Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Letras, Departamento de Linguística, Português e Línguas Clássicas, Programa de Pós-Graduação em Linguística, 2017. / Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2017-07-14T16:18:43Z No. of bitstreams: 1 2017_ClaudiaMurielJustinianodaCruz.pdf: 4820269 bytes, checksum: 863fbe37b592e2bd2bbcd6e55c452fed (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-08-10T17:07:41Z (GMT) No. of bitstreams: 1 2017_ClaudiaMurielJustinianodaCruz.pdf: 4820269 bytes, checksum: 863fbe37b592e2bd2bbcd6e55c452fed (MD5) / Made available in DSpace on 2017-08-10T17:07:41Z (GMT). No. of bitstreams: 1 2017_ClaudiaMurielJustinianodaCruz.pdf: 4820269 bytes, checksum: 863fbe37b592e2bd2bbcd6e55c452fed (MD5) Previous issue date: 2017-08-10 / Este estudo consistiu em uma investigação do tempo gramatical pretérito imperfeito (PI) do português brasileiro. Além disso, buscou-se observar de que forma estudos linguísticos e descritivos do aspecto verbal (COSTA, 2002; COROA, 2005 e CASTILHO, 2010) podem auxiliar o ensino do PI nas aulas de português brasileiro como língua estrangeira (PBLE), bem como na produção de material didático para tal público. Por conseguinte, uma análise foi empreendida por meio da Linguística de Corpus, e observamos que as formas verbais do PI mais frequentes em materiais de PBLE são equivalentes àquelas empregadas em contexto de uso da norma culta do português brasileiro. Essa etapa, de caráter quantitativo, foi realizada a partir da comparação entre um corpus de estudo – formado por materiais didáticos de PBLE – e um corpus de referência – o Corpus Brasileiro –, representativo do português brasileiro culto contemporâneo. Essa primeira análise foi ampliada por uma análise qualitativa na qual foi examinado o espaço, ou seja, o contexto (textos, diálogos, exercícios) em que as formas do pretérito imperfeito são apresentadas nos materiais didáticos de PBLE (Bem-Vindo!, 2004; Novo Avenida Brasil, 2012; Terra Brasil, 2008 e Falar, Ler e Escrever, 2006). Assim sendo, ao final desta dissertação, uma explicação sobre as noções aspectuais do imperfeito foi feita, bem como uma proposta de quatro usos desse tempo gramatical baseados em Coroa (2005): (i) habitualidade no passado, (ii) descrição no passado, (iii) situação passada que inclui um ou mais eventos e (iv) situações que ocorreram concomitantemente. Por fim, esta dissertação, cujo escopo se restringiu ao pretérito imperfeito, serve como exemplo da importância de se trabalhar os tempos gramaticais do português brasileiro considerando seus usos reais devidamente contextualizados. / This paper substantiate into an investigation of a verbal aspect category from Brazilian Portuguese language, particularly, on imperfect past tense (PI - Passado Imperfeito) category. Furthermore, seeking to investigate in what sort of linguistic and descriptive studies of verbal aspect (COSTA, 2002; COROA, 2005 e CASTILHO, 2010) may help the teaching of Portuguese Brazilian as a foreign language (PBLE) and the didactic material production for this specific public. Moreover, an analysis was made through Corpus Linguistic technique, and it was observed that the Imperfect Tenses more frequently shown in PBLE material are tantamount to those attached into the context using of Brazilian Portuguese standard language. This branch, quantitative dealing, was fulfilled from the comparison between a corpus study – composed by PBLE didactic material – and a corpus reference – The Brazilian Corpus –, abettor of the modern Portuguese Brazilian standard language. This first analysis was magnified from other quantitative one, in which space has been examined, i.e, the context (texts, dialogues, exercises) whichever forms imperfect past tenses are shown in PBLE didactic material. (Bem-Vindo!, 2004; Novo Avenida Brasil, 2012; Terra Brasil, 2008 e Falar, Ler e Escrever, 2006). Therefore, at the end of this paper, an explanation about the aspectual notions of the imperfect tense was done, thus how a propose of four uses in this grammatical tense based in Coroa (2005): (i) past habitually, (ii) past description, (iii) past situation including one or more events and (iv) situation happening concomitantly. Lastly, this paper, which purview was strict to the Imperfect Tense, serves as a valid example of working with the grammatical tenses in Brazilian Portuguese considering its real uses properly contextualized.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Letras, Departamento de Línguas Estrangeiras e Tradução, Programa de Pós-Graduação em Linguística Aplicada, 2016. / Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2017-07-19T12:24:06Z No. of bitstreams: 1 2016_CamilaOliveiraMacêdo.pdf: 2607073 bytes, checksum: 2730f57f54ac10c531deaf6857385d7b (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-07-26T19:24:35Z (GMT) No. of bitstreams: 1 2016_CamilaOliveiraMacêdo.pdf: 2607073 bytes, checksum: 2730f57f54ac10c531deaf6857385d7b (MD5) / Made available in DSpace on 2017-07-26T19:24:35Z (GMT). No. of bitstreams: 1 2016_CamilaOliveiraMacêdo.pdf: 2607073 bytes, checksum: 2730f57f54ac10c531deaf6857385d7b (MD5) Previous issue date: 2017-07-26 / O ensino de Línguas (Segundas Línguas ou Línguas Estrangeiras) ganhou ímpeto e mereceu renovação importante no país na década de 30 do século passado (Schmidt, 1935). Nesse contexto, o ensino de Português do Brasil para Estrangeiros (PLE) ganhou envergadura desde a década de 80 do século XX graças à evolução da influência cultural brasileira em nível mundial e aos esforços dos profissionais e pesquisadores dessa área, muitas vezes sem respaldo oficial que atuam em políticas deliberadas de Ensino de Línguas. Os cursos de PLE nas universidades brasileiras foram confrontados em 1998 com um exame não gramatical de talhe comunicativo que começou a questionar bases mais convencionais de uma abordagem gramatical de tipo estruturalista. Nesse cenário, o presente trabalho propõe, com base no conhecimento e análise de planos de curso em vigência ao nível do ensino superior no Distrito Federal, (re)planejar esse programa sistêmico de níveis em PLE de uma instituição pública de ensino superior explicitando pressupostos e avaliar o possível impacto que uma política institucional proposta teria em termos de uma abordagem comunicativa partindo das demandas específicas dos estudantes. Para a realização da pesquisa relatada nesta dissertação foram analisados documentos da instituição, além da elaboração de um novo plano de curso, sendo, ainda, oferecido um protótipo de uma unidade didática para dar munição para a continuidade do projeto na instituição. Trata-se de uma pesquisa de cunho qualitativo (CHIZZOTTI, 2006; LAVILLE; DIONNE, 1999) com método em estudo de caso (STAKE, 1994; FREEBODY, 2003) na modalidade análise documental (MOREIRA, 2010; LUDKE; ANDRÉ, 1989). Para a fundamentação teórica foram utilizados estudos dos seguintes autores Barbirato (2016), Almeida Filho (2015), Rajagopalan (2013), Lopes (2009), Morita (1999), Viana (1997), dentre outros. Os resultados obtidos demonstram que há necessidade de uma política de ensino de língua explícita que demonstre o posicionamento quanto à abordagem adotada na instituição, além de definições referentes às materialidades da Operação Global do Ensino de Línguas (OGEL). / The teaching of Languages (Second or Foreign) gained impetus and underwent major renovation in the country in the 1930s (Schmidt, 1935). In this context, the teaching of Brazilian Portuguese for Foreigners (PLE) has also gained importance since the 1980s thanks to the evolution of the Brazilian cultural influence worldwide and to the efforts of professionals and researchers in this area, often without official support, who act in deliberate policies of Language Teaching. The PLE courses at Brazilian universities were confronted in 1998 with a non-grammatical examination of communicative skills that began to question the most conventional bases of a grammatical approach on a structuralist tradition. In this scenario, the present work proposes, based on the knowledge and analysis of the curricular plan at the level of higher education in the Federal District, to (re)design this systemic program of levels in PLE of a public institution of higher education, explaining assumptions, and evaluating the possible impact that a proposed institutional policy would have in terms of a communicative approach based on the specific demands of students. For the accomplishment of the research reported in this dissertation, documents of the institution were analyzed, besides the elaboration of a new course plan, being also offered a prototype of a didactic unity to give ammunition for the continuity of the project in the institution. It is a qualitative research (CHIZZOTI, 2006; LAVILLE; DIONNE, 1999) with a method in case study (STAKE, 1994; FREEBODY, 2003) in the modality of documentary analysis (MOREIRA, 2010. LUDKE; ANDRÉ, 1989). For the theoretical basis, the following authors were used: Barbirato (2016), Almeida Filho (2015), Rajagopalan (2013), Lopes (2009), Morita (1999) and Viana (1997), among others. The results show that there is a need for an explicit language teaching policy that demonstrates the position regarding the approach adopted in the institution, as well as definitions regarding the materialities of the Global Operations of Language Teaching (OGEL).

Dissertação (mestrado)—Universidade de Brasília, Instituto de Letras, Departamento de Linguística, Português e Línguas Clássicas, Programa de Pós-graduação em Linguística, 2016. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2016-10-14T15:10:46Z No. of bitstreams: 1 2016_VictorAraujoCoutinho.pdf: 11988124 bytes, checksum: ad72e7063e722bd9df8cbd96de944d6e (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-11-21T13:04:51Z (GMT) No. of bitstreams: 1 2016_VictorAraujoCoutinho.pdf: 11988124 bytes, checksum: ad72e7063e722bd9df8cbd96de944d6e (MD5) / Made available in DSpace on 2016-11-21T13:04:51Z (GMT). No. of bitstreams: 1 2016_VictorAraujoCoutinho.pdf: 11988124 bytes, checksum: ad72e7063e722bd9df8cbd96de944d6e (MD5) / Esta dissertação consiste em uma ponte entre os estudos de linguística e os estudos de linguística aplicada. Partindo de uma compreensão sobre a variação linguística (Bagno, 2000, 2001, 2007, 2013) e a abordagem sociointeracionista (Brown, 1994), vamos observar como os livros didáticos (LDs) de português brasileiro como língua estrangeira (PBLE) lidam com fenômenos de variação. A fim de delimitar o escopo de análise, esta pesquisa se dedica a observar como as diferentes estratégias de retomada anafórica de terceira pessoa (Omena, 1978; Duarte, 1986; Bagno, 2000) são trabalhadas nos LDs de PBLE. Para que seja possível compreender não apenas o fenômeno, mas também seus contextos de ocorrência, buscaremos na teoria dos gêneros textuais (Marcuschi, 2008) embasamento teórico para lidar com o texto como evidenciador do fenômeno de variação. Este trabalho segue as diretrizes propostas por Carvalho (2002), ao analisar fenômenos de variação em LDs de PBLE, e Dettoni (2010), que retoma a proposta de Carvalho e sugere parâmetros para um ensino de língua que leve em consideração os fenômenos de variação. Desse modo, selecionamos 5 das obras mais conhecidas no mercado editorial de PBLE, a fim de ilustrar o modo com o qual cada uma delas lida com o fenômeno selecionado. Como nos deparamos com muitas expressões artificiais no PB, recorremos ao corpus Brasileiro em busca de uma referência de língua escrita, nos moldes propostos por Sardinha (2004), para comprovar a artificialidade das estruturas encontradas. Ao final das análises, é proposto não apenas um modelo pedagógico, mas também uma reflexão acerca das inconsistências encontradas durante a elaboração dos materiais didáticos. Portanto, este estudo demonstra, por meio de dados analisados qualitativamente e quantitativamente, a incoerência no ensino de PBLE, que privilegia o ensino de uma norma-padrão distante da realidade dos falantes nativos e, portanto, distante das necessidades dos aprendizes da língua. _________________________________________________________________________________________________ ABSTRACT / This dissertation consists into a bridge between the Linguistics studies and the Applied Linguistics studies. Starting with a definition of linguistic variation (Bagno, 2000, 2001, 2007, 2013) and socio-interactionist approach (Brown, 1994), it will be observed how the didactic books (DB) of Brazilian Portuguese as foreign language (BPFL) deal with variation phenomena. To set out boundaries to this research, we selected the phenomenon of variation on the anaphoric recovery of the direct object in BP (Omena, 1978; Duarte, 1986; Bagno, 2000). Our focus is to understand how the didactic books work this phenomenon throughout its unities. In order to understand it better, considering also the contexts where this variation is inserted. This study has base on the sociolinguistic theory of texts genres (Marcuschi, 2008), understanding the text as the interface through which the linguistic variation will be shown. Also, this study follows the guidelines purposed by Carvalho (2002) and retaken by Dettoni (2010), setting principles into analyzing didactic books in which concerns the linguistic variation and selecting phenomena to be taught to BPFL learners, respectively. Five out of the most known BPFL books on the market were chosen in order to illustrate how each of them considers the phenomenon studied in this dissertation. Since we crossed a lot of artificial constructions throughout the books, we decided to use the Brasileiro corpus as a reference corpus, looking for this constructions on the corpus to check its tokens and frequency on the language (Sardinha, 2004). At the end of the analysis, we purpose not only a pedagogical model to teach this phenomenon to BPFL learners, but we also consider the inconsistences between what is taught and what compose the book’s texts. Therefore, this study assemblies quantitative and qualitative analysis, proving the inconsistences on the BPFL teaching due to its privilege on the normative grammar (based on European Portuguese), despite of its huge difference from the actual Brazilian Portuguese.

À partir de quarante-huit extraits empruntés à dix-huit manuels de Portugais Langue Étrangère (PLE) publiés au Brésil et aux États-Unis entre 1954 et 2008, s’effectuera une analyse de la représentation de la brésilianité – traduction du mot « brasilidade » en portugais, un concept qui condense les principales caractéristiques de la culture et de l’histoire brésiliennes – depuis sa construction à l’époque du Brésil colonial (1500-1815), jusqu’à son utilisation à l’ère de la mondialisation (période considérée à partir des années 1950). En me basant sur le système de conception des auteurs, j’observe que la manuélisation ne se limite pas à rendre compte des ruptures et des continuités qui s’établissent entre le savoir savant et le savoir réellement enseigné, ce qui équivaudrait à un cas spécifique de transposition didactique (Verret 1975). Bien davantage, ces extraits corroborent l’idée selon laquelle les manuels dont ils sont issus expriment un ensemble d’idéologies, en particulier l’idéologie du bonheur (Ellul, 1998), caractéristique fréquemment attribuée aux images d’un certain Brésil ainsi réduit à n’être perçu que comme un lieu de joie, dansant et chantant (Almeida Filho, 2005). / This analysis of the representations of Brazilianness (translation of the word « brasilidade » in Portuguese, a concept that embodies the main features of Brazilian culture and history) – from its construction in colonial Brazil (1500-1815) through to its use in the era of globalization (starting in the 1950s) – is based on forty-eight excerpts taken from eighteen Portuguese as a Foreign Language textbooks published in Brazil and the United States from 1954 to 2008: considering the authors’ process of conception, it can be noted that manualization is not only satisfied by the interruptions and continuities established between scholarly knowledge and knowledge to be taught, which would be classed as a specific case of didactic transposition (Verret 1975). These excerpts confirm the idea that their respective textbooks express certain ideologies, which include the ideology of happiness (Ellul, 1998), a characteristic frequently attributed to images of Brazil as a joyful place where singing and dancing abound (Almeida Filho, 2005). / Por intermédio de quarenta e oito excertos provenientes de dezoito manuais de Português Língua Estrangeira (PLE) publicados no Brasil e nos Estados Unidos entre 1954 e 2008, são analisadas as representações da brasilidade (conceito que concentra as principais características da cultura e da história nacionais) – desde sua construção no período do Brasil colonial (1500-1815) até seu uso na era da globalização (a partir da década de 1950): partindo do sistema de concepções dos autores percebe-se que a manualização não se satisfaz apenas com as rupturas e continuidades estabelecidas entre o saber sábio e o saber a ser ensinado; o que seria um caso específico de transposição didática (Verret 1975). Tais extratos validam a idea de que seus respectivos manuais expressam certas ideologias; dentre elas, a ideologia da felicidade (Ellul, 1998), característica frequentemente atribuída às imagens do Brasil enquanto lugar de alegria cantante e dançante (Ameida Filho, 2005).