Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 105-111). Also available via the Internet.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 73-80). Also available via the Internet.

Made available in DSpace on 2014-06-12T18:06:37Z (GMT). No. of bitstreams: 2 arquivo6316_1.pdf: 1519850 bytes, checksum: e4bd8e3bdb516150584f89ad5cae15a2 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / A atividade larvicida do Bacillus sphaericus depende da ligação da toxina binária (Bin) com um receptor específico presente no epitélio intestinal de larvas de culicídeos. Este trabalho descreve a clonagem e sequenciamento do gene deste receptor em larvas de Culex quinquefasciatus de uma colônia susceptível (CqSF) e de uma resistente (CqRL1/2362) ao Bs, para elucidar o mecanismo molecular da resistência. Ensaios de afinidade in vitro mostraram que uma proteína de 60 kDa é o receptor da toxina na colônia CqSF e nenhuma molécula funcional foi observada em preparações da colônia CqRL1/2362. O sequenciamento do gene obtido da colônia CqSF mostrou uma seqüência de 1925 pb composta por regiões 5 e 3´não traduzidas, sequência codificadora e íntron. A respectiva sequência do gene para a colônia CqRL1/2362 mostrou uma deleção de 19 nucleotídeos, geradora de uma mudança na fase de leitura de 28 aminoácidos e de um códon de terminação da tradução prematuro. A presença deste códon prematuro pode desestabilizar o respectivo mRNA, e impedir sua tradução, bem como leva a síntese de uma proteína truncada sem a âncora GPI necessária a sua localização na membrana epitelial. Em ambos os casos a ausência do receptor no epitélio intestinal seria a causa da resistência. Neste trabalho foi produzida ainda uma proteína recombinante do receptor de 45 kDa que mostrou funcionalidade, demonstrando que o sítio de ligação está localizado na porção N-terminal da molécula. Anticorpos obtidos contra esta proteína recombinante demonstraram ser uma ótima ferramenta de detecção do receptor nativo

No description available.

Techn. Universiẗat, Diss., 2003--Dresden.

Bacillus sphaericus 2362 is highly pathogenic to the larvae of mosquitos. The potential of this bacterium as a biological control agent could be improved by the availability of a stable cloning vector. Such a vector would allow the incorporation of traits into B. sphaericus which could benefit its large scale production or larvicidal characteristics. To evaluate the use of the Staphylococcus aureus plasmid pUB110 (4.5 kb) for this purpose, the stability (ability of the plasmid to remain intact and divide into daughter cells during bacterial cell division) of this plasmid and four plasmids derived from pUB110 was examined. The recombinant plasmids were developed in the laboratory of Dr. William Burke at Arizona State University. Stability was examined under various conditions of cell growth. Throughout spore germination, vegetative growth, and sporulation (spore recycling) in host mosquito larvae cadavers, pUB110 and two of the derivatives, pLDT103 (7.6 kb) and pTST130 (6.5 kb), were found to be stable (100%, 99%, and 96% respectively). Plasmids pLDT117 (9.3 kb) and pTST112 (6.5 kb) were lost from B. sphaericus in 23% (pLDT117) and 35% (pTST112) of the colonies from resulting spores examined. During the construction of these unstable plasmids, a region of DNA which binds the host cell membrane in B. subtilis was interrupted. Stability was also measured after 40 generations of vegetative growth in a chemostat. Each plasmid-bearing strain was grown at both 28°C and 38°C, with generations times of 42 min and 108 min. All plasmids were functionally stable under the conditions of extended vegetative growth that were successfully tested. The strain which carried pTST130 was unable maintain a steady growth rate in the chemostat with a generation time of 42 min. Therefore, reliable stability determinations could not be made at the higher growth rate for this strain. As through spore recycling in mosquito larvae, strains harboring pUB110, pLDT103, and pTST130 were stable throughout the growth cycle in a shaken flask. Plasmid pLDT117 was found to be stable during exponential and early stationary phase. However, 10-15% of the colonies grown from resultant spores lost neomycin resistance (conferred by the plasmid). The strain carrying pTST112 was unstable throughout the growth curve. During exponential and early stationary phases, the plasmid was present in about 85% of the cells tested. The stability reported for spores of B. sphaericus 2362 [pTST112] was 78%. / Master of Science

Mosquito pathogenic strains of Bacillus sphaericus are indistinguishable from nonpathogenic strains based on simple phenotypic tests. DNA-DNA hybridizations performed in 1980 placed the 7 pathogens included in that study in a distinct homology group separate from 5 groups of nonpathogens. The overall homology of the pathogenic strains to the species type strain was only 19% indicating that these pathogens should be a separate species. Since the DNA homology study was published in 1980, many more pathogenic strains have been isolated worldwide. Pathogenic strains have been differentiated from other strains of B. sphaericus by rRNA sequencing, fatty acid analysis, and isozyme analysis. The pathogens have been further classified by type of toxin produced, serotyping, and phage typing. I have used random amplified polymorphic DNA (RAPD) fingerprinting to determine the phenetic relationships among 31 pathogenic and 14 nonpathogenic strains of B. sphaericus. DNA Bands in agarose gel migrating the same distance were verified as being homologous using PCR-generated probes made from the RAPD bands. Band patterns resulting from 8 10-mer primers were examined by three coefficients, Jaccard, Dice, and simple matching. Each coefficient was able to distinguish DNA homology groups, although the relative similarity values differed. In agreement with DNA homology studies, pathogenic strains showed less than 10% similarity to nonpathogens using Jaccard and Dice coefficients. This value was 68% based on the simple matching coefficient. Individual serotypes were clearly indicated among the pathogenic strains by each coefficient. This suggests an overall genetic homogeneity among strains within serotypes. It also parallels the uniform toxicity pattern found within each serotype (unlike the toxin diversity found within B. thuringiensis serotypes). These results together with DNA homology data support the establishment of a new species for the pathogenic strains. / Master of Science

Made available in DSpace on 2015-04-20T12:31:08Z (GMT). No. of bitstreams: 1 eleilza.pdf: 788700 bytes, checksum: 7900cf61a2a7a4d6a517f73e27656735 (MD5) Previous issue date: 2006-04-03 / Larvicidal activity of B. sphaericus from several places in Amazonia, was estimated to Anopheles darlingi and Culex quinquefasciatus. Pobit analysis was used to determine the median lethal concentration (LC50) and potency relative to the 2362 standard strain. Findings pointed out strains IB15 (LC50 = 0.040 ppm), IB19 and S1116 (LC50 = 0.048 ppm), IB16 (LC50 = 0.052 ppm) and S265 (LC50 = 0.057 ppm) as the most effective ones, being IB15 with a potency of close to 50%, higher to strain 2362 in the assays carried out with A. darlingi. IB16 and S1116 were more potent in the experiments with C. quinquefasciatus, showing to be nearly 300-400% higher. Through molecular characterisation were diagnosed the binary toxin gene in all twenty studied stains and MTX1 toxin was found only on fourteen of them. Twenty-three polymorphic sites on the sequences were observed on the basis of the BinA sequences, being only four of them informative for the parsimony. As a whole, ten haplotypes were obtained amongst the Amazonian strains, being haplotype 1 (nine strains) similar to that of 2362 (Type 2) and the rest presenting distinct sequences. The 16 polymorphic sites observed on the aminoacid sequences resulted into 19 variant aminoacids and, on the basis of the genetic distance among the strains, it was not possible to establish a correlation between variations on BinA toxin sequences and toxicity level, as well as precedence of the strains. As B. sphaericus raises biotechnological interest in the production of biolarvicides, IB15 strain microbial growth in NYSM medium during 24h of fermentation, was studied. It was found that IB15 presented a growth profile similar to 2362 strain to prduction of cells, spores, biomass and larvicidal activity throughout fermentation, with no statistically significant differences. At the end, 1.61x109 spores.mL-1 was obtained with IB15 and 6.46x108 spores.mL-1 with 2362. LT50 values were similar, being in average 2.5h in the experiments with one culture of the bacillus at the end of 24 hours. Strain IB15 showed to be suitable for growth in NYSM medium, presenting desirable levels of production of spores and toxins throughout fermentation. Further studies are needed on the large-scale production with that strain for the development of biolarvicides. Therefore, with the findings obtained in this study, one verifies the great importance of mosquito biological control with the use of entomopathogenic bacteria, showing to be a viable complement or alternative on the control of vector mosquitoes in Amazonia. / A atividade larvicida de B. sphaericus, procedentes de diversas localidades da Amazônia, foi estimada por meio de bioensaios com Anopheles darlingi e Culex quinquefasciatus. Pela análise de Probit foi determinada a concentração letal mediana (CL50) e a potência em relação à estirpe padrão 2362. Os resultados apontaram as estirpes IB15 (CL50 = 0,040 ppm), IB19 e S1116 (CL50 = 0,048 ppm), IB16 (CL50 = 0,052 ppm) e S265 (CL50 = 0,057 ppm) como mais efetivas, sendo IB15 com potência cerca de 50% superior à estirpe 2362 nos bioensaios realizados contra A. darlingi e IB16 e S1116 mais potentes nos testes com C. quinquefasciatus, mostrando-se cerca de 300-400% superiores. Na caracterização molecular o gene da toxina binária foi diagnosticado em todas as estirpes estudadas e a toxina MTX1 foi observada em apenas quatorze estirpes. Com base nas sequências de BinA, foram observados 23 sítios polimórficos nas sequências, sendo apenas quatro informativos para parcimônia. Ao total obteve-se dez haplótipos entre as estirpes da Amazônia, sendo o haplótipo 1 (nove estirpes) similar a 2362 (Tipo 2) e o restante com sequências distintas. Os 16 sítios polimórficos observados nas sequências de aminoácidos resultaram em 19 aminoácidos variantes e, com base na distância genética, não foi possível estabelecer uma correlação entre variações nas sequências da toxina BinA e o nível de toxicidade, bem como a procedência das estirpes. Como B. sphaericus desperta um interesse biotecnológico na produção de biolarvicidas para controle de mosquitos vetores, foi estudado o crescimento microbiano da estirpe IB15 em meio NYSM, durante 24 horas de fermentação. IB15 apresentou um perfil de crescimento semelhante à estirpe 2362 quanto às produções de células, esporos, biomassa e em atividade larvicida ao longo da fermentação. Ao final, obteve-se 1,61x109 esporos.mL-1 com IB15 e 6,46x108 esporos.mL-1 com 2362. Os valores de TL50 foram semelhantes, sendo em média 2,5h nos testes com uma cultura do bacilo ao final de 24 horas. A estirpe IB15 demonstrou ser adequada para crescimento em meio NYSM, apresentando níveis desejados de produção de esporos e toxinas ao longo da fermentação. São necessários estudos complementares sobre produção em grande escala com essa estirpe para o desenvolvimento de biolarvicidas. Portanto, com os resultados obtidos nesse trabalho, verifica-se a grande importância do controle biológico de mosquitos com o emprego de bactérias entomopatogênicas, mostrando ser uma alternativa ou complemento viável no controle de mosquitos vetores na Amazônia.

Biocere verkörpern eine neue Klasse von keramischen Funktionswerkstoffen, deren Eigenschaften entscheidend von den konkreten Herstellungsbedingungen beeinflusst werden. Am Beispiel ausgewählter Biocere mit immobilisierten vegetativen Zellen und Sporen von Bacillus sphaericus JG-A12 wurden im Rahmen der vorliegenden Arbeit Zusammenhänge zwischen Gefügeausbildung und Funktionalität, besonders unter dem Gesichtspunkt metallbindender Eigenschaften, untersucht. Mit Hilfe von Fluoreszenzfarbstoffen konnte die räumliche Verteilung der Biokomponente in der Matrix, die Überlebensfähigkeit der immobilisierten vegetativen Zellen und das Verhalten von Bakteriensuspensionen in Gegenwart von verschiedenen Metallionen untersucht werden. Mit ionensensitiven Fluoreszenzfarbstoffen wurde die Aufnahme von Kupfer(II) und Nickel(II) in lebende Bakterien unter Kontrolle des physiologischen Status der Zellen verfolgt. Es wurde nachgewiesen, dass die Aufnahme von Metallionen von der Erhöhung des intrazellulären Ca2+-Spiegels begleitet und die Aufnahmeraten sowie die Ca2+-abhängige Zellantwort stammspezifisch und von der Konzentration der Metallsalzlösung abhängig sind. Durch Immobilisierung bakterieller Sporen in dünnen Sol-Gel-Schichten konnten langzeitlagerfähige Biokeramiken erzeugt werden, die sich durch die Aktivierung mit einem 1:1 Komplex aus Ca2+ und Dipicolinsäure gezielt aus Ruhephasen in biologisch aktive Zustände schalten ließen. Im Durchschnitt führt die vorherige Aktivierung der Sporen mit Ca2+-DPA zu einer fünf- bis achtfach erhöhten Keimungsrate. Mit Hilfe einer kapazitiven Messanordnung wurden darüber hinaus an diesen Schichten während der Keimung auftretende mechanische Spannungszustände nachgewiesen und quantifiziert. Biocere unterliegen während ihrer Nutzung als Filtermaterialien überwiegend durch mikrobielle Einflüsse hervorgerufenen Materialveränderungen, die mit licht- und rasterelektronenmikroskopischen Untersuchungen nachgewiesen wurden. Diese Untersuchungen sind als Teil eines Monitoring-Systems zur kontinuierlichen Überwachung von Filtersystemen denkbar. / Biocers embody a new class of ceramic functional materials in which the properties are mainly determined by the specific production conditions. The interplay between structure formation and functionality of selected biocers containing immobilised vegetative cells and spores of Bacillus sphaericus JG-A12 was investigated under special consideration of metal binding properties. Spatial distribution of the bio-component inside the matrix, the viability of the immobilised vegetative cells, as well as the behaviour of cell suspensions in the presence of different metal ions were examined using fluorescence dyes. The uptake of copper(II) and nickel(II) ions into the living cells while monitoring of their physiological state were detected using different ion-sensitive fluorescence dyes. As expected, the uptake of metal ions was thereby accompanied by an increase of the intracellular Ca2+ level. Further, the uptake rate of metal ions and the Ca2+ dependent cellular reaction are strain-specific and depend on the metal ion concentration. Bioceramics suitable for long-term storage were produced through immobilisation of bacterial spores in thin sol-gel layers. The switch from the metabolic inactive to the active state of the bio-component was achieved by the activation with a 1:1 ratio of the chelate of Ca2+ and dipicolinic acid. The average germination rate after activation with Ca2+-DPA was increased by five- to eightfold. The mechanical stress conditions during the germination of spores inside these silica layers were measured and quantified using a capacitive deflection measurement. As filter materials, biocers are affected by microbial determined degrading impacts. Material alterations could be detected by light and scanning electron microscopic methods. Such investigations should be considered part and parcel of monitoring systems required for continuous checks of filter systems.

Biocere verkörpern eine neue Klasse von keramischen Funktionswerkstoffen, deren Eigenschaften entscheidend von den konkreten Herstellungsbedingungen beeinflusst werden. Am Beispiel ausgewählter Biocere mit immobilisierten vegetativen Zellen und Sporen von Bacillus sphaericus JG-A12 wurden im Rahmen der vorliegenden Arbeit Zusammenhänge zwischen Gefügeausbildung und Funktionalität, besonders unter dem Gesichtspunkt metallbindender Eigenschaften, untersucht. Mit Hilfe von Fluoreszenzfarbstoffen konnte die räumliche Verteilung der Biokomponente in der Matrix, die Überlebensfähigkeit der immobilisierten vegetativen Zellen und das Verhalten von Bakteriensuspensionen in Gegenwart von verschiedenen Metallionen untersucht werden. Mit ionensensitiven Fluoreszenzfarbstoffen wurde die Aufnahme von Kupfer(II) und Nickel(II) in lebende Bakterien unter Kontrolle des physiologischen Status der Zellen verfolgt. Es wurde nachgewiesen, dass die Aufnahme von Metallionen von der Erhöhung des intrazellulären Ca2+-Spiegels begleitet und die Aufnahmeraten sowie die Ca2+-abhängige Zellantwort stammspezifisch und von der Konzentration der Metallsalzlösung abhängig sind. Durch Immobilisierung bakterieller Sporen in dünnen Sol-Gel-Schichten konnten langzeitlagerfähige Biokeramiken erzeugt werden, die sich durch die Aktivierung mit einem 1:1 Komplex aus Ca2+ und Dipicolinsäure gezielt aus Ruhephasen in biologisch aktive Zustände schalten ließen. Im Durchschnitt führt die vorherige Aktivierung der Sporen mit Ca2+-DPA zu einer fünf- bis achtfach erhöhten Keimungsrate. Mit Hilfe einer kapazitiven Messanordnung wurden darüber hinaus an diesen Schichten während der Keimung auftretende mechanische Spannungszustände nachgewiesen und quantifiziert. Biocere unterliegen während ihrer Nutzung als Filtermaterialien überwiegend durch mikrobielle Einflüsse hervorgerufenen Materialveränderungen, die mit licht- und rasterelektronenmikroskopischen Untersuchungen nachgewiesen wurden. Diese Untersuchungen sind als Teil eines Monitoring-Systems zur kontinuierlichen Überwachung von Filtersystemen denkbar. / Biocers embody a new class of ceramic functional materials in which the properties are mainly determined by the specific production conditions. The interplay between structure formation and functionality of selected biocers containing immobilised vegetative cells and spores of Bacillus sphaericus JG-A12 was investigated under special consideration of metal binding properties. Spatial distribution of the bio-component inside the matrix, the viability of the immobilised vegetative cells, as well as the behaviour of cell suspensions in the presence of different metal ions were examined using fluorescence dyes. The uptake of copper(II) and nickel(II) ions into the living cells while monitoring of their physiological state were detected using different ion-sensitive fluorescence dyes. As expected, the uptake of metal ions was thereby accompanied by an increase of the intracellular Ca2+ level. Further, the uptake rate of metal ions and the Ca2+ dependent cellular reaction are strain-specific and depend on the metal ion concentration. Bioceramics suitable for long-term storage were produced through immobilisation of bacterial spores in thin sol-gel layers. The switch from the metabolic inactive to the active state of the bio-component was achieved by the activation with a 1:1 ratio of the chelate of Ca2+ and dipicolinic acid. The average germination rate after activation with Ca2+-DPA was increased by five- to eightfold. The mechanical stress conditions during the germination of spores inside these silica layers were measured and quantified using a capacitive deflection measurement. As filter materials, biocers are affected by microbial determined degrading impacts. Material alterations could be detected by light and scanning electron microscopic methods. Such investigations should be considered part and parcel of monitoring systems required for continuous checks of filter systems.