Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, β-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two β-1,3-endoglucanses while CA whole leaf protein extracts contain at least three β-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 β-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for β-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of β -1,3-endoglucanses.

To identify known genes or expressed sequence tags (ESTs) which are expressed specifically or preferentially in the chicken hypothalamus and pituitary gland related to highly reproductive performance, two reciprocal cDNA libraries were constructed using a subtractive hybridization strategy. Two different strains, L2 (dam line; n=12) and B (sire line; n=12) of Taiwan Country Chickens (TCCs), which were originated from one single strain and further subjected to 40-wk egg production and comb size, body weight, respectively since 1982, were used in our study. A total of 324 and 370 clones were identified from L2-subtract-B and B-subtract-L2 hypothalamus/pituitary cDNA libraries. 311 and 360 single inserted sequences from each cDNA library, 53 and 23 non-redundant candidate genes were identified. Quantitative reverse-transcription (RT)-PCR were used to validate the association of mRNA expression profiles of the identified candidate genes and high-egg yield trait in another 118 hypothalamuses and pituitary glands that were dissected from seven different chicken stocks, including B-, L2-, Black-, Red-feather TCCs, commercial Single-Comb White Leghorn (WL) layer at National Chung-Hsing University (NCHU) and Red-feather TCCs grouped into high eggs (Red-high) & low eggs (Red-low) to 40 wks of age at National Chiayi University (NCYU). Among identified genes including known genes and novel genes, involving 33 screened genes, Inhibitor-1 of protein phosphatase type 2A (ANP32A), 3-hydroxybutyrate dehydrogenase (BDH), Contactin (CNTN1), Deiodinase iodothyronine type II (DIO2), Inhibitor of growth family, member 3 (ING3), Lysosomal-associated transmembrane protein 4 beta (LAPTM4B), Neural cell adhesion molecule 1 (NCAM1), DJ-1 protein (PARK7), Prostaglandin D2 synthase (PGDS), Prolactin (PRL), Protocadherin 1 (PCDH1), Pleiomorphic adenoma gene 1 (PLAG1), GTP-binding protein SAR1a (SAR1A), Secretogranin II (SCG2), Stathmin 2 (STMN2), T-box protein 2 (TBX2) were up-regulated in B-subtract-L2 cDNA library. Among above-mentioned 16 identified genes, there were 9 genes related to high-egg yield in chickens., including BDH, NCAM1, PCDH1, PGDS, PLAG1, PRL, SAR1A, SCG2, STMN2.

This study was carried out for understanding of antioxidant mechanisms of lentil under abiotic stress conditions. For this aim, 14 days old lentil seedlings (Lens culinaris Medik cv. Sultan-1) were subjected to drought (20% PEG 6000), and salt (150 mM NaCl ) stress for 6, 12 and 24 hours, for 3, 5 and 7 days. PCR conditions for Mn SOD, Cu/Zn SOD, chloroplastic/mitochondrial GR, CAT and chloroplast /stromal APX antioxidant enzymes were optimized. Then, total RNA was isolated from stressed and non-stressed plant roots and shoots. The gene expression levels of Mn SOD and Cu/Zn SOD were examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. Arabidopsis 18S rRNA was used as internal control in multiplex PCR technique. Relative expression levels of Mn SOD were lower in shoots and roots under salt stress while no significant change was obtained under drought conditions in both tissues. Relative expression levels of Mn SOD were increased on 5th day of salt and drought applications in both shoots and roots. Relative expression levels of Cu/Zn SOD increased after 5th, and on 1st and 7th days of drough treatment in shoots and roots, respectively. On the other hand, expression levels of Cu/Zn SOD increased on 3rd and 5th days of salt treatment in shoot tissues. Although it is nearly impossible to understand the whole antioxidant mechanism of plants under environmental stresses, this study was the first step to learn about molecular background of antioxidant defence mechanisms in lentil.

名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程） 学位授与年月日:平成20年3月25日

Le sulfure d'hydrogène (H2S) est un métabolite bactérien produit notamment par les bactéries sulfato-réductrices du côlon à partir des acides aminés soufrés, des sulfates/sulfites alimentaires et des sulfomucines. A fortes concentrations, le H2S est un gaz toxique, de par sa capacité à inhiber la cytochome c oxydase, et par conséquent, la respiration mitochondriale. A l'inverse, notre travail montre qu'à faibles concentrations, le H2S induit une énergisation mitochondriale des cellules épithéliales coliques humaines HT 29 Glc-/+ lui conférant aussi un rôle de substrat minéral énergétique. Dans ce travail, nous avons déterminé les concentrations de H2S permettant l'oxydation/détoxication de H2S par les cellules HT 29 Glc-/+ et celles provoquant une inhibition de la consommation d'oxygène. L'oxydation du H2S nécessite la coopération entre la " sulfide oxidation unit " et la chaîne respiratoire. La capacité des cellules HT29 Glc-/+ à oxyder le H2S est associée à la présence des transcrits codant les enzymes constituant la " sulfide oxidation unit " : la sulfure d'hydrogène quinone reductase (SQR), la dioxygenase ETHE1 et la thiosulfate transferase (TST). Nous avons démontré la priorité de l'oxydation du H2S sur les substrats carbonés . En effet, nos résultats suggèrent que les électrons venant de la SQR sont transférés au pool d'ubiquinone aux dépens de ceux venant du complexe I. Nos résultats démontrent que la SQR joue un rôle déterminant pour l'oxydation de H2S. De plus, la détoxication du H2S par les cellules HT29 Glc-/+ augmente au cours de la différenciation spontanée ou induite par un traitement au butyrate. L'augmentation de la détoxication de H2S au cours de la différenciation est associée à une augmentation de la réserve respiratoire soulignant l'importance de la chaîne respiratoire comme composante de la fonction de détoxication du H2S. En situation d'inhibition de la cytochrome c oxydase, la grande capacité des cellules coliques humaines à détoxiquer le H2S pourrait être en partie due à la présence d'un transfert réverse des électrons issus de l'oxydation de H2S de la SQR vers le complexe I. Outre le butyrate, le zinc un autre composé de la lumière colique, exerce un effet protecteur contre la toxicité cellulaire du H2S. Enfin, notre travail a mis en évidence une diminution de l'expression d'un gène codant pour une enzyme de la " sulfide oxidation unit " (la TST) dans le rectum comparée à différents segments du côlon, ce qui pourrait correspondre à des capacités de détoxication du H2S différente en fonctions des segments du gros intestin humain.

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No. of bitstreams: 1 rossi_gam_dr_jabo.pdf: 3246904 bytes, checksum: 2ed62292bd3bfa8f9ec88fe41c5bbaa2 (MD5) Previous issue date: 2017-12-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As bactérias pertencentes ao grupo do Bacillus cereus são importantes para indústrias processadoras de leite e produtos lácteos devido à capacidade de sobrevivência aos tratamentos térmicos utilizados durante os processamentos, e consequente deterioração dos produtos e risco à saúde pública. Assim, inicialmente, objetivou-se compreender a estrutura populacional de isolados pertencentes ao grupo do B. cereus na cadeia produtiva do leite e produtos lácteos. Por meio da utilização de amostragem estruturada e técnicas genômicas comparativas, investigou-se quais linhagens específicas e genes estão significativamente associados a estágios de produção específicos e produtos. Os genomas de 69 isolados obtidos de equipamentos, leite cru e produtos lácteos foram comparados com outros 193 disponíveis de diversas origens. A estrutura populacional incluiu os conhecidos grupos filogenéticos II, III, IV, V e VI, e quase todos os isolados dos produtos lácteos pertenceram ao grupo III. A investigação de genes específicos revelou um grande número de isolados carreando aqueles relacionados à produção de toxinas, como o cytK (53,62%), hblA (59,42%), hblC (44,93%), hblD (53,62%), nheA (84,06%), nheB (89,86%), nheC (84,06%), cesA (2,90%) e cesB (2,90%). Os isolados pertencentes aos grupos IV e V possuíram uma prevalência significativamente maior dos genes hblACD e o grupo IV do gene cytK. Os isolados obtidos de produtos lácteos tiveram uma prevalência significativamente menor dos genes cytK e hblACD comparados aos de equipamentos e leite cru/tanques de refrigeração. A análise genômica populacional demonstrou a diversidade dos isolados e a variedade de funções dentro do grupo do B. cereus da cadeia produtiva leiteira, com grande número de isolados potencialmente capazes de causar doenças alimentares. Posteriormente, objetivou-se avaliar a viabilidade do processo de tindalização de leite cru refrigerado e determinar se o processo de pasteurização do leite influencia na expressão dos genes relacionados à produção das toxinas diarreicas pelo B. cereus s.s. em leite experimentalmente contaminado. Para isso, realizou-se um processo de tindalização de leite cru refrigerado e então foi realizada a contaminação com um isolado potencialmente toxigênico, e, posteriormente, o mesmo foi pasteurizado, envasado e mantido em refrigeração durante 10 dias. Em momentos determinados, foram coletadas amostras do produto afim de avaliar a expressão de genes codificadores de toxinas (hblACD, nheABC e cytK). O protocolo de tindalização utilizado permitiu redução logarítmica da população de bactérias do grupo do B. cereus e foi possível detectar a expressão do gene hblA em amostras de leite 5 e 10 dias após a pasteurização. Conclui-se que o modelo proposto foi adequado e que a expressão do gene hblA não foi inibida após a realização da pasteurização do leite, demonstrando o potencial risco aos consumidores decorrentes do consumo de leite pasteurizado contaminado. / The bacteria belonging to Bacillus cereus group are important to dairy industries due their ability to survive to thermal treatments used during processing, and consequently cause food spoilage and risk to public health. Thus, firstly, this study aimed to better understand the population structure of B. cereus group isolates in dairy production chain. Using structured sampling of B. cereus in dairy production chain and comparative genomics techniques, we investigated if specific lineages and genes are significantly overrepresented in particular production stages. The genomes of 69 isolates obtained from equipments, raw milk and dairy products were compared with others 193 avaiable from several origins. The populational structure included the known phylogenetic groups II, III, IV, V and V, and almost all isolates from dairy products belonged to group III. The genes investigation showed a high number of isolates carrying genes related to toxins production, such as cytK (53.62%), hblA (59.42%), hblC (44.93%), hblD (53.62%), nheA (84.06%), nheB (89.86%), nheC (84.06%), cesA (2.90%) and cesB (2.90%). The isolates belonging to groups IV and V had a significant higher prevalence of genes hblACD an group VI of cytK. The isolates obtained from dairy products had a significant lower prevalence of genes cytK and hblACD compared to those from equipments and raw milk/bulk tanks. The populational genomic analyses showed the diversity of isolates and variability of functions in B. cereus group in dairy production chain, with a high number of isolates potentially able to cause foodborne disease. Posteriorly, this study aimed to evaluate the viability of tyndallizating raw milk and to establish if milk’s pasteurization influences on the expression of genes related to the production of diarrheal toxins by B. cereus s.s. using a milk contamined experimentally. For this purpose, the tyndallization of raw milk was performed and later it was contaminated with a potentially toxigenic isolate, and, posteriorly, it was pasteurized, packaged and kept under refrigeration during 10 days. In established periods, samples of this product were collected in order to evaluate the expression of genes related to toxins production (hblACD, nheABC and cytK). The protocol used for tyndallization allowed a logarithmic reduction of population of B. cereus group and the expression of hblA gene was detected in samples from milk after 5 and 10 days after pasteurization. It was concluded that the proposed model was appropriate and the expression of hblA gene was not inihibited after milk pasteurization, highlighting the potential risk for consumers through consumption of contamined pasteurized milk. / 2016/19214-9 / 2014/13104-1

O vírus da diarreia viral bovina (BVDV) é um dos principais patógenos virais dos bovinos e sua infecção causa importantes perdas na produção dos rebanhos afetados. O presente trabalho contém os estudos realizados para esclarecer determinados aspectos da epidemiologia e da detecção da infecção causada pelo BVDV no Estado do Rio Grande do Sul, Brasil. O primeiro artigo consistiu de um estudo transversal onde os níveis de anticorpos contra o BVDV em amostras de tanque de leite que foram correlacionados com os aspectos produtivos dos rebanhos. As amostras e as informações foram coletadas de 300 criações escolhidas aleatoriamente entre 1.656 associados de uma cooperativa de leite na região central do Estado do Rio Grande do Sul e que não utilizaram vacina contra BVDV nos últimos 12 meses anteriores ao experimento. Dessas, 80 foram consideradas positivas para BVDV pela detecção de anticorpos contra vírus em níveis superiores ao ponto de corte do teste de ELISA comercial utilizado. A prevalência aparente estimada foi de 26,7% e, usando um intervalo de confiança de 95%, apresentou uma variação entre 22 e 31%. Os parâmetros de produção mensal de leite, densidade de bovinos (relação vacas/hectare), tipo de armazenamento do leite e origem do sêmen não apresentaram associação com a infecção pelo BVDV, mas o aumento do número total de animais por rebanho foi correlacionado positivamente com a infecção. A prevalência estimada foi considerada baixa para uma população de rebanhos, criados em sistema semi-intensivo de produção de leite, que não usava medidas específicas de controle contra a infecção. A hipótese proposta no presente artigo é que o pequeno número de animais por rebanho está favorecendo a uma provável limpeza ou erradicação da infecção nas criações e que a prevalência encontrada deve corresponder ao ponto de equilíbrio entre a pressão de infecção e a limpeza da infecção destes rebanhos. A baixa prevalência estimada pode ser uma condição favorável para iniciar um programa de controle para BVDV na população estudada. No segundo artigo, um teste da transcrição reversa seguida da reação em cadeia da polimerase (RT-PCR) foi estabelecido para detecção do vírus nas amostras de tanque de leite dos rebanhos suspeitos de infecção ativa pelo BVDV identificados no primeiro artigo. O teste foi capaz de detectar até 0,001 TCID50 de BVDV por mL de leite. Esses resultados demonstraram uma sensibilidade analítica 100 ou 1000 vezes superior aos testes de RTPCR convencionais descritos anteriormente para leite e são semelhantes aos resultados obtidos em testes de RT-PCR em tempo real para BVDV. Na análise das amostras de campo, o teste detectou RNA viral em dois rebanhos entre os 59 analisados. Esses resultados comprovaram a capacidade do teste para identificar amostras naturalmente infectadas e a presença de vacas adultas produtivas e virêmicas em 3% dos rebanhos suspeitos de infecção. Este teste é uma alternativa rápida e sensível para monitorar a infecção pelo vírus em rebanhos leiteiros, a partir de amostras coletivas. O terceiro artigo tratou da detecção do BVDV em carrapatos Rhipicephalus (Boophilus) microplus alimentados em bovino persistentemente infectado (PI). Seu objetivo foi investigar o papel do carrapato bovino R. microplus na transmissão do BVDV. O RNA viral foi detectado nas teleóginas alimentadas no bovino PI, mas não pode ser identificado em sua progênie (ovos e larvas). Esses resultados sugeriram não haver transmissão transovariana do BVDV nos carrapatos. Por outro lado, a infestação experimental de um segundo bovino com as larvas oriundas das teleóginas contaminadas com BVDV não resultou em manifestação clínica e o animal foi negativo no teste de RT-PCR. O experimento demonstrou que o carrapato R. microplus pode ser contaminado com BVDV durante o repasto sanguíneo, reforçando a idéia de que vetores hematófagos possam estar envolvidos na disseminação da doença e merecem mais investigações. / Bovine viral diarrhea virus (BVDV) is one of the main pathogenic agents in cattle and the infection with the agent causes important production losses in the affected herds. The present work contains studies carried out to clarify some aspects of the epidemiology and detection of the infection caused by BVDV in the State of Rio Grande do Sul, Brazil. The first paper consisted of a transversal study, where antibody levels against BVDV in samples of bulk milk tank were correlated with some aspects of milk production in the herds. Samples and information were collected from 300 herds randomly chosen between 1656 associates of a dairy cooperative in the region of the central area of the state of Rio Grande do Sul, Brazil, that were not using routine vaccination against BVDV in the last 12 months previous to the experiment. Among them, 80 were considered positive to BVDV by detection of antibodies against the virus in levels above the cut off point of the commercial ELISA test used. The apparent estimated prevalence was 26.7% and, using a confidence interval of 95%, showed a range between 22 and 31%. The parameters of average milk production, bovine density (relationship cattle/hectare), milk storing process and origin of the semen were not significantly associated with BVDV infection, but the increase in the total number of cows in the herds was positively correlated with the infection. The prevalence was considered low for a population of herds raised in a semi-intensive milk production system that was not using specific measures to control the viral infection. The hypothesis proposed in the present study is that the small number of animals present in the analyzed herds would favors a likely virus clearance in the herd and that the prevalence found would correspond to a point of balance between the pressure of infection and the virus clearance. The low prevalence detected can be a favorable condition to launch a control program for BVDV in the studied population. In the second paper, a reverse transcription polymerase chain reaction (RT-PCR) was established for BVDV detection in bulk tank milk samples of the herds suspected of active infection with BVDV in the first paper. The test detected up to 0.001 TCID50 of BVDV per mL of milk. These results demonstrated an analytical sensitivity 100 to 1000 times higher than conventional RT-PCR tests described previously for milk and are similar to results obtained by the use of real time RT-PCR already published. In the analysis of field samples, the test detected viral RNA in 2 out of 59 analyzed herds. These results demonstrated the relationship between the ability of the test to identify samples naturally infected and the presence of viremic adult cows in 3% of the herds suspected of the infection. This test represents a fast and sensitive alternative for monitoring virus infection in dairy herds, through the analysis of collective samples. The third article dealt with the detection of BVDV in the tick Rhipicephalus (Boophilus) microplus fed in a bovine persistently infected (PI). The objective was to investigate the role of the cattle tick R. microplus in the transmission of BVDV. Viral RNA was detected in adult females fed on the PI bovine, but could not be identified in its progeny (eggs and larvae). These results suggest that there is no transovarial transmission of BVDV in ticks. On the other side, experimental infestation of a second bovine with larvae derived from adult cattle contaminated with BVDV did not result in clinical manifestations and the animal was negative in the RT-PCR test. The study demonstrated that the tick R. microplus can be contaminated with BVDV during blood feeding, strengthening the idea that haematophagous vectors could be involved in the dissemination of the disease and would deserve further consideration.

Algumas espécies de morcegos têm sido reconhecidas como reservatórios naturais de várias famílias virais, desempenhando um importante papel na trasmissão e manutenção desses micro organismos. Devido à descaracterização e fragmentação de habitats naturais, esses mamíferos buscam alternativas de abrigo e alimento, e assim, ficam cada vez mais expostos aos meios antrópicos e em contato com humanos e animais domésticos. Com exceção do vírus rábico, existem poucos trabalhos realizados na detecção de vírus em morcegos no Brasil. Em virtude disso, o presente estudo objetivou a detecção de vírus (circovírus, astrovírus, coronavírus e lyssavírus relacionados ao vírus da raiva) em amostras de órgãos de morcegos do estado do Rio Grande do Sul. Os ácidos nucléicos foram extraídos das amostras de órgãos de morcegos e submetidos à detecão por PCR e RT-PCR. Após a detecção, os fragmentos obtidos foram sequenciados para realizar análise filogenética dos vírus encontrados. Ao total foram analisadas 108 amostras de diferentes espécies e localidades, das quais dez foram positivas para circovírus, seis para coronavírus e 25 para astrovírus, este último sendo o primeiro registro do vírus em morcegos para o Brasil. Todas as amostras foram negativas para lyssavírus relacionados ao vírus da raiva. Análises filogenéticas revelaram que as sequências de circovírus agruparam em ambos os gêneros Circovirus e Cyclovirus, coronavírus no gênero Alphacoronavirus em dois clados diferentes e astrovírus no gênero Mamastrovirus junto com outros astrovírus de morcegos, o qual formam um clado separado dos outros mamíferos. Os resultados demonstram uma diversidade genética entre os vírus encontrados em diferentes espécies de morcegos, que possuem dietas alimentares e habitats distintos. / Some bat species have been recognized as natural reservoirs of several viral families, playing an important role in the transmission and maintaining of these micoorganism. Due to mischaracterization and fragmentation of natural habitats, these mammals seek shelter alternatives and food, and thus are increasingly exposed to anthropism, which make the contact with humans and domestic animals closer. With the exception of the rabies virus, there are few studies on the detection of viruses in bats in Brazil. Therefore, the present study aimed the detection of viruses (circovirus, astrovirus, coronavirus and rabies-related virus) in bats organs samples from Rio Grande do Sul state. Nucleic acids were extracted from bat organs samples and submitted to detection by PCR and RT-PCR. After detection, the obtained fragments were sequenced to perform phylogenetic analysis of the viruses found. From a total of 108 samples analyzed of different species and locations, ten were positive for circoviruses, six for coronaviruse and 25 for astrovirus, which was the first report of this virus in bats in Brazil. All samples were negative for rabies-related virus. Phylogenetic analyzes revealed that the sequences of circoviruses grouped in both Circovirus and Cyclovirus genus, coronaviruses in Alphacoronavirus genus in two different clades and astroviruses in Mamastrovirus genus along with other bats astrovirus, which form a separate clade from other mammals. Results demonstrate a genetic diversity among viruses found in different species of bats, which have different diets and habitats.

Orientador: Hélio José Montassier / Banca: Liana Brentano / Banca: Manoel Victor Franco Lemos / Resumo: A técnica de imunocaptura associada com a reação de transcrição reversa e reação em cadeia da polimerase (IC-RT-PCR) executadas tanto pelos procedimentos convencional como em tempo real foram testadas para a detecção rápida do gene da glicoproteína de Matriz (M) do vírus de influenza aviária (AIV) em amostras de líquido cório-alantóide (LCA) e em suabes traqueais e cloacais. O presente trabalho teve como objetivo desenvolver e otimizar a técnica de IC-RT-PCR para o diagnóstico do vírus da Influenza aviária. Os resultados obtidos foram comparados com um sistema empregando "beads" magnéticas em microplacas (AMBION), que é o método padrão de extração de RNA usado no laboratório de referência para diagnóstico de influenza aviária, o National Veterinary Services Laboratory - Ames, EUA (USDA), acrescido ainda de outros métodos de extração tradicionalmente usados nos laboratórios de referência para AIV, como os procedimentos com o uso do solvente orgânico Trizol® (Invitrogen) e com um sistema robotizado e que utiliza "beads" magnéticas (MagNA Pure - ROCHE). A técnica de IC-RT-PCR em tempo real neste estudo detectou a estirpe H2N2 do AIV, sem que nenhum outro dos RNA-vírus heterólogos testados fossem detectados (vírus das doença de Gumboro, de Newcastle e da bronquite infecciosa aviária). Os limites de detecção do IC-RTPCR foram iguais aos obtidos na técnica de extração com o kit da AMBION e menores do que aqueles que foram observados para os métodos de extração com Trizol® (Invitrogen) e com o MagNA Pure. O IC-RT-PCR demonstrou ser um sistema de diagnóstico capaz de conciliar simplicidade operacional e um menor custo com sensibilidade e especificidade analíticas iguais às do procedimento padrão atualmente adotado, podendo ser inclusive por laboratórios dotados de uma infra-estrutura mais simples / Abstract: The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), including real-time RT-PCR have been used for the rapid detection of Matrix glycoprotein gene (M gene) Avian influenza virus (AIV). Despite the availability of various RNA extraction methods for using in RT-PCR, isolation and detection of viral RNA are still difficult due to the unstable nature of viral RNA molecules and the presence of PCR inhibitory substances. In this study, a simple method using immune-capture (IC) to recover viral RNA from H2 AIV samples was developed and compared to one standard and two others reference methods used for viral RNA extraction, such as Ambion MagMAXTM kit and Trizol® (Invitrogen) and Magnapure kit (Roche), respectively, with subsequent analysis by real-time RT-PCR. The real-time IC-RT-PCR developed in was able to detect specifically H2N2 AIV strain, without detecting non-related avian RNA-virus pathogens, such as Newcastle disease virus, avian infectious bronchitis virus and Gumboro disease virus. Comparable detection limits were found for IC and the standard RNA extraction method using Ambion MagMAXTM kit, either for the detection of AIV in allantoic fluid suspension or in seeded tracheal and cloacal swab samples by conventional or real time RT-PCR techniques. These methods were less sensitive than Trizol® (Invitrogen) and Magnapure kit (Roche) procedures. Thus, IC was rapid and as sensitive and specific as current standard AIV RNA extraction method for real time or conventional RT-PCR, besides it conciliated simplicity and lower cost and can be applied simultaneously for direct detection of AIV in a large number of samples, including less-equipped laboratories / Mestre

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-10-18T15:38:40Z No. of bitstreams: 1 texto completo.pdf: 16418727 bytes, checksum: 1e4d06e6842f6d8fd2819d47448b314d (MD5) / Made available in DSpace on 2017-10-18T15:38:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 16418727 bytes, checksum: 1e4d06e6842f6d8fd2819d47448b314d (MD5) Previous issue date: 1999-10-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Durante a construção de um mapa de RFLP de soja, na DuPont (EUA), as sondas analisadas foram, em sua maioria, monomórflcas, porém algumas se mostraram altamente polimórticas (A1-10, A2-08, A45-10, ASS-09 e A75-10). A fim de melhor caracterizar esse padrão hipervariável, essas sondas foram seqúenciadas. Duas delas (Al-10 e A2-08) não apresentaram similaridade relevante com nenhuma sequência do “GenBank”; entretanto, uma característica marcante dessas sondas foi a sua riqueza em sequências A:T. As outras três sondas continham sequências homólogas a genes que codificam proteínas de resistência a doenças. Devido ao padrão de hibridização extremamente polimorflco revelado pela sonda A45-10, foram desenhados “primers” flanqueando essa sonda para amplincar regiões homólogas em diversos genótipos de soja, para que esse perfil de amplificação pudesse ser utilizado como “tingerprint”. Os produtos de amplificação obtidos, na faixa de 1,5 a 2,0 kb, foram capazes de identificar os diferentes genótipos analisados. A partir de uma biblioteca genômica da variedade de soja FT-Cristalina, foram isolados quatro clones, usando-se a sonda ASB-09. Um deles, o clone CR44, continha um inserto de aproximadamente 16 kb. Dois fragmentos de EcoRl e Pstl do clone CR44, que hibridizaram com a sonda A53-09, foram subclonados e seqúenciados. Esses dois fragmentos formaram um contíguo, denominado GR, de 4.198 pb. A comparação da sequência de aminoácidos predita com outras existentes no “GenBank” indicou uma homologia entre GR e a proteína “Cf-2.1-Iike” de Arabidopsis tha/iana, proteína de resistência a doenças homóloga à Cf-2/Cf-5 de Hordeum vulgare e outras proteínas de resistência de Lycopersicon. Além disso, foi identificada uma ORF de 789 nucleotídios, que codiôca uma proteína de 262 aminoácidos com domínios LRRs, uma das características estruturais da maioria das proteínas de resistência. Um RT-PCR foi feito para detectar transcritos homólogos a A53-09, A75-1O e A45-10, usando-se “primers” que flanqueavam a região de maior similaridade com genes de resistência. Em amostras de RNA extraídas de folhas, raízes e haste foi detectada a presença de transcritos, usando-se os “primers” derivados de ASB-09, o que indicou que A53-09 não é expresso de modo órgão-específico. Os produtos de amplificação presentes nos três órgãos foram seqúenciados e alinhados perfeitamente com a ORF de 789 nucleotídios. Para A75-10, foi detectada a presença de várias bandas, nos três órgãos examinados, indicando que, possivelmente, os “primers” estariam pareando com mais de um transcrito. Para A45-10, não foi detectada a presença de nenhum transcrito nos três órgãos analisados. / During the construction of one soybean RFLP map at DuPont (USA), the majority of the probes analyzed were monomorphic, however, a few of them were highly polymorphic (A1-10, A2-08, A45-10, ASB-09, and A75-10). To better understand the hypervariable pattern revealed by these probes, they were sequenced. Two of them (A1-1O and A2-08) did not present any similarities with sequences deposited in the GenBank. Their main feature was that they were highly A:T rich. The other three probes contained sequences homologous to known disease resistance genes in plants. Due to the hypervariable pattern revealed by probe A45-10, primers flanking this probe were designed and used to amplify the corresponding region in several soybean genotypes. The amplification products, in the range of 1.5 to 2 kb, allowed the identification of each of the different genotypes analyzed. Probe ASB-09 was used to screen a genomic library prepared with DNA from cultivar FT-Cristalina. Four clones were isolated. One of them, clone CR44 of approximately 16 kb, was further analyzed. Restriction of this clone with EcoRl and Pstl revealed two bands hybrizing with probe A53-09. These were subcloned and sequenced. The two fragments partially overlapped and formed a config of 4,198 bp designated GR. Blast analyses of GR with sequences deposited in the GenBank showed that it potentially encode a protein homologous to “Cf-2.1-Iike” protein of Arabidopsis tha/iana, to disease resistance protein homologous to Cf-2le-5 of Hordeum vulgare and to disease resistance proteins of Lycopersicon. An open-reading frame of 789 nucleotídes was identified in GR. It potentially encode a sequence of 262 amino acid residues with LRR motifs, a structural characteristic of most disease resistance proteins described so far. RT-PCR was performed with primers flanking the regions homologous to disease resistance genes present in probes ASB-09, A75-10, and A45-10. RNA samples extracted from leaves, roots, and stems contained transcripts which were amplitied with primers derived from probe ASB-09, indicating that expression of ASB-09 is not organ-specific. The amplification products from the three organs were sequenced and presented perfect homology with part of the 789 bp ORF present in A53-09. The primers derived from A75-10 amplifled several bands with different sizes in the three organs analyzed indicating that more than one transcript homologous to A75-10 was present in the different organs. The primers derived from A45-1O did not detect any transcripts in the organs analyzed. / CPF do autor não encontrado