Diss. med. dent. Zürich. / Literaturverz.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 78-85).

The lcrF gene of Yersinia pestis, the etiological agent of plague, encodes a transcription activator responsible for inducing expression of several virulence-related proteins (Yops) in response to temperature. The mechanism of this thermoregulation was investigated. Using a yopE::lacZ reporter fusion, lcrF-mediated thermal regulation was observed in Y. pestis and Escherichia coli. The lcrF gene was sequenced, the 30.8 kDa. LcrF protein identified and purified, and LcrF-dependent yopE-specific DNA binding activity was detected. A sequence similarity search revealed that LcrF exhibits 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcription activators. During localization studies, a significant proportion of LcrF was found associated with the membrane fraction in E. coli. However, pulse-chase experiments indicated that this result is an artifact of fractionation. lcrF-mediated thermal induction of the yopE::lacZ reporter fusion remains intact in a Shigella flexneri virR mutant. The virR mutation is known to affect thermal induction of Shigellavirulence genes, which are also controlled by an activator in the AraC family. As a first step toward identifying the temperature-sensitive step in the regulation of yop expression, lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. To confirm these results, attempts were made to identify both the native lcrF message in Y. pestis, and a lcrF-lacZ hybrid message in Y. pestis and E. coli. These attempts were unsuccessful. Examination of LcrF protein production revealed temperature-dependent expression in Y. pestis. Surprisingly, high-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at both 26 and 37°C, suggesting that translation rate or message degradation is thermally controlled. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37°C in E. coli indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented.

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

Symbiosis is the close and protracted interaction between organisms. The molecular interactions that occur during symbiosis are complex with multiple barriers that must be overcome. Many Gram-negative, host-associated bacteria use a type III secretion system to mediate associations with their eukaryotic hosts. This secretion system is a specialized apparatus for the injection of type III effector proteins directly into host cells, which in the case of plant pathogens, are collectively necessary to modulate host defense. The type III secretion system is not a mechanism exclusive to pathogens, however, as many strains of commensal Pseudomonas fluorescens and mutualistic rhizobia demonstrably require a type III secretion system to interact with their host plants. The work presented in this thesis describes genome-enabled approaches for characterizing type III effector genes across the range of plant symbiosis. Using high-throughput sequencing technology, draft genome sequences were generated for the plant pathogen, Xanthomonas hortorum pv. carotae M081, the plant commensal, Pseudomonas fluorescens WH6, and six strains from the plant mutualists Sinorhizobium fredii and Bradyrhizobium japonicum. Analyses of the draft genome sequences and publicly available finished sequences contributed insights into mechanisms of host-association and to increasing the inventory of type III effector sequences as well as developing methods directly applicable for agriculture. Finally, characterization of the genetic diversity of type III effectors from rhizobia shows that collections of type III effectors of mutualists are static, with little diversity in content and sequence variation. This represents the first comprehensive cataloging of type III effector from species of mutualistic bacteria and the first to provide evidence for purifying selection of this important class of genes. / Graduation date: 2012

Thesis (Ph.D. in Bioinformatics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 141-150). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [100]-113).