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Made available in DSpace on 2014-06-11T19:26:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-13Bitstream added on 2014-06-13T19:12:59Z : No. of bitstreams: 1 rossi_jr_me_jabo.pdf: 1016425 bytes, checksum: 885b9804e9660ef2c359c1d1eb71a757 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dentre os microrganismos empregados no controle de mosquitos, a bactéria Bacillus thuringiensis var. israelensis (Bti) se destaca por apresentar atividade tóxica contra insetos da ordem Diptera, na qual produz inclusões cristalinas compostas pelas proteínas Cry, que são codificadas por genes cry presentes em um único plasmídeo. A produção dessas proteínas em grande escala está relacionada a mecanismos transcricionais, como por exemplo, a expressão de um gene sob o controle de um promotor forte. Tendo em vista que a bactéria Bti SPS1 (Patente PI0200228-0) foi isolada do território brasileiro e que possui potencial para o controle de vetores por produzir uma maior quantidade de esporos/cristais em menor tempo, este trabalho teve por finalidade a caracterização da região promotora dos genes cry4Aa, cry4Ba e cry11Aa de Bti SPS1 pelas técnicas de PCR e “Southern blotting”. Em associação a estas técnicas, foi realizado bioensaio para a verificação da mortalidade de larvas de Aedes aegypti. Os resultados das análises moleculares indicaram homologia no perfil de hibridização da região promotora da linhagem padrão Bti T14-001 e do isolado Bti SPS1. A quantificação das suspensões em espectrofotômetro (DO600nm) e a leitura de esporos em câmara de Neubauer, revelaram que o isolado Bti SPS1 produz uma maior quantidade de esporos/cristais em relação à linhagem padrão Bti T14-001. O bioensaio apresentou elevados índices de mortalidade. Estes resultados tornam a bactéria Bti SPS1 uma fonte promissora para novas formulações visando o controle de vetores. / Among the microorganisms used for mosquitoes’ control the bacterium Bacillus thuringiensis var. israelensis (Bti) is frequently considered since it presents toxic activities’ against Diptera, producing crystal inclusions including Cry proteins, which are coded by a sole plasmid borne set of genes. The production of these proteins in large scale is related to transcriptional mechanisms, as an example, a particular gene expression controlled by a strong promoter. Since the Bti bacterial isolate SPS1 (Patent PI0200228-0) was isolated within the Brazilian territory and exhibits potential for the control of vector insects due to a higher crystal production ability in shorter time period, this work had as objective the characterization of the promotion region for the genes cry4Aa, cry4Ba e cry11Aa using PCR and “Southern blotting” techniques. Also some bioassays using Aedes aegypti larvae were carried out. The results from the molecular analysis have indicated homology for the hybridization profile from the promoter region from the type strain of Bti T14-001 and that of the SPS1 isolate. Spectrofotometric (OD600nm) and Neubauer chamber measures have revealed that the SPS1 isolate produces a higher amount of spore/crystal as compared to the Bti T14-001 strain. The bioassay presented higher mortality levels. These results seem to indicate that the isolate SPS1 is a promising bacterial strain to be used on formulations able to control insect vector pests.

Orientador: Manoel Victor Franco Lemos / Banca: Cristina Lacerda Soares Petrarolha Silva / Banca: Sérgio Antonio de Bortoli / Resumo: Dentre os microrganismos empregados no controle de mosquitos, a bactéria Bacillus thuringiensis var. israelensis (Bti) se destaca por apresentar atividade tóxica contra insetos da ordem Diptera, na qual produz inclusões cristalinas compostas pelas proteínas Cry, que são codificadas por genes cry presentes em um único plasmídeo. A produção dessas proteínas em grande escala está relacionada a mecanismos transcricionais, como por exemplo, a expressão de um gene sob o controle de um promotor forte. Tendo em vista que a bactéria Bti SPS1 (Patente PI0200228-0) foi isolada do território brasileiro e que possui potencial para o controle de vetores por produzir uma maior quantidade de esporos/cristais em menor tempo, este trabalho teve por finalidade a caracterização da região promotora dos genes cry4Aa, cry4Ba e cry11Aa de Bti SPS1 pelas técnicas de PCR e "Southern blotting". Em associação a estas técnicas, foi realizado bioensaio para a verificação da mortalidade de larvas de Aedes aegypti. Os resultados das análises moleculares indicaram homologia no perfil de hibridização da região promotora da linhagem padrão Bti T14-001 e do isolado Bti SPS1. A quantificação das suspensões em espectrofotômetro (DO600nm) e a leitura de esporos em câmara de Neubauer, revelaram que o isolado Bti SPS1 produz uma maior quantidade de esporos/cristais em relação à linhagem padrão Bti T14-001. O bioensaio apresentou elevados índices de mortalidade. Estes resultados tornam a bactéria Bti SPS1 uma fonte promissora para novas formulações visando o controle de vetores. / Abstract: Among the microorganisms used for mosquitoes' control the bacterium Bacillus thuringiensis var. israelensis (Bti) is frequently considered since it presents toxic activities' against Diptera, producing crystal inclusions including Cry proteins, which are coded by a sole plasmid borne set of genes. The production of these proteins in large scale is related to transcriptional mechanisms, as an example, a particular gene expression controlled by a strong promoter. Since the Bti bacterial isolate SPS1 (Patent PI0200228-0) was isolated within the Brazilian territory and exhibits potential for the control of vector insects due to a higher crystal production ability in shorter time period, this work had as objective the characterization of the promotion region for the genes cry4Aa, cry4Ba e cry11Aa using PCR and "Southern blotting" techniques. Also some bioassays using Aedes aegypti larvae were carried out. The results from the molecular analysis have indicated homology for the hybridization profile from the promoter region from the type strain of Bti T14-001 and that of the SPS1 isolate. Spectrofotometric (OD600nm) and Neubauer chamber measures have revealed that the SPS1 isolate produces a higher amount of spore/crystal as compared to the Bti T14-001 strain. The bioassay presented higher mortality levels. These results seem to indicate that the isolate SPS1 is a promising bacterial strain to be used on formulations able to control insect vector pests. / Mestre

Das in der vorliegenden Arbeit vorgestellte Verfahren der nicht-radioaktiven Southern Blot Hybridisierung unter Verwendung einer Digoxigenin Markierung hat sich für die Darstellung von Rekombinationen im TEL-Genlokus genomischer DNA als sensitive Vergleichsmethode bewiesen. Es wurden insgesamt 122 Kinder mit dieser Methode auf das Vorliegen der Translokation t(12;21) untersucht. Bei einer nur relevante Faktoren berücksichtigenden Beschränkung des PatientInnenkollektivs auf protokollgerecht nach ALL-REZ BFM behandelte B-Vorläufer-Zell-ALL und Erstrezidive konnte eine Rekombination von TEL in 5 von 65 PatientInnen (7,7%) nachgewiesen werden. Lässt man die Einschlusskriterien der ALL-REZ BFM Studie unberücksichtigt, handelt es sich sogar um 7 von 71 (9,9%) PatientInnen. Damit bestätigen die hier vorliegenden Ergebnisse den Trend der aktuellen Diskussion über die Häufigkeit des Vorliegens des Fusionsgenes TEL-AML1 bei Erstrezidiven, die eine kumulative Inzidenz bei einem 10-jährigen Untersuchungszeitraum von 9 ± 5% angeben. Weiterhin konnte mit ereignisfreien Beobachtungszeiträumen nach dem 1. Rezidiv im Median von 8,6 Jahren ein Trend zu späten Rezidiven und somit eine mittelfristig günstige Prognose für die TEL-Rearrangement positiven Rezidivfälle konstatiert werden. Zusammenfassend kann gesagt werden, dass TEL-AML1 positive PatientInnen eine Subgruppe darstellen, die lange erkrankungsfreie Intervalle mit zur Zeit üblichen Therapieprotokollen erreichen, nach Therapie der Ersterkrankung aber auch nach dem ersten Rezidiv. Auf Grund der sich aus dem retrospektiven PatientInnekollektiv ergebenen Selektion war es nicht möglich, statistisch signifikante Aussagen zur unabhängigen prognostischen Bedeutung für die langfristige Prognose von ALL im Kindesalter mit TEL-Rekombinationen zu treffen. / The presence of TEL/AML1 fusion gene in childhood acute lymphoblastic leukaemia (ALL) defines a subgroup of patients with better than average outcome. Around 20% of the patient at point of initial ALL diagnosis are characterised by this fusion transcript from translocation t(12;21)(p12;q22). However, the prognostic significance of this aberration has recently been disputed by the Berlin-Frankfurt-Munster (BFM) study group due to its relatively high incidence found in relapsed patients (19.6% and 21.9%, in two cohorts). Here we wanted to get more data in a long term follow up retrospect investigation by analysing DNA from frozen conserved bone marrow samples of 65 children. In the study presented here only five out of 65 (7.7%) patients selected as childhood B cell precursor acute lymphoblastic leukaemia only treated according to Berlin-Frankfurt-Munster (BFM) ALL relapse trial protocols (ALL-REZ BFM 82-96) (excluding T-lineage and Philadelphia chromosome (Ph)-positive leukaemia) carry this fusion. The detection was done due to a new developed non-radioactive Southern blotting with a Digoxigenin marked template. We could confirm the still good middle term prognosis in the relapsed TEL-AML1 positive subgroup. The TEL-AML1-positive and negative patients differed with respect to duration of last remission and age at initial diagnosis. At a median follow-up time of 8.6 years, children positive for TEL-AML1 had a higher probability of disease-free survival. Because of the small number of patients in this study it was not possible to get statistical significant facts about the independent prognostic impact for the long term prognosis of ALL in childhood with TEL rearrangement.