Orientador: Manoel Victor Franco Lemos / Banca: Edgar Augusto de Toledo Picoli / Banca: Fernando Hercos Valicente / Banca: Jaime Maia dos Santos / Banca: Flávia Maria de Souza Carvalho / Resumo: A produção de alimentos é prejudicada devido a incidência de diversas pragas e doenças. A fim de poder controlá-las, utilizam-se uma grande quantidade e variedade de produtos químicos. Em muitos casos, esses produtos incidem sobre uma vasta gama de organismos, fato este que causa um grande impacto no meio ambiente. A fim de minimizar os efeitos negativos do uso de produtos químicos com grande espectro de ação, há grande empenho no desenvolvimento de produtos cujos alvos sejam específicos. Neste sentido, o uso do controle biológico tem se apresentado como uma alternativa bastante interessante. Dentre os vários sistemas utilizados no controle biológico de pragas, a bactéria Bacillus thuringiensis é a opção de maior destaque. No entanto, inúmeros casos de resistência têm surgido ao longo destes últimos anos, devido aos produtos codificados pelos genes com ação entomopatogênica estarem sendo utilizados de maneira descontrolada. Desse modo, a busca par novas proteínas Cry é tarefa importante dentro da estratégia de controle biológico de pragas agrícolas. Com o intuito de se identificar novas genes cry par meio de análises de PCR Múltiplas, seguida de análise por RFLP, identificaram-se 3 isolados de B. thuringiensis cujos fragmentos de genes analisados diferiram entre si e das linhagens padrão conhecidas até o momento. Ao analisar essas sequências gênicas depositadas no GenBank, verificou-se que dois desses fragmentos de DNA pertencentes aos isolados 113 e 133 possuíam alta similaridade com os genes efetivos contra nematoides, uma terceira sequência caracterizada a partir do isolado 123 não foi similar a nenhuma outra sequência depositada no GenBank atualmente. Os isolados cuja sequência de aminoácidos de suas proteínas Cry predita "in silica" mostraram-se similares a genes cry... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Food production is strongly harmed by a large amount of plant pests and diseases. Aiming to control these problems, a large quantity and variety of chemical compounds have been used. In many cases, these products cause a severe environmental impact over a wide range of non-target organisms. In order to minimize the negative effects of using chemical products with great action spectrum, there is a great effort to develop products of which targets are specific. Therein, the biological control has been presenting itself as a very interesting alternative. Among the various systems used in biological control, the bacteria Bacillus thuringiensis is the option pinpointed as the best notability. However, some resistance towards its use as a biological controlling agent have been reported recently due to the uncontrolled use of products decoded by genes of entomopathogenic action. Thus, the search of new Cry proteins is an important task inside the strategy of biological control of field pests. In order to overcome these situations efforts on the isolation and characterization of new B. thuringiensis isolates using molecular techniques such as PCR and RFLP were adopted and three B. thurigiensis isolates were compared among them and were considered different among the known and described standard type strains up to the present time. When part of the obtained gene sequences were compared to those deposited on the GeneBank, it was possible to observe that for two of them, isolate 113 and 133, a high degree of similarity with sequences described as active against nematodes; the third isolate, 123, had no similarity to any deposited sequence up to date. The isolates, which aminoacid sequence of their Cry proteins "in silico" were similar to cry genes earlier deposited on GenBank, had their respective gene fully characterized. To this, several... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T18:44:30Z : No. of bitstreams: 1 goncalves_jf_dr_jabo.pdf: 1447890 bytes, checksum: 213f62c5c0d5c7deaaea4b29c3aa4e09 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A produção de alimentos é prejudicada devido a incidência de diversas pragas e doenças. A fim de poder controlá-las, utilizam-se uma grande quantidade e variedade de produtos químicos. Em muitos casos, esses produtos incidem sobre uma vasta gama de organismos, fato este que causa um grande impacto no meio ambiente. A fim de minimizar os efeitos negativos do uso de produtos químicos com grande espectro de ação, há grande empenho no desenvolvimento de produtos cujos alvos sejam específicos. Neste sentido, o uso do controle biológico tem se apresentado como uma alternativa bastante interessante. Dentre os vários sistemas utilizados no controle biológico de pragas, a bactéria Bacillus thuringiensis é a opção de maior destaque. No entanto, inúmeros casos de resistência têm surgido ao longo destes últimos anos, devido aos produtos codificados pelos genes com ação entomopatogênica estarem sendo utilizados de maneira descontrolada. Desse modo, a busca par novas proteínas Cry é tarefa importante dentro da estratégia de controle biológico de pragas agrícolas. Com o intuito de se identificar novas genes cry par meio de análises de PCR Múltiplas, seguida de análise por RFLP, identificaram-se 3 isolados de B. thuringiensis cujos fragmentos de genes analisados diferiram entre si e das linhagens padrão conhecidas até o momento. Ao analisar essas sequências gênicas depositadas no GenBank, verificou-se que dois desses fragmentos de DNA pertencentes aos isolados 113 e 133 possuíam alta similaridade com os genes efetivos contra nematoides, uma terceira sequência caracterizada a partir do isolado 123 não foi similar a nenhuma outra sequência depositada no GenBank atualmente. Os isolados cuja sequência de aminoácidos de suas proteínas Cry predita in silica mostraram-se similares a genes cry... / Food production is strongly harmed by a large amount of plant pests and diseases. Aiming to control these problems, a large quantity and variety of chemical compounds have been used. In many cases, these products cause a severe environmental impact over a wide range of non-target organisms. In order to minimize the negative effects of using chemical products with great action spectrum, there is a great effort to develop products of which targets are specific. Therein, the biological control has been presenting itself as a very interesting alternative. Among the various systems used in biological control, the bacteria Bacillus thuringiensis is the option pinpointed as the best notability. However, some resistance towards its use as a biological controlling agent have been reported recently due to the uncontrolled use of products decoded by genes of entomopathogenic action. Thus, the search of new Cry proteins is an important task inside the strategy of biological control of field pests. In order to overcome these situations efforts on the isolation and characterization of new B. thuringiensis isolates using molecular techniques such as PCR and RFLP were adopted and three B. thurigiensis isolates were compared among them and were considered different among the known and described standard type strains up to the present time. When part of the obtained gene sequences were compared to those deposited on the GeneBank, it was possible to observe that for two of them, isolate 113 and 133, a high degree of similarity with sequences described as active against nematodes; the third isolate, 123, had no similarity to any deposited sequence up to date. The isolates, which aminoacid sequence of their Cry proteins in silico were similar to cry genes earlier deposited on GenBank, had their respective gene fully characterized. To this, several... (Complete abstract click electronic access below)

The diptericidal $\textit{Bacillus thuringiensis}$ (Bt) ssp. $\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\textit{am1}$ and $\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\delta$-endotoxins (Cry19A from Bt ssp. $\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\textit{240A}$ from Bti and an insertion sequence associated with the $\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\alpha$/$\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\alpha$/$\beta$-type SASP from $\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\textit{Bacilli}$.

Submitted by Natalia de Souza Gonçalves (natalia.goncalves@ufpe.br) on 2016-09-23T13:38:34Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Paulo Geovani Silva Martins - 2016.pdf: 11199647 bytes, checksum: e9d2a3a59d3656debcafdd36c106889f (MD5) / Made available in DSpace on 2016-09-23T13:38:34Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Paulo Geovani Silva Martins - 2016.pdf: 11199647 bytes, checksum: e9d2a3a59d3656debcafdd36c106889f (MD5) Previous issue date: 2016-03-11 / CAPES / A cultura da cana-de-açúcar possui importância econômica para o Brasil pela produção de açúcar e etanol. No entanto, parte da produção é ameaçada pelo ataque de insetos-praga, destacando-se a broca da cana-de-açúcar (Diatraea saccharalis), que causa graves perdas econômicas. O controle biológico é alternativo ao controle químico e consiste no emprego de inimigos naturais como patógenos, que regulam a população de insetos-praga em níveis nãoprejudiciais. Dentre os patógenos destaca-se a bactéria Bacillus thuringiensis (Bt) que produz toxinas, codificadas pelos genes cry, no formato de cristais proteicos com atividade entomotóxica para diversas ordens de insetos. O presente trabalho teve como objetivo o isolamento e caracterização de novas estirpes de Bt com potencial entomotóxico para controle biológico da D. saccharalis em cana-de-açúcar. Amostras de solo coletadas possibilitaram o isolamento de 97 colônias bacterianas com características fenotípicas compatíveis para Bt. O DNA extraído foi amplificado com primers para os genes cry1, cry2 e cry9, ativos contra dípteros e lepidópteros. Em 11,4% e 12,4% dos isolados foram constatados os genes cry2 e cry9, respectivamente, e 10 destes patogenicidade variando entre 3,47% a 20,05% para D.saccharalis. Após comparação dos perfis proteômicos dos isolados Bt.Pri 4.7, Bt.Pri 4.29, Bt.CDi 1.3 e Bt.CDi 1.11, de maior e menor potencial patogênico de cada classe gênica, foram identificadas proteínas como a shikimato quinase, envolvida na produção de toxinas em Bt, e que auxiliam o entendimento da patogênese deste microorganismo. / Sugarcane production has economical importance to Brazil, especially due to production of sugar and ethanol. However, a proportion of the production is lost owing to damage of pest insects, including sugarcane borer (Diatraea saccharalis), which cause severe economical lost in the sugar/ethanol industry. Biological control, alternatively to chemical control, which cause damage to human health and environment, is based on use of natural enemies such as pathogens that regulated pest insects population to a non-prejudicial level. Among pathogens, especially bacteria Bacillus thuringiensis (Bt) that produce toxins, encoding by cry genes, which are protein crystal with entomotoxicity activity for diverse insect orders. The present work aims to isolate and characterize new strains of Bt with potential entomotoxicity to biological control of D. saccharalis in sugarcane. Soil samples collected allowed the isolation of 97 bacterial colonies phenotypically compatible to Bt. DNA extracted was used in PCR reaction using primers to amplify cry1, cry2 and cry9 genes, with activity against Lepidoptera and Diptera. In 11.4 and 12.4% of the strains was observed fragments with pattern expected for cry2 and cry9 genes, respectively, and ten isolated shown pathogenicity ranging from 3.47% to 15.62% for D.saccharalis. After comparing the proteomic profiles of the isolates Bt.Pri 4.7 Bt.Pri 4.29, Bt.CDi 1.3 and Bt.CDi 1.11 of major and minor pathogenic potential of every gene class proteins were identified as shikimate kinase, involved in the production of Bt toxins, and assist the understanding of the pathogenesis of this microorganism.

Les parasporines sont des toxines Cry du bacille de Thuringe actives contre des cellules tumorales. Ce travail montre que la parasporine PS1Aa2 (Cry31Aa2) forme des pores dans des membranes artificielles, comme de nombreuses toxines Cry. Ceux-ci ont plusieurs niveaux de conductance dont les plus fréquents étaient de 11, 16 et 21 pS dans une solution de 150 mM KCl. Nos résultats de microspectrofluorométrie avec la sonde Fura-2 montrent que la présence de la PS1Aa2 peut produire des augmentations du calcium intracellulaire, la plupart du temps sous la forme d’oscillations calciques et parfois des augmentations soutenues. Ces réponses ont été observées en présence et en absence de calcium extracellulaire, dans les lignées tumorales HeLa et HepG2 et dans la lignée non tumorale HEK 293. Bien que quelques études aient montré que le calcium semble intervenir dans leur mode d’action, de telles oscillations calciques n’ont jamais été décrites auparavant pour des toxines Cry. Les expériences ont dû être faites à des concentrations beaucoup plus élevées de toxine que prévues sur la base des résultats publiés de cytotoxicité. Malgré la présence des fragments identifiés auparavant comme actifs, sa faible efficacité semble liée à la présence d’ADN dans les préparations qui entraîne la précipitation de la protéine. Les travaux futurs sur cette toxine seraient donc grandement facilités par une amélioration de sa méthode de préparation. / Parasporins are Cry toxins from Bacillus thuringiensis that are active against tumor cells. This work shows that parasporin PS1Aa2 (Cry31Aa2) forms pores in artificial membranes like many Cry toxins. These pores have several levels of conductance; the most frequently seen in 150 mM KCl solutions were of 11, 16 and 21 pS. Our microspectrofluorometric results with the Fura-2 probe showed that the presence of PS1Aa2 can induce changes in intracellular calcium levels, most often in the form of calcium oscillations and sometimes as sustained increases. Such responses were observed in the presence and absence of extracellular calcium, with the tumor cell lines HeLa and HepG2, and with the non-tumorous cell line HEK 293. Calcium oscillations have not been described previously for Cry toxins even though some studies have shown that calcium appears to be involved in their mode of action. Our experiments required the use of much higher concentrations of toxin than suggested from the published cytotoxicity results. Despite the presence of fragments previously identified as active, its low efficacy appears to be related to the presence of DNA in the preparations causing the protein to precipitate. Future work on this toxin would therefore be greatly facilitated by an improvement in its method of preparation.