No description available.

<p>Ribosome biogenesis is a major metabolic expense of bacteria and a promising target for antibacterial drug discovery. <em>Trans-</em>acting proteins, called ribosome biogenesis factors, aid this complex and cooperative process. EngA (YfgK, Der) is a widely distributed bacterial GTPase that is shown here to be important for normal ribosome biogenesis. EngA is an attractive antibacterial target because it is essential for viability in bacteria but is absent in humans.</p> <p>The GTPase activity and cellular function of EngA was investigated in <em>Escherichia coli</em>. Depletion of EngA caused accumulation of 30S and 50S ribosomal subunits at the expense of 70S ribosomes, showing for the first time that EngA is important for normal ribosome biogenesis. Mutation of either of the tandem GTPase domains of EngA led to abnormal ribosome profiles, cell death and loss of GTPase activity, revealing that the two GTPase domains act cooperatively to carry out an essential function. EngA bound the 50S subunit of the ribosome in cells and <em>in vitro</em>. Depletion of EngA resulted in sensitization to aminoglycoside antibiotics, which bind at the aminoacyl-tRNA binding site of ribosomes. To search for an inhibitor of ribosome biogenesis, a high-throughput screen of the GTPase activity of EngA was developed. A specific inhibitor was not identified, however, this robust screen can be extended to other compound libraries. Thus, we showed that the GTPase domains of EngA have a cooperative function in ribosome biogenesis, probably in maturation of the 50S subunit, and that EngA is an amenable target for further inhibitor screens.</p> / Doctor of Philosophy (PhD)

A non-replicating recombinant adenovirus, Ad5MCMVlacZ, which expresses the 13-galactosidase (l3-gal) reporter gene, was used to examine both constitutive and inducible repair of UVC-damaged DNA in Chinese hamster ovary (CHO) cells. Host cell reactivation (HCR) of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated nucleotide excision repair (NER) proficient parental CHO-AA8 and m mutant CHO-UV61 cells which are deficient in the transcription-coupled repair (TCR) pathway of NER. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for 13-gal activity 24 h later. HCR of 13-gal activity for UVC-irradiated Ad5MCMVlacZ was significantly reduced in non-irradiated CHO-.UV61 cells compared to that in non-irradiated CHO-AA8 cells suggesting that repair in the transcribed strand of the UVC-damaged reporter gene in untreated CHO-AA8 cells utilizes TCR. Prior UVC-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR for expression of the UVC-damaged reporter gene in CHO-AA8 cells but not m TCR deficient CHO-UV61 cells. Pre-UVC-treatment of cells resulted also in an enhanced expression of 13 -gal for unirradiated Ad5MCMVlacZ in both CHO-AA8 and CHO-UV61 cells. However, compared to CHO-AA8 cells, the CHO-UV61 cells exhibited comparable levels of enhanced 13-gal activity following significantly lower UVC exposures to cells suggesting that persistent damage in active genes plays a direct role in enhancing 13-gal activity driven by the MCMV promoter in CHO cells. These results suggest that prior UVC treatment results in a transient enhancement in repair of UVC-damage DNA in the transcribed strand of the active reporter gene in CHO-AA8 cells through an enhancement of TCR or a mechanism that involves the TCR pathway and that the upregulation of reporter gene expression alone is not sufficient for enhanced repair of the reporter gene in CHO-UV61 cells. The HCR assay was used also to examine both constitutive and inducible repair of UVC-damaged DNA in mouse embryonic fibroblast (MEF) cells. HCR of B-gal activity for UVC-irradiated Ad5MCMVlacZ was examined in non-irradiated and UVC-irradiated NER proficient parental wild type MEF cells and in MEF cells with specific knockouts in the p53 (p53-/-), pRb (pRb-/-), and p107 (p107-/-) genes. Cells were infected with either UVC-irradiated or non-irradiated Ad5MCMVlacZ and scored for ~-gal activity 24 h later. HCR of ~-gal activity for UVC-irradiated Ad5MCMVlacZ did not show a significant difference in non-irradiated cells for any of the MEF knockouts cells compared to the parental strain suggesting that p53, pRb and p107 does not play a role in repair of the UV -damaged reporter gene in untreated MEF cells. Prior UVC-irradiation of cells with low UVC fluences resulted in an enhancement of HCR for expression of the UV C-damaged reporter gene in MEF wild type cells, low passage pRb-/-and p 1 07 -I-MEF cells but not in p53-/-MEF cells or in high passage pRb-/-and p107-/-MEF cells. These results suggest that prior UVC treatment MEF cells results in an induced repair of UVC-damaged DNA that is dependent on p53. The presence of an enhancement of HCR for the UVC-damaged reporter gene in pre-UVC treated cells in low passage, but not in high passage, pRb-/-and p 1 07-I-cells suggests that the lack of pRb or pI 07 expression per-se does not result in a deficiency in inducible DNA repair. However, these results suggest that the lack of pRb or p 1 07 expression results in alterations in MEF cells at high passage number that abrogate inducible repair of UVC-damaged DNA. UVA produces predominantly single base damage that is repaired through base excision repair (BER), whereas UVC and UVB produce predominantly cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PP) that are repaired through NER. The colony survival following exposure to various UV sources was examined in cells proficient and deficient in (NER). The UV sources were a UVC source from a germicidal lamp emitting predominantly at 254 nm. and a UVA source from a lKW Hg-Xe arc lamp using either a Band pass filter (BPF) or a 335 Cut-off-filter (335COF). NER deficient CHO-UV5 and CHO-UV61 cells were more sensitive to UVC exposure compared to NER proficient CHO-AA8 cells, consistent with the production of UVC-induced DNA damage predominantly in the form of CPDs and 6-4PPs which are repaired through the NER pathway. NER deficient xeroderma pigmentosum cells from complementation group D (XPD) were more sensitive compared to NER proficient normal human cells following exposure to the UVA-BPF source. In addition XPDdenV cells, which express the denY gene from bacteriophage T4, were more resistant than XPD cells following exposure to the UVA-BPF source. Since the denY protein is specific for excision ofCPDs these results indicate a substantial proportion of the induced DNA damage resulting from the UV A-BPF is in the form of CPDs, presumably due to a significant UVB component in the beam. In contrast, the NER deficient CHO-UV5 and CHO-UV61 cells showed a similar sensitivity compared to the NER proficient CHO-AA8 cell line following UVA-335COF exposures up to 60 KJ/m2• However, for UVA-335COF exposures greater than 60 KJ/m2 the NER deficient cells were more sensitive compared to the NER proficient CHO-AA8 cells, although the difference in sensitivity between NER deficient and NER proficient cells was less than that detected following UV A-BPF exposure. These results suggest that the UVA-335COF exposure produces predominantly DNA damage of the single base type for exposures less 60 KJ/m2. This is consistent with the calculated spectral distribution, which showed a 5.62% UVB component for the UVA-BPF, but only 0.14% UVB component for the UVA-335COF. / Thesis / Master of Science (MS)

[pt] O foco do estudo são os estilos conversacionais de repórteres aéreos em rádios da cidade do Rio de Janeiro durante a transmissão de notícias em tempo real sobre o fluxo do trânsito na cidade. O objetivo consiste em avaliar como os repórteres aéreos alternam seus estilos em função da audiência e do contexto situacional do trânsito em um grande centro urbano. A pesquisa parte da perspectiva teórica da sociolinguística interacional, em interface com a teoria da acomodação e do design da audiência, em contextos de ordem micro e macro. A discussão do conceito de estilo conversacional é fundamental, no âmbito das teorias em articulação. Conceitos também importantes são os de tipos de atividade, avaliação, enquadre, alinhamento, pistas de contextualização e conversa cotidiana. A pesquisa se pauta pela investigação qualitativa, de natureza interpretativa. A análise baseia-se em dados gerados mediante gravação de notícias sobre o fluxo do trânsito com quatro repórteres aéreos, em seis emissoras de rádio FM do Rio de Janeiro. Os dados foram transcritos de acordo com convenções da análise da conversa e foram analisados no curso da fala-eminteração dos repórteres aéreos com locutores das rádio e com foco na audiência no trânsito. O estudo mostra que os mesmos repórteres aéreos variam seus estilos conversacionais em diferentes rádios, com diferentes tipos de discurso, variando entre um estilo conversacional informativo de baixo envolvimento interpessoal em algumas rádios e um estilo conversacional informativo de alto envolvimento interpessoal em outras. / [en] The present study focuses on the conversational styles of the aerial reporters on radio stations during the transmission of real-time news about the traffic flow in the city of Rio de Janeiro. The study aims to evaluate how the aerial reporters shift their styles depending on the audience and the situational context of traffic on a great city. The theoretical perspective lies on interactional sociolinguistics and its relation to the social accommodation theory and audience design, in micro and macro contexts. The discussion on the concept of conversational style is crucial, on the scope of the related theories. Other important concepts deal with speech activity and evaluation; framing, alignment, contextualization cues and discourse strategies. The research are characterized on the qualitative and interpretative investigation. The analysis is based on the recording of news about the traffic flow from four aerial reporters in six FM radio stations in Rio de Janeiro. The data were transcribed according to the conventions of conversation analysis and analyzed on the scope of talk-in-interaction from the aerial reporters and radio announcers focusing on the audience on traffic. The study shows that the same aerial reporters shift their conversational styles in different radios, performing different discourse types, varying from a low involvement interpersonal conversational informative style on some radio stations to a high involvement interpersonal conversational informative style on other radio stations.

Der G Protein-gekoppelte Rezeptor 30 (GPR30) wurde vornehmlich im Kontext von schnellen Östrogeneffekten auf zelluläre Signaltransduktionskaskaden untersucht und stellt möglicherweise einen neuen Östrogenrezeptor dar. Die physiologische Funktion von GPR30 in vivo konnte jedoch bisher nicht ermittelt werden. Daher wurde in dieser Arbeit ein Gpr30-defizientes Mausmodell charakterisiert, bei dem ein Teil der kodierenden Sequenz durch einen LacZ-Reporter ersetzt wurde (Gpr30-lacZ). Die Integration des Konstruktes in den Gpr30-Locus wurde mittels Southern blotting und Real-time PCR verifiziert. Gpr30-positive Zelltypen wurden durch Kolokalisation von LacZ mit zelltyp-spezifischen Markerproteinen identifiziert. Weitere Versuche dienten der Aufklärung des Phänotyps von Gpr30-lacZ Mäusen. Zur Identifizierung von Proteinen des GPR30-Signalkomplexes wurden Yeast-Two-Hybrid Analysen mit der N- bzw. C-terminalen Domäne des Rezeptors durchgeführt. Die wesentlichen LacZ-positiven Zellpopulationen waren (i) Endothelzellen in kleinen arteriellen Gefäßen, (ii) glatte Muskelzellen, Perizyten und neuronale Subpopulationen im Gehirn, (iii) Hauptzellen in der Magenschleimhaut, (iv) Zellpopulationen in der Adenohypophyse und dem Hypophysenzwischenlappen sowie (vi) chromaffine Zellen im Nebennierenmark. Während der Phenotypisierung des Mausmodells wurde eine Reduktion der CD62L+ T-Zellen von ca. 50% im peripheren Blut festgestellt. Mittels Yeast Two-Hybrid Analyse wurden Pals1-associated tight junction protein (PATJ) und FUN14 domain-containing 2 (FUNDC2) als mögliche Interaktionspartner identifiziert. Zusammenfassend wurde in dieser Arbeit eine zelluläre Basis für die Funktion von Gpr30 in vivo ermittelt. Der Phänotyp in Gpr30-lacZ Mäusen ist wahrscheinlich durch eine verringerte Produktion von naiven T-Zellen im Thymus bedingt. PATJ bindet die C-terminalen Aminosäuren von GPR30 mit einer PDZ-Domäne und könnte ein Gerüst-protein des GPR30-Signal¬komplexes darstellen. / The orphan G protein coupled receptor 30 (GPR30) was predominantly analyzed in the context of membrane-initiated estrogen signaling suggesting that GPR30 represents a novel estrogen receptor. However, the physiological function of GPR30 in vivo remained unknown. To unravel the physiological role of murine Gpr30 in vivo, a Gpr30-deficient mouse model was analyzed that harbors a LacZ reporter (Gpr30-lacZ) within the Gpr30 locus. The targeting of Gpr30 was verified by Southern blotting and real-time PCR. Gpr30-expressing cell types were identified by colocalization of LacZ along with cell type-specific markers. Further experiments aimed to decipher the phenotype of Gpr30-lacZ mice. To gain information about the signaling complex of human GPR30, yeast two-hybrid screenings were performed with the N- and C-terminal domains as bait. The main LacZ-positive cell populations were (i) endothelial cells in small arterial vessels of various tissues, (ii) smooth muscle cells, pericytes, and neuronal subpopulations in the brain, (iii) gastric chief cells in the stomach, (iv) cells in the intermediate and anterior pituitary, and (v) chromaffin cells in the adrenal glands. Extensive phenotype screening at the German Mouse Clinic revealed reduced numbers of T cells in the peripheral blood of Gpr30-lacZ mice. Especially the proportion of CD62L+ cells was decreased by approx. 50%. Yeast two-hybrid screening led to the identification of Pals1-associated tight junction protein (PATJ) and FUN14 domain-containing 2 (FUNDC2). In conclusion, this study provides a cellular basis for the function of Gpr30 in vivo. Since CD62L+ cells represent the naive T cell compartment, the phenotype of Gpr30-lacZ mice suggests an impaired production of T cells in the thymus. PATJ likely binds the C-terminus of GPR30 with one of its PDZ domains and may represent a scaffolding protein of the GPR30 signaling complex.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

Orientador: Fernão Vitor Pessoa de Almeida Ramos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Artes / Made available in DSpace on 2018-08-14T11:12:46Z (GMT). No. of bitstreams: 1 Vargas_Heidy_M.pdf: 2014463 bytes, checksum: bb1bace353c62c55d3b3e8afa368481a (MD5) Previous issue date: 2009 / Resumo: Este trabalho busca identificar as condições internas e externas à Rede Globo de Televisão que permitiram o surgimento dos programas Globo-Shell Especial e Globo Repórter e discutir a influência da linguagem e da estética do Cinema Novo no documentário televisivo. O Globo-Shell Especial foi criado em 1971 e, em 1973, foi substituído pelo Globo Repórter, cuja exibição foi suspensa pela primeira vez em 1983. Para mapear essa história, recorreu-se à bibliografia existente sobre o assunto - ainda pouco estudado -, às notícias e críticas publicadas e aos boletins de programação da emissora, bem como a entrevistas realizadas com profissionais envolvidos nesses projetos. Elaborou-se também, a partir de um mapeamento da produção dos doze anos que delimitam a pesquisa, uma planilha com informações primárias (nome, data de exibição, ficha técnica), subdividida em segmentos temáticos, que serve de base para uma análise preliminar do modo de produção, da linguagem audiovisual e da estética adotadas. Essa análise é cotejada com a tradição narrativa documental, a questão da autoria e as revoluções tecnológicas que permitiram a construção do discurso do documentário televisivo. Em ambos os programas, cineastas e jornalistas fizeram parte das equipes, somando competências e gerando contradições. O resultado desse encontro foi a criação de documentários únicos, diferenciados do restante da produção televisiva e cinematográfica do período, tanto nos planos da estética e da linguagem - uma preocupação intrínseca ao trabalho dos cineastas - quanto no da informação - uma marca do trabalho dos jornalistas. / Abstract: This paper seeks to identify the conditions inside and outside of the Globo Television Network that allowed the creation of Globo-Shell Especial and Globo Repórter programs and discuss the influence of Cinema Novo's language and aesthetics on TV documentary. Globo-Shell Especial was established in 1971 and replaced in 1973 by Globo Repórter, which had its transmission suspended for the first time in 1983. To map that history, we appealed to literature on the subject - which is still little studied -, news, reviews and published reports of the broadcast programming, as well as interviews with professionals involved in these projects. Based on a survey of production from the twelve years that are subject of research, we also produced a spreadsheet of primary information (name, date display, fact sheet), divided into thematic segments, which serves as the basis for a preliminary analysis of the adopted mode of production, audiovisual language and aesthetics. This analysis is compared to the traditional documentary narrative, the question of authorship and the technological revolutions that enabled the construction of the television documentary discourse. In both programs, filmmakers and journalists were part of the team, adding expertise and creating contradictions. The result of such a meeting was the creation of unique documentaries, different from the rest of the TV and film production of the period both in terms of aesthetics and language - a concern intrinsic to filmmaking - and in terms of information - a mark of the work of journalists. / Mestrado / Mestre em Multimeios

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied. CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease. Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter. The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system.

No description available.

Made available in DSpace on 2016-04-26T18:15:36Z (GMT). No. of bitstreams: 1 Edson_Capoano_Mestrado.pdf: 1327952 bytes, checksum: 109b0c3d624f47d0d5e639ced6348b2b (MD5) Previous issue date: 2006-06-12 / O trabalho analisa as imagens da natureza produzidas por um programa de TV jornalístico, o Globo Repórter, que trata do meio ambiente, uma de suas principais pautas. Para tanto, propomos discutir quais são os discursos culturais vigentes sobre a natureza e como eles são alterados pela intermediação de suportes eletrônicos de comunicação. Buscaremos as origens e os formatos consagrados de GR para, em seguida, analisar um de seus episódios, como consideraremos seus aspectos audiovisuais, o roteiro de edição e as falas do repórter. Para instrumentarmos a análise, tomaremos como base a Agenda 21, carta mundial de intenções sobre os recursos naturais, bem como um breve histórico sobre o movimento ambientalista brasileiro e alguns conceitos desenvolvidos durante a modernidade, que alteraram o modo de pensar e agir sobre o meio ambiente. Igualmente embasador serão os conceitos que demonstrarão outras formas de mediação cultural do homem com seu meio, como o descolamento das representações de suas bases materiais e o conseqüente culto às imagens de natureza produzidas pela cultura, conceitos retirados dos estudos de Harry Pross e Dietmar Kamper, entre outros autores ligados à Teoria da Mídia e à Teoria da Cultura. O resultado da análise apontará as imagens ambientais se desligam do objeto real e originário, e funcionam segundo outros padrões e referências. O objetivo é o de reter a atenção do telespectador por meio de vinculação emocional com as imagens de natureza. E, assim, a atenção do público é desviada a outros discursos culturais, como o culto à beleza e ao medo das perdas ambientais, que geram uma ameaça à vida na Terra. As imagens, consideradas janelas para o mundo, tornam-se véus que chamam a atenção para si mesmas e não estimulam a reflexão do público sobre a temática