Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 116-125.

Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i_b), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i_a) antigen, had little to no biological activity. When fractions containing i_a and i_b were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i_a or i_b, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i_a and i_b, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i_a and i_b were mixed. Monospecific antisera against purified i_a and i_b neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i_a and i_b proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i_a protein is heat stable (85° C/15 min) while i_b lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i_a, but not i_b, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor. / Ph. D.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The biobutanol production by fermentative process has a great importance to increase the global supply of biofuel and becomes these able to replace the use of fossil fuel. The main difficulty associated to this production occurs due the not economic viability of applied production process. The aspects that have more contribution to this are the product inhibition at low concentration, low titer and the use of expensive substrates. This work is divided in four scientific articles which are focused in question involved to this solvent production. The first two are review papers about the topic, whereas the last two are research papers related to development of analytical methods and production process. The first paper reports to the main process development since 1980 year, by analyses of registered patents in relation to butanol production worldwide. The second paper presents a review from scientific articles about butyric fermentation published in recent years. The central characteristic of it is show the main troubles related to production, exhibiting the importance of the used substrate, as well as the choice of microorganism and separation process. Third paper presents a methodology to solvents determination from fermentation medium. This technique proposes a linear relationship between the density variation, sugar and solvents concentrations. The method proposed showed good results being promising to predict the ABE concentration in an easy and fast procedure. Fourth paper reports the development of the process to production of biobutanol by clostridial fermentation from sweet sorghum juice. Butanol is produced from substrate and small addition of yeast extract and tryptone, using 12.5% of initial inoculums size, at initial pH pH value equal to 5.5. In this work was demonstrated the possibility to produce biobutanol from sweet sorghum. / A produção de butanol a partir de processos fermentativos é de fundamental importância para aumentar a oferta mundial de biocombustíveis e permitir que estes substituam o uso de combustíveis fósseis. A principal dificuldade em relação a esta produção ocorre devido a não viabilidade econômica dos processos de produção aplicados. Os aspectos que mais contribuem para isto são a inibição pelo produto a baixas concentrações, baixa produtividade e uso de substratos caros. Este trabalho está dividido em quatro artigos científicos que estão voltados a questões envolvidas com a produção deste biocombustível. Os dois primeiros fazem uma revisão da literatura científica sobre o tópico, já os últimos são trabalhos científicos de desenvolvimento de metodologias e processos. O artigo 1 traz um ponto de vista em relação ao desenvolvimento do processo, desde o ano de 1980, através da análise das patentes registradas sobre produção de butanol no mundo. Além disso, a partir dos resultados é possível prever, parcialmente, como a tecnologia deverá avançar nos próximos anos. O artigo 2 faz uma revisão dos artigos científicos publicados sobre o fermentação butílica nos últimos tempos. A principal característica do mesmo é apontar os principais problemas relacionados à produção, mostrando a importância dada ao substrato utilizado, o microorganismo e os processos de separação. No artigo 3 uma metodologia para determinação de solventes no meio de fermentação é desenvolvida. Esta técnica propõe uma relação linear entre a variação da densidade, a concentração de açúcar e a concentração de solventes. Com sua aplicação o cromatógrafo pode ser dispensado e há somente a necessidade de um densímetro. O ajuste se mostrou bastante promissor e aparentemente capaz de predizer os resultados. Por fim, no artigo 4 é desenvolvido um processo para a produção de biobutanol via fermentação por clostridium a partir de sorgo sacaríneo. Butanol é produzido a partir do substrato sendo necessário um acréscimo pequeno de extrato de levedura e triptona com apenas 12,5% de volume de inóculo, com pH inicial ajustado em 5,5. Para a execução dos experimentos em meio anaeróbio foram elaborados aparatos alternativos e de baixo custo, que demonstraram ser eficientes na sua função. O principal ponto observado durante o trabalho é que é possível produzir biobutanol a partir de sorgo sacaríneo utilizando artefatos fabricados no laboratório para manutenção do meio anaeróbio.

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD), and spontaneous diarrhea (SD). In enterotoxigenic type A isolates, the cpe gene is found on the chromosome in food poisoning isolates, but is present on a large virulence plasmid in AAD and SD type A isolates. Food poisoning cases typically exhibit shorter duration of infection and less severe GI symptoms than AAD or SD. Since previous epidemiological evidence has linked the newly discovered beta2-toxin (CPB2) to gastroenteritis in pigs, horses, and chickens, we hypothesize that the CPB2 toxin may be an accessory toxin when cpe positive type A isolates cause human AAD or SD. In the current study, the presence and expression of CPE and CPB2 were assessed in 44 C. perfringens type A human fecal isolates associated with GI diseases in the United Kingdom. Polymerase chain reaction (PCR) and restriction fragment length polymorphisim (RFLP) confirmed the presence of the cpe (32%) and cpb2 (39%) genes. Furthermore, pulsed field gel electrophoresis (PFGE) and I-CeuI RFLP PFGE Southern blot analysis was used to show the localization of the cpe and cpb2 genes, as well as to determine that there was no clonal relationship between the isolates. All surveyed cpb2-positive isolates were determined to carry their cpb2 gene on a large plasmid that was estimated to be the similar size of the cpe large plasmid. Finally, CPE and CPB2 Western blotting demonstrated that all cpe-positive isolates expressed CPE and that all cpb2-positive isolates expressed CPB2. This study identified, for the first time, the C. perfringens non-food-borne human GI disease isolates carrying both the cpe and cpb2 genes (18%), and these isolates all actively expressed both CPE and CPB2. It was also shown that, although CPE expression occurs only under sporulation conditions, CPB2 expressed both in vegetative and sporulation conditions. The CPB2 made by two of these cpe /cpb2 - positive isolates was determined to be very (-99%) similar to the deduced amino acid sequence of the biologically-active CPB2 made by the original type C isolate CWC245. Finally, the expression of CPB2 by only type A isolates carrying the cpe gene on a plasmid and not the isolates carrying a chromosomal cpe gene, could possibly explain the increased GI symptoms and disease duration associated with these non-food-borne GI diseases. Collectively, the current results support a significant association between cpb2-positive C. perfringens isolates and non-food-borne GI disease in human. / Graduation date: 2004

The increased demand for an alternative form of fuel has raised a great interest towards exploring various metabolic pathways and enzymes in several microbial species for hydrocarbon production. In recent years, cyanobacteria have emerged as an attractive microbial host and cyanobacterial metabolic pathways were targeted for engineering to produce "drop in" fuels such as propane and butane. Whilst appealing, practicalities for producing biofuels in cyanobacteria remain challenging, requiring the identification and engineering of natural biocatalysts and their integration into metabolic processes. Cyanobacterial hydrocarbon biosynthesis arises from fatty acid metabolism involving a potential enzyme, aldehyde deformylating oxygenase (ADO), which catalyses the decarbonylation of long-chain fatty aldehydes to alkanes, mainly in the conversion of octadecanal (C17H35CHO) to heptadecane (C17H36) and formate. The substrate specificity and preferences for long-chain aldehyde by ADO necessitates a detailed kinetic and structural characterisation in order to optimise/engineer this enzyme for future biotechnological applications. Thus, the main objective was to identify a potential ADO enzyme that can be optimised for shorter chain alkane production. By studying the substrate specificity and reaction kinetics of different ADO enzymes, it was found that ADO from Prochlorococcus marinus MIT 9313 (PmADO) is a potential target for short chain alkane production. The crystal structural of PmADO was solved and further GC-MS analysis was carried out to identify the chemical origin of a mixture of long-chain fatty acid in the active site, originated from E. coli cells during recombinant over-expression and purification. It was suggested that the structure-guided protein engineering for short-chain alkane production should be carried out along with the removal of this adventitious ligand from the active site in order to increase the alkane production. Four important residues present at the entrance of the ligand-binding cavity were targeted and saturated mutagenesis was performed on PmADO to identify variants that excluded the long fatty acid ligands from the active site but have specificity and higher conversion rates for shorter chain aldehydes. This identified two variants, V41Y and A134F, with the A134F variant that not only exhibiting an improved activity and turnover value of PmADO by four-fold but also improved binding affinity for butyraldehyde by 2 times. Finally the improved variants were incorporated in a host organism (E. coli) and the possibilities for the development of a microbial platform for renewable propane synthesis based on a fermentative clostridial butanol pathway were explored. Four pathways were designed namely atoB-adhE2, atoB-TPC7, nphT7-adhE2 and nphT7-TPC7 routes, which utilise CoA intermediates selected to incorporate ADO as the terminal enzyme. When PmADO was co-expressed with these pathways, the engineered E. coli host produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed A134F variant ofPmADO with an enhanced specificity towards short-chain substrates, and (iii) including a ferredoxin-based electron supply system, the propane titre was increased up to 3.40 ± 0.19 mg/L. It was also shown that the best propane producing pathways are scalable in a 250 mL flask and in a large-scale (up to 30 L) fermentor setup. This thesis focuses on the detailed kinetics and structure-guided characterisation and engineering studies on the ADO enzyme for the development of a renewable microbial biofuel platform.

Orientador: Oswaldo Durival Rossi Junior / Banca: Luiz Augusto do Amaral / Banca: Ana Maria Centola Vidal Martins / Resumo: A deterioração "blown pack" é caracterizada por abundante produção de gás, induzindo a completa distensão da embalagem durante o processo de estocagem sob refrigeração. Quando a embalagem é aberta, há um odor desagradável, levemente fecal. O gás presente na embalagem é composto por dióxido de carbono e hidrogênio e por vários tipos butíricos do metabolismo fermentativo. O objetivo deste experimento foi determinar possíveis causadores deste tipo de deterioração, quantificando as populações de bactérias da família Enterobacteriaceae, e caracterizando-as nos principais gêneros e espécies encontradas, o número de bactérias ácido-lácticas, a freqüência de Clostridium estertheticum e do Clostridium gasigenes, em carnes próprias para o consumo e em carnes que apresentaram a deterioração "blown pack". Para contagem e identificação dos membros da família Enterobacteriaceae e contagem de bactérias ácido-lácticas utilizou-se de técnicas microbiológicas clássicas. Já para pesquisa do C. estertheticum e C. gasigenes fez-se uso de técnicas de biologia molecular. Os microrganismos da família Enterobacteriaceae e bactérias ácido-láticas estavam presentes em populações elevadas e em maior número nas carnes com deterioração "blown pack". A espécie mais freqüentemente encontrada foi a Hafnia alvei. As amostras com deterioração "blown pack' apresentaram maior positividade para o C. estertethicum que amostras não deterioradas. Não houve diferença estatística de positividade para a presença do C. gasigenes entre amostras com deterioração "blown pack" e carnes não deterioradas. A principal forma de controle desta deterioração é a prevenção da contaminação da carne por material fecal. / Abstract: The "blown pack" spoilage is characterised by abundant gas production, leading to complete gross distention pack during refrigerated storage. When the packaging is opened, there is an unpleasant smell, lightly fecal. The gas present in the package is composed of carbon dioxide and hydrogen and also of several butyric types of metabolism fermentation. The purpose of this experiment was to determine possible causes of this spoilage type, quantifying the populations of bacteria of the family Enterobacteriaceae, and characterizing them in the major genera and species found, the number of lactic acid bacteria, the frequency of Clostridium estertheticum and Clostridium gasigenes in meat proper for consumption and meat which showed the "blown pack" spoilage. In order to enumerate and identify the members of the Enterobacteriaceae family, and to enumerate the lactic acid bacteria the procedure was classical microbiological techniques. However to search the C. estertheticum and C. gasigenes the procedure was molecular biology techniques. The microorganisms of the family Enterobacteriaceae and lactic acid bacteria were present in large populations and in greater numbers in meat with "blown pack" spoilage. The species which were found more often was the Hafnia alvei. Samples of "blown pack" spoilage had greater positive features for C. estertethicum than samples not damaged. There was no statistical difference of positive features for the presence of C. gasigenes between samples of "blown pack" spoilage and not damaged meat. The main way to control this spoilage is the prevention of contamination of meat by fecal material. / Mestre

Determina la asociación entre FTP y la mortalidad por enterotoxemia en alpacas neonatas. La toma de muestras se realiza en comunidades y centro de producción de los departamentos de Cusco y Puno. Se realiza la cuantificación de niveles de IgG de 17 animales casos y 26 controles usando el test de Inmunodifusión Radial (IDR) mediante el método de Mancini y se confronta con una curva estándar de cinética de degradación de IgG en crías de alpaca normales. Mediante la prueba de Odds Ratio se determina que no hay una asociación estadísticamente significativa entre FTP y Enterotoxemia. / Tesis

Made available in DSpace on 2014-06-11T19:27:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-06Bitstream added on 2014-06-13T19:35:10Z : No. of bitstreams: 1 felipe_lm_me_jabo.pdf: 356871 bytes, checksum: 16d6d5f606db8c2a71fd32fc4c6570dc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A deterioração “blown pack” é caracterizada por abundante produção de gás, induzindo a completa distensão da embalagem durante o processo de estocagem sob refrigeração. Quando a embalagem é aberta, há um odor desagradável, levemente fecal. O gás presente na embalagem é composto por dióxido de carbono e hidrogênio e por vários tipos butíricos do metabolismo fermentativo. O objetivo deste experimento foi determinar possíveis causadores deste tipo de deterioração, quantificando as populações de bactérias da família Enterobacteriaceae, e caracterizando-as nos principais gêneros e espécies encontradas, o número de bactérias ácido-lácticas, a freqüência de Clostridium estertheticum e do Clostridium gasigenes, em carnes próprias para o consumo e em carnes que apresentaram a deterioração “blown pack”. Para contagem e identificação dos membros da família Enterobacteriaceae e contagem de bactérias ácido-lácticas utilizou-se de técnicas microbiológicas clássicas. Já para pesquisa do C. estertheticum e C. gasigenes fez-se uso de técnicas de biologia molecular. Os microrganismos da família Enterobacteriaceae e bactérias ácido-láticas estavam presentes em populações elevadas e em maior número nas carnes com deterioração “blown pack”. A espécie mais freqüentemente encontrada foi a Hafnia alvei. As amostras com deterioração “blown pack’ apresentaram maior positividade para o C. estertethicum que amostras não deterioradas. Não houve diferença estatística de positividade para a presença do C. gasigenes entre amostras com deterioração “blown pack” e carnes não deterioradas. A principal forma de controle desta deterioração é a prevenção da contaminação da carne por material fecal. / The blown pack spoilage is characterised by abundant gas production, leading to complete gross distention pack during refrigerated storage. When the packaging is opened, there is an unpleasant smell, lightly fecal. The gas present in the package is composed of carbon dioxide and hydrogen and also of several butyric types of metabolism fermentation. The purpose of this experiment was to determine possible causes of this spoilage type, quantifying the populations of bacteria of the family Enterobacteriaceae, and characterizing them in the major genera and species found, the number of lactic acid bacteria, the frequency of Clostridium estertheticum and Clostridium gasigenes in meat proper for consumption and meat which showed the blown pack spoilage. In order to enumerate and identify the members of the Enterobacteriaceae family, and to enumerate the lactic acid bacteria the procedure was classical microbiological techniques. However to search the C. estertheticum and C. gasigenes the procedure was molecular biology techniques. The microorganisms of the family Enterobacteriaceae and lactic acid bacteria were present in large populations and in greater numbers in meat with blown pack spoilage. The species which were found more often was the Hafnia alvei. Samples of blown pack“ spoilage had greater positive features for C. estertethicum than samples not damaged. There was no statistical difference of positive features for the presence of C. gasigenes between samples of blown pack spoilage and not damaged meat. The main way to control this spoilage is the prevention of contamination of meat by fecal material.

Erst kürzlich wurde der Lipolyse-stimulierte Lipoproteinrezeptor (LSR, engl. lipolysis-stimulated lipoprotein receptor) als der zelluläre Oberflächenrezeptor von CDT und Iota-Toxin, zweier Vertreter der Iota-Toxin-Familie der clostridialen Aktin-ADP-ribosylierenden Toxine, identifiziert. In dieser Arbeit sollte geprüft werden, ob CST, ein weiterer Vertreter der Iota-Toxin-Familie, ebenfalls LSR für den Zelleintritt nutzt. Zunächst wurden die Toxinkomponenten CSTa und CSTb erstmals rekombinant hergestellt. Dazu wurden die für CSTa und CSTb codierenden Genabschnitte mittels PCR amplifiziert und anschließend in einen Expressionsvektor kloniert. Als Expressionsvektor wurde in dieser Arbeit der pHis1522-Vektor verwendet. Zur Amplifizierung wurden die Plasmide in E. coli transformiert und anschließend aufgereinigt. Die Proteinexpression erfolgte in B. megaterium, weil dieses Bakterium sich bereits zur Expression anderer clostridialer Toxine bewährt hatte. Zur Aufreinigung der 6xHis-getaggten Proteine wurde die Nickel-Affinitätschromatographie eingesetzt. Als nächstes wurde gezeigt, dass die rekombinant hergestellten Toxinkomponenten CSTa und CSTb biologisch aktiv waren. Dazu wurden CaCo2-Zellen mit CST behandelt und anschließend die Morphologie der Zellen untersucht. CaCo2-Zellen, die mit CSTa und CSTb behandelt wurden, wiesen Vergiftungserscheinungen wie eine typische Zellabrundung auf. Mit dem „Aktin-Nach-ADP-Ribosylierungs-Assay“ und der fluoreszenzmikroskopischen Untersuchung von TRITC-Phalloidin-gefärbtem Aktin wurde gezeigt, dass das rekombinant hergestellte CST Aktin-ADP-ribosylierende Eigenschaften besaß. Nachdem gezeigt war, dass rekombinant hergestelltes CST sich wie ein biologisch aktives, binäres Aktin-ADP-ribosylierendes Toxin verhält, konnte mithilfe der Vergiftung von H1-HeLa(+LSR)-Zellen und nativen H1-HeLa-Zellen, die kein LSR exprimierten, nachgewiesen werden, dass die Wirkung des Toxins LSR-abhängig ist. FACS-Analysen und Kolokalisationsstudien mit Alexa488-gefärbtem CSTb und Antikörper-gefärbtem LSR erbrachten zusätzlich den Beweis, dass CSTb auf der Zelloberfläche an LSR bindet und bei der Aufnahme in die Zellen mit LSR in endozytischen Vesikeln kolokalisiert. Die Ergebnisse dieser Arbeit zeigen, dass das C. spiroforme Toxin (CST) ebenfalls LSR als Rezeptor für den Zelleintritt verwendet.