Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 201-223).

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 116-129.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 155-182.

Made available in DSpace on 2014-08-13T14:50:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-12-16Bitstream added on 2014-08-13T18:01:02Z : No. of bitstreams: 1 000748612_20150116.pdf: 262543 bytes, checksum: 83687670798f532aa71d1023029e6d35 (MD5) Bitstreams deleted on 2015-01-23T10:59:20Z: 000748612_20150116.pdf,Bitstream added on 2015-01-23T10:59:52Z : No. of bitstreams: 1 000748612.pdf: 1126755 bytes, checksum: d94522b75e5a7b2b4fd318e70207535a (MD5) / Os inseticidas químicos são amplamente utilizados no controle de insetos-praga, no entanto, causam enormes prejuízos ambientais. Uma alternativa a esses inseticidas é a utilização de microrganismos entomopatogênicos, que controlam seletivamente os insetos e preservam o meio ambiente. A bactéria Bacillus thuringiensis Berliner é considerada a espécie de maior interesse neste cenário, pois é responsável pela produção de proteínas inseticida. Genes de B. thuringiensis vêm sendo amplamente estudados e utilizados na construção de culturas transgênicas no mundo todo. No entanto, quando não associadas a áreas de refúgio podem acelerar a seleção de organismos resistentes em populações de insetos-praga. Estudos relatam a necessidade em retardar a evolução da resistência a insetos-praga e dentre as possibilidades, a utilização de mais de um gene na construção de plantas transgênica mostra-se eficiente. Desta forma, o presente estudo avaliou a suscetibilidade de Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae) e Chrysodeixis includens (Walker, 1857) (Lepidoptera: Noctuidae) às proteínas de B. thuringiensis, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Ea, Vip3Aa, Vip3Ae e Vip3Af. Após estimar a CL50 de cada proteína para cada espécie avaliou-se a interação entre as combinações de Vip3A+Cry1 e Cry1+Cry1. Os resultados sugerem que todas as proteínas foram eficientes no controle de ambas as espécies, destacando-se a proteína Cry1Ac (0,75 ng.cm-2) para A. gemmatalis e a proteína Vip3Af (1,4 ng.cm-2) para C. includens. O padrão geral de comparação da suscetibilidade das espécies frente às proteínas avaliadas indicou que há uma diferença significativa na toxicidade das proteínas para cada espécie. Verificou-se uma grande inibição do desenvolvimento larval de lagartas sobreviventes a CL50 de cada proteína. As interações das proteínas indicaram ... / Chemical insecticides are widely used to control insect pests, however, cause enormous environmental damage. An alternative to these insecticides is the use of entomopathogenic microorganisms which selectively control insects and preserve the environment. The Bacillus thuringiensis Berliner is the species of greatest interest responsible for production of insecticidal proteins. Genes of B. thuringiensis have been widely studied and used in the construction of transgenic crops worldwide. However, when not associated with refuge areas can accelerate the selection of resistant organisms in populations of insect pests. Studies have reported the need to slow the evolution of resistance to insect pests and among the possibilities, the use of more than one gene in the construction of transgenic plants proves efficient. Thus, the present study evaluated the susceptibility of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae) and Chrysodeixis includens (Walker, 1857) (Lepidoptera: Noctuidae), from the proteins isolated from B. thuringiensis Cry1Aa , Cry1Ab , Cry1Ac , Cry1Ca , Cry1Ea , Vip3Aa , Vip3Ae and Vip3Af . After estimating the LC50 of each protein for each species evaluated the interaction between combinations of Vip3A + Cry1 and Cry1 + Cry1. The results suggest that all proteins were effective in controlling both species, emphasized the Cry1Ac protein (0.75 ng.cm-2) to A. gemmatalis and Vip3Af protein (1.4 ng.cm-2) to C. includens. A comparison of the susceptibility of the species to proteins indicated that there is a significant difference in toxicity for each species. There was a large inhibition of larval development of caterpillars surviving the LC50 of each protein. The interactions of proteins indicated the synergistic combinations Vip3Aa + Cry1Ea, Cry1Aa + Cry1Ab and Cry1Ea + Cry1Ac as alternatives for the control and management of resistance in A. gemmatalis and C. includens ...

Orientador: Janete Apparecida Desidério / Coorientador: Odair Aparecido Fernandes / Banca: Manoel Victor Franco Lemos / Banca: Ricardo Antonio Polanczyk / Banca: Alexandre de Sene Pinto / Banca: Ana Maria Guidelli Thuler / Resumo: Os inseticidas químicos são amplamente utilizados no controle de insetos-praga, no entanto, causam enormes prejuízos ambientais. Uma alternativa a esses inseticidas é a utilização de microrganismos entomopatogênicos, que controlam seletivamente os insetos e preservam o meio ambiente. A bactéria Bacillus thuringiensis Berliner é considerada a espécie de maior interesse neste cenário, pois é responsável pela produção de proteínas inseticida. Genes de B. thuringiensis vêm sendo amplamente estudados e utilizados na construção de culturas transgênicas no mundo todo. No entanto, quando não associadas a áreas de refúgio podem acelerar a seleção de organismos resistentes em populações de insetos-praga. Estudos relatam a necessidade em retardar a evolução da resistência a insetos-praga e dentre as possibilidades, a utilização de mais de um gene na construção de plantas transgênica mostra-se eficiente. Desta forma, o presente estudo avaliou a suscetibilidade de Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae) e Chrysodeixis includens (Walker, 1857) (Lepidoptera: Noctuidae) às proteínas de B. thuringiensis, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry1Ea, Vip3Aa, Vip3Ae e Vip3Af. Após estimar a CL50 de cada proteína para cada espécie avaliou-se a interação entre as combinações de Vip3A+Cry1 e Cry1+Cry1. Os resultados sugerem que todas as proteínas foram eficientes no controle de ambas as espécies, destacando-se a proteína Cry1Ac (0,75 ng.cm-2) para A. gemmatalis e a proteína Vip3Af (1,4 ng.cm-2) para C. includens. O padrão geral de comparação da suscetibilidade das espécies frente às proteínas avaliadas indicou que há uma diferença significativa na toxicidade das proteínas para cada espécie. Verificou-se uma grande inibição do desenvolvimento larval de lagartas sobreviventes a CL50 de cada proteína. As interações das proteínas indicaram ... / Abstract: Chemical insecticides are widely used to control insect pests, however, cause enormous environmental damage. An alternative to these insecticides is the use of entomopathogenic microorganisms which selectively control insects and preserve the environment. The Bacillus thuringiensis Berliner is the species of greatest interest responsible for production of insecticidal proteins. Genes of B. thuringiensis have been widely studied and used in the construction of transgenic crops worldwide. However, when not associated with refuge areas can accelerate the selection of resistant organisms in populations of insect pests. Studies have reported the need to slow the evolution of resistance to insect pests and among the possibilities, the use of more than one gene in the construction of transgenic plants proves efficient. Thus, the present study evaluated the susceptibility of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae) and Chrysodeixis includens (Walker, 1857) (Lepidoptera: Noctuidae), from the proteins isolated from B. thuringiensis Cry1Aa , Cry1Ab , Cry1Ac , Cry1Ca , Cry1Ea , Vip3Aa , Vip3Ae and Vip3Af . After estimating the LC50 of each protein for each species evaluated the interaction between combinations of Vip3A + Cry1 and Cry1 + Cry1. The results suggest that all proteins were effective in controlling both species, emphasized the Cry1Ac protein (0.75 ng.cm-2) to A. gemmatalis and Vip3Af protein (1.4 ng.cm-2) to C. includens. A comparison of the susceptibility of the species to proteins indicated that there is a significant difference in toxicity for each species. There was a large inhibition of larval development of caterpillars surviving the LC50 of each protein. The interactions of proteins indicated the synergistic combinations Vip3Aa + Cry1Ea, Cry1Aa + Cry1Ab and Cry1Ea + Cry1Ac as alternatives for the control and management of resistance in A. gemmatalis and C. includens ... / Doutor

Pseudomonas is a diverse genus of Gram-negative bacteria that includes pathogens of plants, insects, and humans as well as environmental strains with no known pathogenicity. Pseudomonas fluorescens itself encompasses a heterologous group of bacteria that are prevalent in soil and on foliar and root surfaces of plants. Some strains of P. fluorescens suppress plant diseases and the genomic sequences of many biological control strains are now available. I used a combination of bioinformatic and phylogenetic analyses along with mutagenesis and biological assays to identify and compare the TonB-dependent outer-membrane proteins (TBDPs) of ten plant-associated strains of P. fluorescens and related species. TBDPs are common in Gram-negative bacteria, functioning in the uptake of ferric-siderophore complexes and other substrates into the cell. I identified 14 to 45 TBDRs in each strain of P. fluorescens or P. chlororaphis. Collectively, the ten strains have 317 TBDPs, which were grouped into 84 types based upon sequence similarity and phylogeny. As many as 13 TBDPs are unique to a single strain and some show evidence of horizontal gene transfer. Putative functions in the uptake of diverse groups of microbial siderophores, sulfur-esters, and other substrates were assigned to 28 of these TBDP types based on similarity to characterized orthologs from other Pseudomonas species. Redundancy of TBDP function was evident in certain strains of P. fluorescens, especially Pf-5, which has three TBDPs for ferrichrome/ferrioxamine uptake, two for ferric-citrate uptake and three for heme uptake. Five TBDP types are present in all ten strains, and putative functions in heme, ferrichrome, cobalamin, and copper/zinc uptake were assigned to four of the conserved TBDPs. The fluorescent pseudomonads are characterized by the production of pyoverdine siderophores, which are responsible for the diffusible UV fluorescence of these bacteria. Each of the ten plant-associated strains of P. fluorescens or P. chlororaphis has three to six TBDPs with putative roles in ferric-pyoverdine uptake (Fpv). To confirm the roles of the six Fpv outer membrane proteins in P. fluorescens Pf-5, I introduced deletions into each of the six fpv genes in this strain and evaluated the mutants and the parental strain for heterologous pyoverdine uptake. I identified at least one ferric-pyoverdine that was taken up by each of the six Fpv outer-membrane proteins of Pf-5. By comparing the ferric-pyoverdine uptake assay results to a phylogenetic analysis of the Fpv outer-membrane proteins, I observed that phylogenetically-related Fpv outer-membrane proteins take up structurally-related pyoverdines. I then expanded the phylogenetic analysis to include nine other strains within the P. fluorescens group, and identified five additional types of Fpv outer-membrane proteins. Using the characterized Fpv outer-membrane proteins of Pf-5 as a reference, pyoverdine substrates were predicted for many of the Fpv outer-membrane proteins in the nine other strains. Redundancy of Fpv function was evident in Pf-5, as some pyoverdines were recognized by more than one Fpv. It is apparent that heterologous pyoverdine recognition is a conserved feature, giving these ten strains flexibility in acquiring iron from the environment. Overall, the TBDPs of the P. fluorescens group are a functionally diverse set of structurally-related proteins present in high numbers in many strains. While putative functions have been assigned to a subset of the proteins, the functions of most TBDPs remain unknown, providing targets for further investigations into nutrient uptake by P. fluorescens spp.. The work presented here provides a template for future studies using a combination of bioinformatic, phylogenetic, and molecular genetic approaches to predict and analyze the function of these TBDPs. / Graduation date: 2012

Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins. This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems. A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.