A intersecção do webjornalismo com a web 2.0 faz emergir uma nova prática jornalística em rede – o webjornalismo participativo. As notícias publicadas em sites ou canais dessa natureza são produzidas por “cidadãos comuns” e, em alguns casos, passam pela mediação de jornalistas. Esta dissertação propõe-se a traçar o perfil das notícias veiculadas no canal de webjornalismo participativo vc repórter, do Portal Terra. O canal funciona com a intervenção de jornalistas que selecionam e editam o material enviado pelos internautas. Parte-se do pressuposto de que este modelo de produção jornalística acarreta novas formas de apresentação das notícias, tanto conteúdo quanto formato. Os dois meses de coleta (julho e agosto de 2007) totalizaram um corpus de 139 matérias, que foram submetidas à análise de conteúdo. Investigou-se os seguintes itens: valores-notícia; fontes consultadas; editorias mais procuradas; extensão do texto; utilização ou não de fotos e links complementares; sexo e localização geográfica dos colaboradores. / The intersection of webjournalism with Web 2.0 prompts the emergence of a new network journalistic practice – participative webjournalism. News published on sites or channels of this nature are produced by “common citizens” and, in some cases, go through the mediation of journalists. This dissertation is set to delineate the profile of the news items featured in the participative webjournalism channel vc reporter, Portal Terra. The channel functions through the intervention of journalists who select and edit the materials sent by the internet navigators. One parts from the presupposition that this journalistic production model calls for new manners to present news items, both content and format. The two months of collections (July and August) totalled a body of 139 subject matters, which were submitted to content analysis. The following items were investigated: news-values; sources consulted; most looked-after editorials; text extension; the use, or not, of photos and complementary links; gender and geographic location of the collaborators.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-03T14:55:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação final_bbc.pdf: 741010 bytes, checksum: 2a9032403833869b2c43a2062da8fcd6 (MD5) / Made available in DSpace on 2017-04-03T14:55:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) dissertação final_bbc.pdf: 741010 bytes, checksum: 2a9032403833869b2c43a2062da8fcd6 (MD5) Previous issue date: 2015-09-02 / FACEPE / A Influenza é uma doença infecciosa aguda, causada por um vírus pertencente à família Orthomyxoviridae. As drogas antivirais e a vacinação são importantes no controle da disseminação da doença, porém alguns vírus adquirem resistência a certas drogas, alertando a necessidade de novas drogas. Triagem de antivirais através de ensaios biológicos são laboriosos e demorados. Com intuito de facilitar a triagem de drogas foram desenvolvidas duas linhagens celulares repórteres distintas. A linhagem Vero-Gluc-NS-Neo é específica para o vírus da influenza, expressa o gene Gaussia luciferase na presença do vírus, e foi desenvolvida através da tranfecção de células Vero com o plasmídeo pGluc-NSNeo. A segunda linhagem, denominada de A549-ISRE-Luc-Hygro, foi desenvolvida a partir da transfecção de células A549 com o plasmídeo pISRE-Luc-Hygro, o qual expressa o gene repórter Firefly luciferase na presença do interferon do tipo (IFN-I). Seguida da transfecção, ambas linhagens foram selecionadas e submetidas a uma clonagem biológica por diluição limitante e os clones selecionados foram então caracterizados quanto à sua especificidade e sensibilidade no ensaio. Resultados importantes e promissores foram obtidos com a linhagem A549-ISRE-Luc-Hygro, a qual se mostrou eficiente para a triagem de antivirais para influenza e drogas indutoras do IFN-I. Em relação à linhagem Vero-Gluc-NS-Neo, apesar do plasmídeo construído se mostrar funcional e específico, não foi possível observar a expressão do gene repórter após a infecção viral, trazendo à tona questionamentos e mostrando ser necessária a realização de ensaios complementares / Influenza is an acute infectious disease caused by viruses belonging to the Orthomyxoviridae family. Antiviral drugs are vital in controlling the spread of the disease, but some viruses become resistant to certain drugs, prompting the need for new drugs. Antiviral screening through biological tests are laborious and time consuming. In order to facilitate the screening of drugs, it was developed two distinct cell lineages reporters. The Vero-Gluc-Neo-NS cells line is specific for the influenza virus expresses the Gaussia luciferase gene in the presence of the virus, and it was developed by transfection of Vero cells with pGluc-NS-Neo plasmid. The second cell line, A549-called ISRE-Luc-Hygro, was developed from the transfection of A549 cells with pISRE-Hygro-Luc plasmid, which expresses the Firefly luciferase reporter gene in the presence of type one interferon (IFN-I). Followed by transfection, both cell lines were selected and subjected to a biological cloning by limiting dilution and selected clones were then characterized for specificity and sensitivity in the assay. Important and promising results were obtained with A549-Hygro-ISRE-Luc cells, which proved to be efficient for screening of antiviral drugs for influenza and IFN-I inducing drugs. Regarding the Vero-Gluc-NS-Neo cell line, despite the plasmid constructed to show functional and specific, it was not possible to observe the reporter gene expression after viral infection, bringing up questions and shown to be necessary to carry out further testing.

Esta tese trata de aspectos da narrativa no jornalismo, mais especificamente na reportagem. Ao assumir a condição inequívoca de autoria no texto, o jornalista abre-se para possibilidade de dar maior complexidade ao ato narrativo, abre-se para articular vozes, visões de mundo e experiências. Nesta complexidade, é possível estabelecer dois sujeitos distintos: o autor (repórter) e o narrador (sujeito elaborado pelo repórter para contar uma história). O embasamento teórico da pesquisa procura articular as noções de complexidade e dialogia (Cremilda Medina) filosofia do diálogo (Martin Buber); ponto de vista (Norman Friedman); experiência e comportamento face a face (Erving Goffman). O autor no jornalismo é um mediador cultural, tem um lugar social demarcado por sua formação profissional e está diretamente ligado ao exercício de um ofício. Já o narrador, é uma criação do autor. É um sujeito que existe para narrar, que adota perspectivas e estratégias para contar uma história, um fato/acontecimento. A constituição do(s) narrador(es) do jornalismo está sob a batuta do autor jornalista. Estabelecemos conexões entre as referências teóricas e a experiência prática. Foram observadas um total de 20 reportagens publicadas/veiculadas em jornal, revista, rádio, televisão e web. Além da observação das reportagens, foram entrevistados os dez repórteres responsáveis por estes textos jornalísticos / This thesis treats aspects of narrative in journalism, more specifically in the report. When assuming an unequivocal condition of authorship in the text, the journalist opens to the possibility of giving more complexity to the narrative act, opens to articulate voices, world views and experiences. In this complexity, it is possible to establish two distinct subjects: the author (reporter) and the narrator (subject developed by the reporter to tell a story). The theoretical basis of the research intends to articulate the notions of complexity and dialogy (Cremilda Medina) ; philosophy of dialogue (Martin Buber); point of view (Norman Friedman); experience and behavior face to face (Erving Goffman). The author in journalism is a cultural mediator and has a social place demarcated by his professional qualification and is directly linked to the exercise of a craft. Yet, the narrator in journalism is a creation of the author. It is a subject that exists to narrate, which adopts perspectives and strategies to tell a story, a fact /event. The constitution of narrator(s) of journalism is under the baton of the author journalist . We established connections between theoretical references and practical experience. It was observed a total of 20 stories published/aired in newspapers, magazines, radio, television and web. In addition to observing the reports, ten reporters responsible for these journalistic texts were interviewed.

Abstract Clarification of the mechanisms of cardiac-specific gene expression provides not only basic knowledge about how the gene expression is regulated in the heart, but also about the changes in the gene expression during the development of cardiovascular diseases. The purpose of this study was to analyze the mechanisms of cardiac chamber-specific gene expression and cardiac gene activation induced by mechanical load. In the present study, the experiments were carried out by using two cardiac genes, salmon cardiac peptide (sCP) and rat B-type natriuretic peptide (BNP) genes as models. sCP was discovered previously in our laboratory and turned out to be extremely cardiac-specific, representing A-type natriuretic peptide characters in an exaggerated way. In neonatal rat cardiomyocytes, the sCP promoter activity was shown to be strictly restricted to atrial cells and the promoter to be inert to cardiac hypertrophy-inducing factors. In order to find out the mechanisms of earlier proved BNP gene activation by mechanical load, BNP promoter activity was studied in vivo in adult rat hearts. The tandem GATA transcription factor binding site at position -80/-91 was shown to be essential for the BNP gene induction by angiotensin II. To clarify the possiblity to transfer the characters of the BNP gene into the sCP gene, short BNP fragments were inserted to the sCP gene promoter. The otherwise atrial-restricted sCP promoter was shown to be switched on in rat ventricular cardiomyocytes by adding a short BNP proximal promoter element to the sCP promoter, preferably near to the transcription start site. This activity was partly dependent on the -80/-91 GATA sites in the BNP promoter. Thus, A-type natriuretic peptide regulation can be switched to B-type regulation by a short proximal BNP promoter element. In conclusion, these studies reveal certain basic differences in cardiac atrial and ventricular gene expression.

The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis-acting elements, trans-acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA) sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes.

A two-stage mixed methods approach was used to examine how foreign correspondents stationed in the United States use World Wide Web technology to maintain their news perspectives remotely. Despite emerging technology playing an increasingly significant role in the production of international journalism, the subject under investigation has been subject to little empirical research. This is the case even though it is an important topic since what and how the foreign press corps report about the United States to their home audiences affects the way the rest of the world sees America. Open-ended observations and interviews were used in the first stage to collect qualitative data, which was analyzed to inform the development of a quantitative questionnaire. Six full-time foreign correspondents participated in the first stage and 173 completed the survey in the second stage. The results of the qualitative data analysis led to the development of seven themes regarding foreign journalists' use of the Web to maintain their news perspectives. They are, "substitution: a goal-specific alternative," "function: few social needs fulfilled," "competency: Internet use," "self-efficacy: a valid perspective regardless of location," "scanning: a major strategy," "intention: actively seeking currency," and "blind spot: further augmentation necessary." Statistical analysis of the quantitative data in the second stage confirmed the existence of these seven themes in reporters' Web use. Bivariate analysis also discovered relationships between seven journalistic characteristics and these seven dimensions. Those who spent more time online, for instance, were found to associate with higher scores in their intention to seek actively currency of their news perspective with the home office. Additionally, an updated portrait of the foreign press corps was also identified through the quantitative data set, including heavier Web use than has previously been documented, suggesting journalists are spending more time online than on the front line. Based on these investigations into foreign correspondence, online activities such as Web information retrievals alone seem insufficient to replace physical proximity in the shaping of the user's overall value structure. While this research investigated an issue within a specific context, research findings pertain to Web users in general. However, further research is necessary to operationalize these qualitative themes, with findings in such exploratory research as here subject to additional empirical confirmation.

<div>The family Flaviviridae includes important genera such as flavivirus and hepacivirus which comprise significant human pathogens that affect hundreds of millions annually. The understanding of these viruses, the viral life cycle, and pathogenicity is vital when it comes to developing therapeutics. Flavivirus virions undergo major conformational rearrangements during the life cycle, including the assembly and maturation steps. In order to create a reagent to investigate these processes, luminescent reporter viruses have been constructed. Luminescent reporter tags have yet to be incorporated into the structural proteins of dengue virus (DENV) without significantly affecting replication or infectivity and successful tagging would allow for targeted studies examining access to specific structural epitopes. Engineering tags in DENV structural proteins is particularly difficult because most reporter tags involve large insertions which may create steric hindrance and inhibit proper protein folding. However, the reporter system described here, developed by Promega, is much smaller than a full-size luciferase protein. It involves an eleven amino acid subunit (HiBiT) tagged to a viral protein that creates measurable luminescence when incubated with the larger subunit (LgBiT). Using the structure of the virion as a guide, the HiBiT reporter tag was incorporated into the structural region of the DENV genome including sites in capsid (C) as well as the glycoproteins membrane (M) and envelope (E). Resulting recombinant viruses were characterized and tag sites within the C protein membrane anchor as well as the transmembrane domain of M protein were found to tolerate HiBiT insertion and produce infectious particles. The recombinant virus possessing HiBiT in C protein was found to be stable over three rounds of serial passaging while virus containing the M protein tag site was found to be unstable. HiBiT activity of the capsid tagged virus was also found to directly correlate with purified infectious particles, suggesting the capsid membrane anchor may remain associated with the virus even after polyprotein processing. Additionally, insert composition was found to be a key determinant for the production of infectious virus. The lessons learned from engineering HiBiT in the DENV system were then applied to hepatitis C virus (HCV). </div><div>The highly lipophilic and pleiomorphic nature of HCV has made structural studies particularly difficult. However, by constructing multi-tagged reporter viruses containing both HiBiT and various purification tags, researchers will save time and resources in preparation for structural studies which are vital for vaccine development. In this study, HiBiT was incorporated into sites within HCV previously shown to tolerate tags of various sizes. Different insert compositions were engineered within the genome and the construct containing both FLAG and HiBiT tags within the N-terminus of E2 yielded highly infectious and quantifiable, luminescent virus. The recombinant HCV containing FLAG and HiBiT displayed similar peak titer as compared to WT while also demonstrating HiBiT activity. Furthermore, the FLAG peptide was found to be partially surface exposed and capable of being used for virus purification purposes. The multi-tagged reporter virus characterized in this study provides a robust platform for quantification and purification of HCV, two facets of research that are critical for the determination of viral structure via cryo-EM and other imaging techniques. The findings from both the DENV and HCV studies provide a robust foundation for future tagging of viruses within the family Flaviviridae and offer insight on the structural proteins that compose the virion.</div>

Diploma thesis is based on monitoring the succes of transfection of cell linie HEK293. In theoretical part are described principles of transfection methods, cell lines, vectors and reporter genes. HEK293 cells EBNA1 were used for practical part. It was studied the difference between GFP and EGFP plasmids. As well as using various transfection reagents under different culture conditions.

No description available.