changes to make parliamentary expression more meaningful. Firstly, the recording of all important votes within the Parliament and secondly, registering Party whips with the Minister of Parliamentary Affairs so that the voter knows the clear stand of every Parliamentary continuum. The focus of the paper is thus to bring back the attention of the legislators to their central function, which is deliberation on and the passage of legislation.

Reading between Librarians’ lines

It is a well known fact that librarians, as professionals go, are not among the world's most demonstrative people. I can say that with impunity because I am one. It's one thing for a man to slight his own profession; quite another when someone else presumes to do it. But given that librarians are a placid sort, just mention the phrase "intellectual freedom," or utter the word "censorship," and the usually calm demeanor of the librarian becomes as agitated as the water between Charybdis and Scylla! This article addresses three aspects of the issue of intellectual freedom and attempts to define the difference between the phrases "free speech" and "free expression." First to be explored will be the nature of intellectual freedom as defined by the American Library Association's Intellectual Freedom Manual; second, the underlying philosophy implicit in that expression; and third, an alternative to both the manual, and its philosophical presuppositions.

As repercussões dos serviços hospitalares em diferentes contextos: uma análise de profissionais de saúde

This study has as general object: to verify as the health professionals that work in different hospitals evaluate the hospital services; and specific objectives: identify how health professionals evaluate the work conditions in different hospitals and verify the central nucleus and the peripheral elements of the social representations elaborated by these professionals about these institution. This is a descriptive study realized in Natal, capital of Rio Grande do Norte. Were collected 213 questionnaires. Concerning the evaluation of the health services, high averages had been verified in the philanthropic hospital, for example, quality of the customer service. At the state hospital was observed lowest ones: respect to the privacy of the patients. Similar results were found about work conditions. It was observed as central nucleus the categories Overcrowded and Humanized care, in the state and philanthropic hospital respectively, and as peripheral elements Low wages and Overcrowded. Being thus the conflicts in this scene are inevitable because of the poor structure of some public hospitals, however, these problems can be foresee and solvable if the hospital has a free expression channel accessible to all agents. The evaluation has to become integrant part of the culture of the organization, a time that this will guide the steps in direction to the best quality in the hospital assistance

Livre expressão :para que te quero? o interesse público no ensino do Jornalismo

The main objective of this research was studying the meanings of the freedom of expression and what professors of Journalism think about the way those meanings are used in pedagogical practices. The term freedom of expression is commonly used in journalism even though it is not so studied, consequently we don t have a word to define it. Therefore, we related the term freedom of expression in Journalism teaching to the condition as the object of this study, aiming to establish a connection among the term, the right, free expression and the endurance of the right. The theorical support to understand the dynamic of the meanings of the freedom of expression in the social practice of Journalism teaching was based in the Hegeliana dialetic theorical principles and in the language philosophy more specifically in Bakhtin s perspective from which we can mention the social auditorium, immediate conditions of production and a wider social horizon as the main categories of analysis. This study is a qualitative research with an interacionist perspective anchored in a semi-structured interview as a privileged method of data collecting made with ten professors from Journalism graduation. The analysis reveals that, in the interviewers perspective, there would be a deceiving practice from freedom of expression in the journalism teaching in which emerges either in speeches with handbills spreading the fear and/or under a stimulus form to concealment

Nas asas de Ícaro: uma análise da Pedagogia Freinet do 2º ao 5º ano do Ensino Fundamental na perspectiva da ação docente continuada

Célestin Freinet was one of the most memorable educators of the twentieth century. He presented some educational alternatives that had the objective of stimulating the construction of pedagogic actions to promote the social development of the student based upon a work centered on the free expression as the way to self-structure the knowledge. With a permanent proposal of research based on the enquiry-based learning, Freinet set human capacity (cognitive, social-affective, psychometrical) taking cooperation in consideration on the processes of knowledge construction. Based on this referential, this present work has the objective to show Freinet s pedagogy in a continuous teaching action from 2nd to 5th grade focusing the teacher s discourse and also the educative practice of the students of a city public school in Natal. The observed work revealed the teacher s discourse and educative experience and delineated the students development while immersed into Freinet s educational practice. By virtue of the nature and specificity of the theme that guided our research, we chose a qualitative approach to it, as a way of conducting ourselves during our investigative process. We observed and analyzed the method that was used to conduct the activities in the classroom, as well as the ways of expression that the children used through drawings, words (oral text), or through writing. We highlight, among the written texts, the individual and collective texts, and also letters and notes, which during some moments served as a base to express a diversity of languages. The results, after a analyzing the research data, point towards a teaching practice that favors the construction of a significant learning process through the grasping of the school environment, on which all these factors are based: society, knowledge acquisition, abilities, attitudes and values. This learning process also strengthens human solidarity bonds and mutual tolerance, on which the student s social life is seated.

DANÇA: linguagem do corpo na educação infantil

This production is a reflection about the practices/experiences involving the teaching of the dance developed by the teachers in the Núcleo de Educação Infantil (NEI/UFRN), with the children from 2 to 7 years old, having as objectives: describe and interpret the lived experiences with the dance focusing on the meaning of the dance and organization of the pedagogic job, identify blanks in the pedagogic practices of dance and appoint possible perspectives to the teaching of dance on the infant education. The way from the investigation has begun with the following question: Who do the teachers understand the dance and live it on the Núcleo de Educação Infantil? The research of phenomenological orientation toke as methodological reference the qualitative approach from the placed phenomenon type, this one has as beginning the interest on the phenomenon by the way as it happens on the lived experience from where comes the knowledge with we can present about the world, trying to interpret it, understand it on its essence/existence. The interviews showed that the researched subjects give to the dance different meanings and consequently present variations on the manner to organize the work around this knowledge. The most of the teachers recognizes the dance as a culture expression, being a priority on the school the job with the folkloric dances. They recognize too the dance as knowledge/content coming from the Arts and Physical Education areas and its relation with the knowledge from others areas on the pedagogic action. There were found different process on the way to conduct the job with the dance in relates from experiences done with dance at NEI and, therefore, of teach/learn this knowledge. Its perceptible that exists a systematic work with the objective of develop the dance and its various educative possibilities, starting from the research about its origins, the exploration of the movement, the contextualization until the artistic practice. Those possibilities are reinforced on the school s curricular propose. Some experiences have as a priority the free expression, the dance s vision without context or the reproduction of movements as stand manners of teaching dance, situations like those observed on the investigation as products from the blanks on the academic formation from the teachers with must be fixed. The interpretation of the experiences with dance, described by the teachers from NEI, connected with the theoretical referential from the investigation, allowed to appoint three perspectives to the work with dance in the infant education, having as main interlocutors Merleau-Ponty and Rudolf Laban: the dance as the body s language; the dance and its movement factors; imitate, improvise and play: manners of draw ways to dance. On those process are emphasized the dance on the scholar context, the elements that constitute this language and the forms of appropriate from it supported in a ludicrous, poetic and educative vision, having as focus the children s education on its peculiarities and possibilities

O processo de apropriação da escrita e a formação da capacidade produtora de textos

Pós-graduação em Educação - FFC

Contribuições da arteterapia no cuidado com mulheres em tratamento do câncer de mama

Esta dissertação, fruto de uma experiência pessoal e profissional com a arte e do interesse por estudos e ações na área da oncologia, consistiu numa pesquisa-intervenção baseada em fundamentos da fenomenologia e da arteterapia gestáltica, que teve a finalidade de identificar contribuições da arteterapia no cuidado de mulheres em tratamento do câncer de mama, a partir dos significados atribuídos pelas participantes à experiência com o fazer criativo. A importância desse estudo esteve relacionada à necessidade de ampliação de estratégias terapêuticas voltadas para a promoção da saúde de indivíduos em tratamento do câncer. Por isso foi levada em consideração a existência de fatores psicossociais geradores de conflitos e sofrimentos para as mulheres durante o período de diagnóstico e tratamento da doença, bem como a condição feminina de submissão e enfrentamento do câncer de mama durante a história. Além disso, o trabalho fez referência às funções terapêuticas da arte e a importância da arteterapia em oncologia. A pesquisa foi realizada no Núcleo de Acolhimento do Enfermo Egresso - NAEE, onde se aplicou a metodologia denominada de Oficina Criativa, constituída pelas etapas de sensibilização, expressão livre, elaboração da expressão, transposição da linguagem e avaliação. Para a coleta de dados foram utilizados os discursos e as criações das mulheres. Participaram 7 mulheres que estavam em tratamento para o câncer de mama, de 8 sessões de arteterapia, nas quais realizaram vivências corporais e atividades expressivas. A análise do tipo qualitativa constituiu-se com base nas expressões manifestas pelas informantes através da linguagem verbal e plástica durante a criação. O ambiente permeado pela escuta e acolhimento, favorecidos durante as oficinas, interpôs o processo terapêutico desta pesquisaintervenção, contribuindo para a resposta das mulheres em relação à experiência nas oficinas. Foi possível identificar uma riqueza de contribuições da arteterapia para o cuidado de mulheres com câncer de mama, no que se referiu a possibilidade de interação com o outro, ao desenvolvimento da capacidade de expressão, significação e ressignificação do vivido através do contato com o próprio potencial criativo e da aquisição de uma postura mais ativa das participantes para enfrentar as dificuldades da vida.

A expressão do pesar nas atividades ocupacionais quando alguém querido morre

O luto pela morte de um ente querido compreende um processo de reorganização pessoal frente à perda e que por isso tende a desdobramento singulares, conforme a natureza e intensidade do vínculo. A pessoa então pode experimentar uma variedade de reações que , conforme a ocorrência e a severidade das manifestações , irão repercutir na qualidade de viver. Esta pesquisa teve o objetivo de compreender a expressão do pesar nas atividades ocupacionais quando da morte de uma pessoa significativa. A estratégia metodológica fundamentou-se na abordagem qualitativa do tipo "estudo de caso", de duas pessoas (colaboradores) que chegaram em Serviço de Pronto Atendimento a Pessoas que Sofreram Perdas. Para a coleta dos dados foram definidos três encontros, sendo que nos dois primeiros foram realizadas às entrevistas abordando aspectos da vida pessoal e ocupacional e no terceiro uma oficina de atividades, sendo disponibilizados materiais plásticos; como papel , lápis de cor, canetas esferográficas, tesoura, revistas, cola branca, cola colorida, purpurina, entre outros, com o objetivo de favorecer a livre expressão nas atividades ocupacionais, estando de acordo com a hipótese de que, em situações de perdas e luto, as pessoas experiência um período de retraimento e afastamento das relações sociais e das atividades habituais, indicando que a perda interfere significativamente no cotidiano das suas ocupações, incluindo falta de prazer em desempenhar o trabalho, em ter cuidados pessoais e nas atividades da vida diária (AVD' S), sugerindo a ocorrência do luto ocupacional pela perda das atividade desempenhadas com e para o ente querido falecido. Neste sentido, pode ser observada alteração nas funções ocupacionais em que padrões habituais de atividades são rompidos, remetendo a difícil tarefa de renunciar, excluir e incluir novos papéis. Por outro lado, a estratégia proposta a coleta dos dados revelou-se como um recurso favorável à compreensão e expressão do enlutado, estimulando a aceitação da perda, a avaliação do vinculo co falecido (a) e a elaboração do luto. O uso do recurso material foi favorável a expressão dos pensamentos, sentimentos e necessidades, bem como de competência, habilidades, funções ocupacionais e outros aspectos da existência, ressaltando a importância da compreensão biopsicossocial e ocupacional da pessoa em situação de luto, em que se destaca a assistência Terapêutica Ocupacional agregando esforços na prevenção e promoção a saúde.

Functional characterisation of in vitro synthesised G-protein coupled receptors in polymersomes

Membrane proteins play an indispensable role in physiological processes. It is, therefore, not surprising that many diseases are based on the malfunction of membrane proteins. Hence membrane proteins and especially G-protein coupled receptors(GPCRs)- the largest subfamily- have become an important drug target. Due to their high selectivity and sensitivity membrane proteins are also feasible for the detection of small quantities of substances with biosensors. Despite this widespread interest in GPCRs due to their importance as drug targets and biosensors there is still a lack of knowledge of structure, function and endogenous ligands for quiet a few of the previously identified receptors.rnBottlenecks in over-expression, purification, reconstitution and handling of membrane proteins arise due to their hydrophobic nature. Therefore the production of reasonable amounts of functional membrane proteins for structural and functional studies is still challenging. Also the limited stability of lipid based membrane systems hampers their application as platforms forrnscreening applications and biosensors.rnIn recent years the in vitro protein synthesis became a promising alternative to gain better yields for expression of membrane proteins in bio-mimetic membrane systems. These expression systems are based on cell extracts. Therefore cellular effects on protein expression are reduced. The open nature of the cell-free expression systems easily allows for the adjustment of reactionrnconditions for the protein of interest. The cell-free expression in the presence of bio-mimetic membrane systems allows the direct incorporation of the membrane proteins and therefore skips the time-consuming purification and reconstitution processes. Amphiphilic block-copolymers emerged as promising alternative for the less stable lipid-based membrane systems. They, likernlipids, form membraneous structures in aqueous solutions but exhibit increased mechanical and chemical stability.rnThe aim of this work was the generation of a GPCR-functionalised membrane system by combining both promising alternatives: in vitro synthesis and polymeric membrane systems. This novel platform should be feasible for the characterisation of the incorporated GPCR. Immunodetection of Dopamine receptor 1 and 2 expressed in diblock- and triblock-polymersomes demonstrated the successful in vitro expression of GPCRs in polymeric membranes. Antibodyrnbinding studies suggested a favoured orientation of dopamine receptors in triblockpolymersomes.rnA dopamine-replacement assay on DRD2-functionalised immobilised triblockpolymersomes confirmed functionality of the receptor in the polymersomes. The altered binding curve suggests an effect of the altered hydrophobic environment presented by the polymer membrane on protein activity.

O ARMÁRIO DE DEUS NO ARMÁRIO DOS HOMENS. ALTERNATIVAS TEOLÓGICAS PARA O ACOLHIMENTO E LIVRE EXPRESSÃO DA PESSOA HOMOSSEXUAL NA COMUNIDADE CRISTÃ BRASILEIRA DA CONTEMPORANEIDADE

Esta tese parte de uma análise que alia elementos do método histórico-crítico com o histórico-social, privilegiando a análise do discurso, com a finalidade de apresentar alternativas teológicas que dêem conta de pensar a possibilidade e viabilidade de que pessoas homossexuais possam expressar sua espiritualidade de forma livre e completa nas comunidades cristãs brasileiras. A investigação ensejada, procura apresentar um panorama da homossexualidade no Brasil, pontuando momentos diversos, valores, projetos e colocando em relevo a questão da identidade homossexual, principalmente, uma referência à mesma como perversão, anormalidade, crime e doença, reservando às pessoas homossexuais, o estigma, marginalização e exclusão. A investigação proposta apresentará também a busca por uma identidade alternativa por parte das pessoas homossexuais, tanto do ponto de vista teórico, procurando novas possibilidades conceituais, quanto do ponto de vista político, articulando-se em busca de seus direitos como cidadãos. Procurar-se-á perceber de que maneira o cristianismo no Brasil, em sua vertente católica e protestante, compreende a homossexualidade, quais os seus argumentos, ensinos e posturas principais. A partir de tal análise, buscar-se-á perceber quais os principais pilares erguidos pelas alternativas teológicas no sentido de entender como viável, legítimo e possível a livre expressão da pessoa homossexual nas comunidades cristãs. Destacar-se-á a desconstrução de gênero em sua referência binária masculino/feminino, como prejudiciais ao entendimento e convivência com a diversidade e complexidade de identidades sociais e, a extensão desse projeto a uma desconstrução necessária da concepção de Deus numa e sob uma caracterização masculina.(AU)