Orientador : Raphael Lucio Andreatti Filho / Abstract: Native intestinal lactic acid bacteria from poultry and mammals provide an adverse environment to development of pathogenic microorganisms. Recently, beyond its use as probiotics, the application in food conservation has been researched. Species like Lactobacillus, Lactococcus, Streptococcus, Pediococcus e Leuconostocs are used in fermented food, presenting antagonistic properties to same species and/or pathogenic bacterias. Lactobacillus spp. species used in probiotics and competitive exclusion products have benefic effects due direct action on particular groups of pathogenic microorganisms. Just by these advantages, lactic acid bacteria have been exhaustively studied allowing the detection of antibiose promoters substances as organic acids, bacteriophags, hydrogen peroxide, and bacteriocins. In the present study, 474 lactic acid bacteria from Lactobacillus species were isolated from the crop and ceca of chicken. Two hundred sixty five samples presented inhibitory activity against indicator microorganisms (Gram-positive and Gram-negative bacterias) using the spot-on-the-lawn assay. Fifty three samples identified as Lactobacillus reuteri, L. salivary and L. spp. confirmed inhibition of Enterococcus faecalis, E. faecium, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. but not on Lactobacillus casei, L. delbrureckii, L. fermentum and L. helveticus by bacteriocins action by antagonistic the simultaneons methods of well diffusion assay on anaerobiosis. The antagonism was detected by inhibition zones to various indicators microorganisms. / Mestre

Made available in DSpace on 2014-06-11T19:30:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T18:06:46Z : No. of bitstreams: 1 lima_et_me_botfmvz.pdf: 1900368 bytes, checksum: 0ed40eb2152ee863edc771400939912f (MD5) / Native intestinal lactic acid bacteria from poultry and mammals provide an adverse environment to development of pathogenic microorganisms. Recently, beyond its use as probiotics, the application in food conservation has been researched. Species like Lactobacillus, Lactococcus, Streptococcus, Pediococcus e Leuconostocs are used in fermented food, presenting antagonistic properties to same species and/or pathogenic bacterias. Lactobacillus spp. species used in probiotics and competitive exclusion products have benefic effects due direct action on particular groups of pathogenic microorganisms. Just by these advantages, lactic acid bacteria have been exhaustively studied allowing the detection of antibiose promoters substances as organic acids, bacteriophags, hydrogen peroxide, and bacteriocins. In the present study, 474 lactic acid bacteria from Lactobacillus species were isolated from the crop and ceca of chicken. Two hundred sixty five samples presented inhibitory activity against indicator microorganisms (Gram-positive and Gram-negative bacterias) using the spot-on-the-lawn assay. Fifty three samples identified as Lactobacillus reuteri, L. salivary and L. spp. confirmed inhibition of Enterococcus faecalis, E. faecium, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. but not on Lactobacillus casei, L. delbrureckii, L. fermentum and L. helveticus by bacteriocins action by antagonistic the simultaneons methods of well diffusion assay on anaerobiosis. The antagonism was detected by inhibition zones to various indicators microorganisms.

Orientador: Janete Apparecida Desidério / Banca: Odair Aparecido Fernandes / Banca: Agda Paula Facincani / Banca: Paula Cristina Brunini Crialesi Legori / Banca: Juliana Regina Rossi / Resumo: As proteínas Vip3Aa e Cry1Ia apresentam potencial no controle de lepidópteros-praga e surgem como alternativa promissora no manejo da resistência de pragas as proteínas Cry1A, que tem sido altamente utilizada na formulação de bioinseticidas comerciais à base de Bacillus thuringiensis (Bt) e em plantas transgênicas. Assim sendo, o presente trabalho teve como objetivo a clonagem e expressão das proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 em Escherichia coli, a fim de se analisar a correlação entre a união aos receptores por meio de análises de competição entre as diferentes toxinas Vip3Aa e a toxina Cry1Ia10, e a toxicidade a lepidópteros-praga, inferindo-se quais as combinações que poderiam ser utilizadas na produção de plantas transgênicas, contendo múltiplos genes, as quais vêm sendo empregadas para contornar a evolução da resistência dos insetos às toxinas Bt. Para tanto, os genes vip3Aa e cry1Ia10 foram clonados no vetor pET SUMO, expressos em E. coli e a toxicidade das proteínas foram testadas em bioensaios com lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalis e Heliothis virescens. As BBMVs ("Brush Border Membrane Vesicles") foram preparadas a partir dos intestinos das três espécies e ensaios de competição homóloga e heteróloga foram realizados. As proteínas Vip3Aa42 e Vip3Aa43 apresentaram toxicidade para S. frugiperda e A. gemmatalis. Já a proteína Cry1Ia10 apresentou toxicidade apenas para A. gemmatalis e, as proteínas não se mostraram tóxicas para H. virescens. Os ensaios de ligação às BBMVs demonstraram que as proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 se unem aos receptores presentes no intestino médio de forma efetiva nas três espécies e que, portanto, houve correlação entre a toxicidade e a união aos receptores para as populações de S. frugiperda e A. gemmatalis, porém para H. virescens não houve relação entre a toxicidade e a união aos receptores. Sendo assim ... / Abstract: Vip3Aa and Cry1Ia proteins have potential in control of Lepidopteran pest and emerge as a promising alternative in the pest resistance management the Cry1A proteins, which has been highly used in the formulation of commercial insecticides based on Bacillus thuringiensis (Bt) and in transgenic plants. Therefore, this study aimed to cloning and expression of Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins in Escherichia coli, in order to analyze the correlation between the binding to receptors through competition assays between the different Vip3Aa toxins and toxin Cry1Ia10, and toxicity to lepidopteran pests inferring up which combinations that could be used to produce transgenic plants containing multiple genes, which have been used to circumvent the development of insects resistance to Bt toxins. Therefore, vip3Aa and cry1Ia10 genes were cloned into the pET SUMO vector, expressed in E. coli and toxicity of proteins were tested in bioassays with neonate larvae of Spodoptera frugiperda, Anticarsia gemmatalis and Heliothis virescens. The BBMVs (Brush Border Membrane Vesicles) were prepared from the gut of the three species, and homologous and heterologous competition assays were performed. The Vip3Aa42 and Vip3Aa43 proteins were toxic to S. frugiperda and A. gemmatalis. Already Cry1Ia10 protein showed toxicity only for A. gemmatalis and proteins were not toxic to H. virescens. Binding assays to BBMVs demonstrated that Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins binding effectively to receptors present in the midgut in three species and, therefore, was correlation between toxicity and the binding to receptors for the populations of S. frugiperda and A. gemmatalis, but for H. virescens there was no relationship between toxicity and the binding to receptors. Thus, the combination of Cry1Ia10 and Vip3Aa42 or Vip3Aa43 proteins is indicated for the production of biological insecticidal, as well as for the production of transgenic plants to circumvent ... / Doutor

Made available in DSpace on 2015-06-17T19:33:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-06. Added 1 bitstream(s) on 2015-06-18T12:47:49Z : No. of bitstreams: 1 000829635_20160806.pdf: 54783 bytes, checksum: 1f71baca5b42308b10e653a6d3b40058 (MD5) Bitstreams deleted on 2016-08-08T11:55:58Z: 000829635_20160806.pdf,. Added 1 bitstream(s) on 2016-08-08T11:56:33Z : No. of bitstreams: 1 000829635.pdf: 1135736 bytes, checksum: 5f82225f1067c07c36424e25cc5c44f1 (MD5) / As proteínas Vip3Aa e Cry1Ia apresentam potencial no controle de lepidópteros-praga e surgem como alternativa promissora no manejo da resistência de pragas as proteínas Cry1A, que tem sido altamente utilizada na formulação de bioinseticidas comerciais à base de Bacillus thuringiensis (Bt) e em plantas transgênicas. Assim sendo, o presente trabalho teve como objetivo a clonagem e expressão das proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 em Escherichia coli, a fim de se analisar a correlação entre a união aos receptores por meio de análises de competição entre as diferentes toxinas Vip3Aa e a toxina Cry1Ia10, e a toxicidade a lepidópteros-praga, inferindo-se quais as combinações que poderiam ser utilizadas na produção de plantas transgênicas, contendo múltiplos genes, as quais vêm sendo empregadas para contornar a evolução da resistência dos insetos às toxinas Bt. Para tanto, os genes vip3Aa e cry1Ia10 foram clonados no vetor pET SUMO, expressos em E. coli e a toxicidade das proteínas foram testadas em bioensaios com lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalis e Heliothis virescens. As BBMVs (Brush Border Membrane Vesicles) foram preparadas a partir dos intestinos das três espécies e ensaios de competição homóloga e heteróloga foram realizados. As proteínas Vip3Aa42 e Vip3Aa43 apresentaram toxicidade para S. frugiperda e A. gemmatalis. Já a proteína Cry1Ia10 apresentou toxicidade apenas para A. gemmatalis e, as proteínas não se mostraram tóxicas para H. virescens. Os ensaios de ligação às BBMVs demonstraram que as proteínas Vip3Aa42, Vip3Aa43 e Cry1Ia10 se unem aos receptores presentes no intestino médio de forma efetiva nas três espécies e que, portanto, houve correlação entre a toxicidade e a união aos receptores para as populações de S. frugiperda e A. gemmatalis, porém para H. virescens não houve relação entre a toxicidade e a união aos receptores. Sendo assim ... / Vip3Aa and Cry1Ia proteins have potential in control of Lepidopteran pest and emerge as a promising alternative in the pest resistance management the Cry1A proteins, which has been highly used in the formulation of commercial insecticides based on Bacillus thuringiensis (Bt) and in transgenic plants. Therefore, this study aimed to cloning and expression of Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins in Escherichia coli, in order to analyze the correlation between the binding to receptors through competition assays between the different Vip3Aa toxins and toxin Cry1Ia10, and toxicity to lepidopteran pests inferring up which combinations that could be used to produce transgenic plants containing multiple genes, which have been used to circumvent the development of insects resistance to Bt toxins. Therefore, vip3Aa and cry1Ia10 genes were cloned into the pET SUMO vector, expressed in E. coli and toxicity of proteins were tested in bioassays with neonate larvae of Spodoptera frugiperda, Anticarsia gemmatalis and Heliothis virescens. The BBMVs (Brush Border Membrane Vesicles) were prepared from the gut of the three species, and homologous and heterologous competition assays were performed. The Vip3Aa42 and Vip3Aa43 proteins were toxic to S. frugiperda and A. gemmatalis. Already Cry1Ia10 protein showed toxicity only for A. gemmatalis and proteins were not toxic to H. virescens. Binding assays to BBMVs demonstrated that Vip3Aa42, Vip3Aa43 and Cry1Ia10 proteins binding effectively to receptors present in the midgut in three species and, therefore, was correlation between toxicity and the binding to receptors for the populations of S. frugiperda and A. gemmatalis, but for H. virescens there was no relationship between toxicity and the binding to receptors. Thus, the combination of Cry1Ia10 and Vip3Aa42 or Vip3Aa43 proteins is indicated for the production of biological insecticidal, as well as for the production of transgenic plants to circumvent ...

Orientador: Manoel Victor Franco Lemos / Coorientador: Baltasar Escriche / Banca: Jackson Antonio Marcondes de Souza / Banca: Odair Aparecido Fernandes / Banca: Bergmann Morais Ribeiro / Banca: Norton Rodrigues Chagas Filho / Resumo: O presente trabalho objetivou avaliar a toxicidade de diferentes proteínas Cry1 e Vip3Aa de Bacillus thuringiensis em larvas de Diatraea saccharalis, verificar o modo de união dessas proteínas aos receptores do inseto alvo e analisar a interação dessas proteínas no controle larval, buscando informações para subsidiar o uso de plantas geneticamente modificadas com genes cry1 e vip3Aa de forma segura e duradoura. A análise de suscetibilidade larval revelou a proteína Cry1Ab como mais efetiva no controle, seguida das proteínas Cry1Ac, Vip3Aa, Cry1Ca e Cry1Fa. A população testada não foi suscetível à proteína Cry1Ea. A proteína Cry1Aa apresentou baixa toxicidade. Nos ensaios de união específica, as proteínas Cry1 ligaram-se a receptores presentes no intestino médio de D. saccharalis e um modelo com três diferentes receptores foi proposto com base nos ensaios de competição heteróloga. Um receptor comum para Cry1Aa, Cry1Ab e Cry1Ac, outro para Cry1Fa e Cry1Ab e um receptor diferente para a proteína Vip3Aa. Dentre as interações avaliadas por bioensaios, as combinações proteicas: Cry1Ab:Cry1Ca e Cry1Fa:Cry1Ca apresentaram efeito sinérgico; as demais combinações revelaram efeitos antagônicos / Abstract: The aim of this research was to evaluate the toxicity of different Cry1 and Vip3A proteins from Bacillus thuringiensis to Diatraea saccharalis, verify the proteins binding to receptors of the target insect and to analyze the protein interactions in larval control, seeking information to support safe and sustainable the use of transgenic Bt plants. The most toxic protein assayed was Cry1Ab followed by Cry1Ac, Vip3Aa, Cry1Ca and Cry1Fa. The population tested was not susceptible to Cry1Ea protein. Cry1Aa showed very low toxicity. Biotinylated Cry1 proteins showed specific binding to the midgut brush border membrane vesicles of the larvae. Heterologous competitive binding assays suggested a model of three receptors, a common receptor for Cry1Aa and Cry1Ab another one for Cry1Fa and Cry1Ab. Vip3Aa did not compete for binding with any of the Cry proteins tested. Among interactions bioassays, the combinations between Cry1Ab:Cry1Ca and Cry1Fa:Cry1Ca showed synergistic effect, whereas the other combinations showed antagonistic effects / Doutor

Orientador: Raghuvir Krishnaswamy Arni / Banca: Ljubica Tasic / Banca: Marcos Roberto de Mattos Fontes / Banca: Fábio Rogério de Moraes / Banca: Lisandra Marques Gava Borges / Resumo: Corynebacterium pseudotuberculosis (C. pseudotuberculosis), é uma bactéria gram positiva anaeróbia facultativa, pleomórfica, que não esporula, não forma cápsula, e que apresenta 2 biotipos ou biovares, sendo o biovar equi capaz de infectar preferencialmente equinos, enquanto o biótipo denominado ovis acomete pequenos ruminantes. Esta bactéria faz parte do grupo CMNR (Corynebacterium, Mycobacterium, Nocardia e Rhodococcus), que demonstra grande importância veterinária e médica, uma vez que estes micro-organismos comumente infectam animais, podendo infectar o homem, causando perdas econômicas pela ineficácia ou alto custo de terapias existentes. Um exemplo seria a linfadenite caseosa (LC) causada por C. pseudotuberculosis, que afeta particularmente a pecuária de caprinos e ovinos, com a condenação da carcaça e redução da produção de lã e de carne. A transmissão da doença e contágio dos animais é direta, muitas vezes através da alimentação e ingestão de água em local contaminado por animais doentes. Essa doença possui incidência na pecuária mundial, principalmente de caprinos e ovinos, havendo registros de ocorrência no Brasil, Europa, Oriente Médio, Austrália, Nova Zelândia, África do Sul, Canadá e Estados Unidos e mesmo com todos os avanços tecnológicos, ainda não há métodos de prevenção totalmente eficazes, como vacinas e medicamentos, tampouco para o tratamento de animais infectados, que geralmente são de custo elevado, por longos períodos e sem a garantia de cura ou de... / Abstract: Corynebacterium pseudotuberculosis (C. pseudotuberculosis), is a gram-positive, facultative anaerobe, pleomorphic, non-sporulating bacterium with two biotopes or biovars, being the biovar equi capable of infecting horses, whereas the biotype called ovis infects small ruminants. It is part of the CMNR group (Corynebacterium, Mycobacterium, Nocardia and Rhodococcus), which demonstrates great veterinary and medical importance, since these common microorganisms infect animals and can infect humans, causing economic losses due to the inefficiency or high cost of existing therapies. An example is a caseous lymphadenitis (LC) caused by C. pseudotuberculosis, which particularly affects the goats and sheep livestock, with carcass condemnation and reduction of wool and meat production. The transmission of disease and the contagion of animals is direct, often through feeding and drinking water in places contaminated by sick animals. This disease has an incidence in the world livestock, mainly of goats and sheep, with occurrence records in Brazil, Europe, the Middle East, Australia, New Zealand, South Africa, Canada and the United States and even with all technological advances, still there are no totally effective prevention methods, such as vaccines and medications, nor for the treatment of infected animals, which are usually of a high cost, for long periods and without guarantee of cure or exemption from recurrence of LC. In this way, faster and easier techniques for the detection of ... / Doutor

Made available in DSpace on 2014-06-11T19:26:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-11-05Bitstream added on 2014-06-13T19:13:03Z : No. of bitstreams: 1 horta_ab_me_jabo.pdf: 129365 bytes, checksum: a43ecf940bba3f7b21533be60728e859 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Bioinseticidas a base de Bacillus thuringiensis são utilizados há décadas no controle de Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae) em plantios de eucalipto no Brasil. No entanto, não há relatos de quais genes cry são eficientes para o controle desta praga. Neste sentido, 74 isolados de B. thuringiensis foram caracterizados por PCR para a detecção dos genes cry1, cry1Ab e cry1Ac e sua eficiência no controle de T. arnobia verificada por bioensaios. Os isolados Br9, Br69 Br74, Br81 e R26 apresentaram elevada mortalidade e sua CL50 foi determinada. Os genes cry1Ab e cry1Ac ocorreram isoladamente em 16% e 45% dos isolados, respectivamente, quando presentes em conjunto o controle das lagartas foi superior a 50%, e os valores de CL50 foram os mais elevados. Ambos os genes ocorreram em 11% dos isolados, os níveis de controle foram superiores a 80% e a CL50 era mais baixa, o que sugere que sua presença em conjunto confere maior toxicidade. É evidente que mais estudos em relação à detecção dos genes presentes em isolados de B. thuringiensis e sua contribuição no controle para T. arnobia são fundamentais para esclarecer quais genes conferem maior toxicidade e como eles atuam isoladamente ou em conjunto no controle dessas lagartas, visando a construção de plantas geneticamente modificadas, formulação de bioinseticidas ou desenvolvimento de programas de manejo de resistência destes insetos para a cultura do eucalipto no Brasil / Biopesticides based on Bacillus thuringiensis have been used for decades to control Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae) in eucalyptus plantations in Brazil. However, there are no reports of which cry genes are efficient to control this insect pest. In this study, 74 strains of B. thuringiensis were characterized by PCR for detection of cry1, cry1Ab and cry1Ac genes and its efficiency in controlling T. arnobia was verified by bioassays. The strains Br9, Br69, Br74, Br81 and R26 showed the highest mortality rates and their LC50 was determined. The cry1Ab and cry1Ac genes occurred separately in 16% and 45% of the isolates, respectively, and when present together the larvae control was above 50%, and the LC50 values were the highest. Both genes occured in 11% of the isolates, the control levels exceeded 80% and the LC50 was lower, suggesting that their presence together confers higher toxicity. It became evident that further studies regarding the detection of the genes present in isolates of B. thuringiensis, and its contribution to control T. arnobia are essential to clarify which genes confer higher toxicity and how they act individually or together to control these caterpillars, aiming the construction of genetically modified plants, biopesticides formulation or development of more effective resistance management programs for these insects for the cultivation of eucalyptus in Brazil

Made available in DSpace on 2015-06-17T19:34:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-26. Added 1 bitstream(s) on 2015-06-18T12:49:23Z : No. of bitstreams: 1 000829428_20170301.pdf: 100482 bytes, checksum: 894af24aad4ca7d75f9898581c07f398 (MD5) Bitstreams deleted on 2017-03-03T11:01:31Z: 000829428_20170301.pdf,. Added 1 bitstream(s) on 2017-03-03T11:02:40Z : No. of bitstreams: 1 000829428.pdf: 275587 bytes, checksum: 79098a3390015f4449a4b64413d932a8 (MD5) / Estudos que visam a interação das proteínas Cry de Bacillus thuringiensis, a fim de encontrar combinações adequadas para o desenvolvimento de plantas Bt são ferramentais fundamentais no controle de lepidópteros-praga. A lagarta H. armigera causa danos severos nas culturas agrícolas e sua introdução no Brasil levou a busca de formas de controle eficientes e nesse contexto B. thuringiensis pode ser um bom agente de controle. Diante do exposto o presente trabalho objetivou avaliar a toxicidade das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca de B. thuringiensis à H. armigera, assim como interação dessas proteínas aos receptores do mesêntero do inseto. A toxicidade foi estimada com bioensaios de dose resposta com as proteínas testadas e a interação das proteínas com os receptores foram verificadas em análise de união entre a proteína ativada e marcada com a vesícula da borda em escova da membrana apical das células do intestino (brush border mambrane vesicle- BBMV) do mesêntero larval de H. armigera, e ensaios de competição heteróloga. Dentre as proteínas testadas, a Cry1Ac destacou-se como a mais efetiva, seguida das proteínas Cry1Ab e Cry1Aa. A proteína Cry1Ca não apresentou toxicidade. As proteínas Cry1Aa, Cry1Ab e Cry1Ac se ligaram aos receptores da membrana do intestino médio das lagartas de H. armigera de forma especifica. Os ensaios de competição heteróloga revelaram que as proteínas Cry1Aa, Cry1Ac e Cry1Ab competem entre si pelo mesmo receptor / Studies attempting interaction of Bacillus thuringiensis Cry proteins in order to find combinations for developing Bt plants are fundamental in controlling lepidopteran pests. H. armigera causes severe damage to agricultural crops and their introduction in Brazil has led the search for efficient control and B. thuringiensis may be a good control agent. The aim of this research was to evaluate the toxicity of Cry1Aa, Cry1Ab, Cry1Ac and Cry1Ca proteins from B. thuringiensis to H. armigera, as well as interaction of these proteins with the receptors present in insect midgut. Toxicity was estimated from the lethal concentration LC50 of the tested proteins and protein interactions with the receptors were found in a binding analysis between activated and biotinylated protein with the midgut brush border vesicle membrane (BBMV) of H. armigera, and heterologous competitive binding assays. Among the tested proteins, Cry1Ac protein was the most toxic, followed by the Cry1Ab and Cry1Aa proteins. The Cry1Ca protein showed no toxicity. The Cry1Aa, Cry1Ab and Cry1Ac proteins showed specific binding to the midgut membrane receptors of H. armigera caterpillars. Heterologous competitive binding assays revealed that Cry1Aa, Cry1Ab, Cry1Ac compete for a common receptor in the midgut larvae

Made available in DSpace on 2014-12-02T11:16:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-16Bitstream added on 2014-12-02T11:21:18Z : No. of bitstreams: 1 000794481_20150112.pdf: 297360 bytes, checksum: 35434c048800285fde551478cff3cbe1 (MD5) Bitstreams deleted on 2015-01-16T10:37:56Z: 000794481_20150112.pdf,Bitstream added on 2015-01-16T10:38:38Z : No. of bitstreams: 1 000794481.pdf: 812954 bytes, checksum: 0c134a7804e377522f7167f4a88d6aff (MD5) / O presente trabalho objetivou avaliar a toxicidade de diferentes proteínas Cry1 e Vip3Aa de Bacillus thuringiensis em larvas de Diatraea saccharalis, verificar o modo de união dessas proteínas aos receptores do inseto alvo e analisar a interação dessas proteínas no controle larval, buscando informações para subsidiar o uso de plantas geneticamente modificadas com genes cry1 e vip3Aa de forma segura e duradoura. A análise de suscetibilidade larval revelou a proteína Cry1Ab como mais efetiva no controle, seguida das proteínas Cry1Ac, Vip3Aa, Cry1Ca e Cry1Fa. A população testada não foi suscetível à proteína Cry1Ea. A proteína Cry1Aa apresentou baixa toxicidade. Nos ensaios de união específica, as proteínas Cry1 ligaram-se a receptores presentes no intestino médio de D. saccharalis e um modelo com três diferentes receptores foi proposto com base nos ensaios de competição heteróloga. Um receptor comum para Cry1Aa, Cry1Ab e Cry1Ac, outro para Cry1Fa e Cry1Ab e um receptor diferente para a proteína Vip3Aa. Dentre as interações avaliadas por bioensaios, as combinações proteicas: Cry1Ab:Cry1Ca e Cry1Fa:Cry1Ca apresentaram efeito sinérgico; as demais combinações revelaram efeitos antagônicos / The aim of this research was to evaluate the toxicity of different Cry1 and Vip3A proteins from Bacillus thuringiensis to Diatraea saccharalis, verify the proteins binding to receptors of the target insect and to analyze the protein interactions in larval control, seeking information to support safe and sustainable the use of transgenic Bt plants. The most toxic protein assayed was Cry1Ab followed by Cry1Ac, Vip3Aa, Cry1Ca and Cry1Fa. The population tested was not susceptible to Cry1Ea protein. Cry1Aa showed very low toxicity. Biotinylated Cry1 proteins showed specific binding to the midgut brush border membrane vesicles of the larvae. Heterologous competitive binding assays suggested a model of three receptors, a common receptor for Cry1Aa and Cry1Ab another one for Cry1Fa and Cry1Ab. Vip3Aa did not compete for binding with any of the Cry proteins tested. Among interactions bioassays, the combinations between Cry1Ab:Cry1Ca and Cry1Fa:Cry1Ca showed synergistic effect, whereas the other combinations showed antagonistic effects

Orientador: Manoel Victor Franco Lemos / Coorientador: Ricardo Antonio Polanczyk / Banca: Janete Apparecida Desidério / Banca: Camila Chiaradia Davolos / Resumo: Estudos que visam a interação das proteínas Cry de Bacillus thuringiensis, a fim de encontrar combinações adequadas para o desenvolvimento de plantas Bt são ferramentais fundamentais no controle de lepidópteros-praga. A lagarta H. armigera causa danos severos nas culturas agrícolas e sua introdução no Brasil levou a busca de formas de controle eficientes e nesse contexto B. thuringiensis pode ser um bom agente de controle. Diante do exposto o presente trabalho objetivou avaliar a toxicidade das proteínas Cry1Aa, Cry1Ab, Cry1Ac e Cry1Ca de B. thuringiensis à H. armigera, assim como interação dessas proteínas aos receptores do mesêntero do inseto. A toxicidade foi estimada com bioensaios de dose resposta com as proteínas testadas e a interação das proteínas com os receptores foram verificadas em análise de união entre a proteína ativada e marcada com a vesícula da "borda em escova" da membrana apical das células do intestino ("brush border mambrane vesicle"- BBMV) do mesêntero larval de H. armigera, e ensaios de competição heteróloga. Dentre as proteínas testadas, a Cry1Ac destacou-se como a mais efetiva, seguida das proteínas Cry1Ab e Cry1Aa. A proteína Cry1Ca não apresentou toxicidade. As proteínas Cry1Aa, Cry1Ab e Cry1Ac se ligaram aos receptores da membrana do intestino médio das lagartas de H. armigera de forma especifica. Os ensaios de competição heteróloga revelaram que as proteínas Cry1Aa, Cry1Ac e Cry1Ab competem entre si pelo mesmo receptor / Abstract: Studies attempting interaction of Bacillus thuringiensis Cry proteins in order to find combinations for developing Bt plants are fundamental in controlling lepidopteran pests. H. armigera causes severe damage to agricultural crops and their introduction in Brazil has led the search for efficient control and B. thuringiensis may be a good control agent. The aim of this research was to evaluate the toxicity of Cry1Aa, Cry1Ab, Cry1Ac and Cry1Ca proteins from B. thuringiensis to H. armigera, as well as interaction of these proteins with the receptors present in insect midgut. Toxicity was estimated from the lethal concentration LC50 of the tested proteins and protein interactions with the receptors were found in a binding analysis between activated and biotinylated protein with the midgut brush border vesicle membrane (BBMV) of H. armigera, and heterologous competitive binding assays. Among the tested proteins, Cry1Ac protein was the most toxic, followed by the Cry1Ab and Cry1Aa proteins. The Cry1Ca protein showed no toxicity. The Cry1Aa, Cry1Ab and Cry1Ac proteins showed specific binding to the midgut membrane receptors of H. armigera caterpillars. Heterologous competitive binding assays revealed that Cry1Aa, Cry1Ab, Cry1Ac compete for a common receptor in the midgut larvae / Mestre