Orientador: Wanderley Dias da Silveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T17:38:26Z (GMT). No. of bitstreams: 1 Stehling_ElianaGuedes_D.pdf: 4279256 bytes, checksum: 060a8c82e3c0a43c76a25979525911e0 (MD5) Previous issue date: 2003 / Doutorado

Orientadores: Antonio Fernando Pestana de Castro, Jorge Blanco Alvarez / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T00:21:47Z (GMT). No. of bitstreams: 1 Ugrinovich_LeilaAidar_D.pdf: 8373928 bytes, checksum: aa5a6acc9829d07ea484dd4d083b5fa2 (MD5) Previous issue date: 2004 / Resumo: Foram isoladas 98 amostras de Escherichia coli, portadoras dos genes eae, stx, ou ambos, entre 264 bezerros diarréicos e 282 bezerros sadios, com idade entre 4 a 5 meses. Uma vez positivas em PCR para 1 ou ambos os genes, as amostras foram estudadas quanto presença de genes que codificam para enterotoxinas e citotoxinas: Termo lábil I (LT-I), Termo lábil li (LT-lI), Termo estável a (Sta), Termo estável b (STh), Fator necrotizante citotóxico 1 (CNF1), Fator necrotizante citotóxico 2 (CNF2), EaggEC heat-stable toxin 1 (EAST1) e Enterohemolisina (Eh1y), sendo que Eh1y foi testado também quanto a sua expressão, em ágar sangue. Entre os animais diarréicos, obtivemos 27 animais (10,2%) stx positivos e 15 animais (5,68%) eae positivos e entre os animais sadios, obtivemos 22 (7,8%) stx positivos e 17 (6,03%) eae positivos. Genes para CNF foram encontrados entre 5 (1,9%) animais diarréicos e 4 (1,4%) animais não diarréicos. LT-lI foi encontrada em uma única amostra isolada de um animal diarréico. Ehly foi encontrada em 20 (7,6%) animais diarréicos e 22 (7,8%) animais sadios. O gene ast1 foi encontrado em apenas 2 animais, sendo um diarréico e um sadio. Dos genes que codificam para as adesinas pesquisadas (BFP e Saa), somente saa foi encontrado (22mnostras),sendo-queoplasmídio EAF não esteve presente. As amostras eae+ foram pesquisadas através de PCR quanto ao subtipo de intimina apresentado, sendo que 17 amostras foram positivas para o subtipo 131, 15 amostras foram positivas para o subtipo y2, 3 amostras foram positivas para E, 2 amostras apresentaram-se ç+ e 1 amostra apresentou-se µ1+. As 10 amostras restantes apresentaram intimina Não Tipável. Também testamos estas amostras quanto à produção da lesão AlE, através do teste de F AS, sendo que 33 amostras apresentaram-se positivas neste teste, 10 apresentaram-se negativas e 5 não puderam ser testadas, pois não aderiram à linha celular HEp-2. Quando testamos estas amostras eae+ quanto à inserção de LEE, a grande maioria (37 amostras) apresentou pheU interrompido, indicando uma provável presença de LEE inserido neste locus. Todas as amostras foram testadas quanto à aderência em células HEp-2. Dez amostras apresentaram aderência Difusa (10,20%), 14 apresentaram aderência Agregativa (14,3%), 17 apresentaram aderência Localizada-like (17,3%), 26 apresentaram aderência Não Característica (26,5%) e 25 (25,5%) foram negativas para este teste. Uma amostra demonstrou aderência do tipo Cover Slip (1%) e 5 amostras provocaram descolamento do tapete celular (5%). O teste de Separação Imuno-Magnética (SIM) não favoreceu o isolamento de nenhuma amostra de E. coZi do sorogrupo 0157 entre as 50 amostras de carne bovina crua e 50 amostras fecais testadas / Abstract: Ninety eight strains of Escheríchía colí fTom 264 diarrheic calves and 282 hea1thy ones were studied. Screening was made based on the presence of genes eae, stx or both. These 98 strains were also tested for presence of genes encoding toxins: LT-I, LT-II, STa, STh, CNFl, CNF2, EASTl and Ehly. Twenty seven diarrheic calves (10,2%) were positive for stx genes and 15 (5,7%) were positive for eae gene. Among the hea1thy calves twenty two (7,8%) were positive to stx genes and 17 (6,0%) were positive to eae gene. The genes for CNF were isolated fTom 5 (1,9%) diarrheic calves and 4 (1,4%) non diarrheic ones calves. Only one strain Itll+ was isolated. The Eh/y was founded in 20 (7,6%) diarrheic calves and in 22 (7,8%) healthy caIves. When the 98 strains were tested for presence b.fp, eaf or saa genes, we only found 22 saa positive strains. From eae+ strains studied,17 were positive togene for _1 intimin subtype, 15 were positive to y2 subtype, 3 were positive to & subtype, 2 were positive to ç; subtype and only one strain was positive to the gene encoding to J.I. subtype. The 10 remaining strains were non-typable. Among the eae+ strains, 33 were positive in the Fluorescence Actin Staining test (FAS) and 14 strains react with anti intimin antisera used in Westem blot. These 48 eae+ strains were tested by PCR to determine the local of LEE insertion into the E. colí chromosome. pheU interrupted was observed in 37 strains indicating that LEE is probably inserted in this locus. By the cell culture assay in HEp-2 cells, 10 strains (10,2%) showed diffuse adesion (DA), 14 enteroaggregative adhesion (14,3%), 17 (17,3%) localized like adhesion (LAL), 26 (26,53%) strains showed no characteristic adhesion (NC), 1 strain showed cover slip (CS) adhesion and 25 strains were no adherent to HEp-2 cells. No 0157 strains were isolated by Imunno Magnetic Separation (IMS), either fTom the 50 raw meat or fecaI specimens examined. / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Orientador: Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-25T11:53:01Z (GMT). No. of bitstreams: 1 Catani_CleideFerreira_D.pdf: 5873013 bytes, checksum: 5656176a69a0675e7a4b73dab8537c20 (MD5) Previous issue date: 1999 / Resumo: As enterohemolisinas (EHly) foram inicialmente detectadas em amostras de Escherichia co/i enteropatogênicas clássicas (EPEC), isoladas a partir de fezes de crianças com diarréia, sendo posteriormente detectada em amostras de E. co/i isoladas de animais. Esta hemolisina, diferentemente da a-hemolisina, não é liberada espontaneamente no meio de cultivo, sendo necessária sua extração da célula bacteriana. Entre os métodos de extração utilizados, a sonicação mostrou ser o método mais eficiente para a liberação da enterohemolisina. Para a detecção da atividade hemolítica, os diferentes materiais a serem testados foram incubados a 37°C por lh, em microplacas ou em tubos, com 1% de eritrócitos de carneiro lavados com tampão PBS. A amostra padrão de E. co/i C3888 foi utilizada para a produção e purificação da enterohemolisina. Esta cultura foi cultivada em meio TSB, acrescido de quelante de ferro EDDA, por 22 horas em shaker a 37°C. Após o cultivo, a cultura foi sonicada e precipitada com sulfato de amônio com 60% de saturação. O material precipitado foi cromatografado em coluna de DEAE FastFlow. As frações com atividade hemolítica foram recromatografadas em coluna de exclusão molecular Superdex 200 e posteriormente em coluna Mono Q no sistema HPLC. A fração com atividade hemolítica obtida da coluna Mono Q apresentou apenas uma banda de 60kDa em gel de SDS-P AGE. No processo de purificação foi obtido um aumento na atividade específica de 2800 vezes, e aproximadamente 100 vezes na atividade relativa. A atividade da EHly se manteve estável por diversos meses no sonicado de cultura, quando este foi mantido a baixas temperaturas (-20°C e -70°C). Entretanto, não foi possível manter a atividade da hemolisina pura nos processos finais de purificação. Apesar da utilização de três diferentes métodos de inoculação em coelhos, para a obtenção de antissoro específico anti-enterohemolisina, não foram obtidos resultados satisfatórios nos testes de neutralização realizados, já que os títulos de soroneutralização obtidos foram os mesmos com soros pré e pós-imunização. Em testes com diferentes culturas celulares, a enterohemolisina semi-purificada foi citotóxica para as culturas de células Hep-2, HeLa, Caco-2, Vero, MDBK e 3T3, com arredondamento celular e descolamento do tapete. As células CHO apresentaram um efeito citopático, com alongamento das células e aparente condensação do núcleo. Nos ensaios com camundongos, não foi observado letalidade ou toxicidade nos animais adultos ou nos recém-nascidos testados / Abstract: Enterohaemolysins (EHly) were ftrst detected in classical enteropathogenic Escherichia coZi strains (EPEC), isolated from feces of infants with diarrhea, and also detected in strains isolated from animaIs. Differring from a-haemolysin, EHly is not spontaneously released to culture medium. Sonication was the most efficient method to extract EHly. In order to detect haemolitic activity, the material was incubated for lh at 37°C in microplates or in tubes, with 1 % sheep erytrocytes, washed with PBS buffer. E. coZi strain C3888 was used to carry out production and puriftcation trials. Bacteria was cultivated in TSB medium with EDDA iron chelant, for 22h at 37°C. The cells were sonicated and the pellet submitted to cromatography in DEAE Fast-Flow column. The fractions that showed 8;ctivity were applied to Superdex-200 and Mono Q HPLC columns. The fraction with haemolitic activity obtained from Mono Q cromatography showed just one 60kDa band in SDS-P AGE electrophoresis gel. Increases of 2800 times in specrnc activity and aproximately 100 times in relative activity were obtained. The activity remained stable for several months in sonicated material when it was kept at low temperatures (-20°C to -70°C). However, it was not possible. to maintain the activity of pure haemolysin in the final purification procedures. Neutralization tests with specific anti-EHly antiserum did not present satisfactory results, despite the three different inoculation methods we tested in rabbits. Titles obtained with pre- and post-immunized sera were the same. In tests with different cell cultures, semipurified EHly proved to be citotoxic to Hep-2, HeLa, Caco-2, Vero, MDBK and 3T3 cells, showing cell rounding and detachment of the cell layer. CHO cells presented a cytopathic effect, with elongation and apparent condensation of the nuclei. No lethality or toxicity were observed in tests carried out in mice / Doutorado / Bioquimica / Doutor em Ciências

Orientador: Antonio Fernando Pestana de Castro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-14T16:52:34Z (GMT). No. of bitstreams: 1 Leonardo_MartaBeatriz_M.pdf: 4436936 bytes, checksum: 8df9211405166fd05a0c6d0c564d6248 (MD5) Previous issue date: 1981 / Resumo: Com objetivo de oferecer a laboratórios menos equipados, e, portanto, sem infraestrutura para realizar a detecção de colibacilos enterotoxigênicos produtoes de enterotoxina termolábil (LT), fez-se uma avaliação da prova de Imunohemólise Radial (SRIH) para este fim. Os principais resultados e conclusões obtidos à partir deste estudo podem ser assim resumidos: Utilizando deferentes diluições de sobrenadantes de culturas provenientes da amostra de E. coli 40T (incluída no grupo I, fortemente enterotoxigênica) frente a diferentes diluições de antitoxina colérica, realizou-se um estudo comparativo entre os testes da SRIH e da Imunohemólise Passiva (PIH). Verificou-se, através deste estudo, que o teste da SRIH é tão sensível quanto o da PIH, para a detecção de enterotoxina LT, quando se correlacionou o diâmetro (em mm) do halo de hemólise com a absorbância, a 420 nm, ou com a quantidade de hemoglobina liberada ('mu¿g/ml), sendo que os valores de r, para n = 25, foram, respectivamente, 0,748 e 0,755. Pode-se considerar como resultado positivo, no teste da SRIH, valores sod diâmetros dos halos de hemólise, superiores a 12,43 mm; valores negativos, aqueles inferiores a 11,33 mm, e duvidosos, aqueles compreendidos entre 11,33 e 12,43mm, conforme estabelecido pelo cálculo do intervalo de Confiança. O teste não apresentou nenhuma reação falso-positivo nas diluições de antitoxina colérica maiores (1:40 e 1:80), sendo, portanto, específico. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Not informed. / Mestrado / Mestre em Ciências Biológicas

Orientadores: Antonio Fernando Pestana de Castro, Jorge Blanco Alvarez / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:25:24Z (GMT). No. of bitstreams: 1 Aidar_Leila_M.pdf: 4916322 bytes, checksum: 5ce886241ad2f530966d97c38fe5219c (MD5) Previous issue date: 1999 / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

No description available.

Escherichia coli (E. coli) frequently experiences changes in environmental osmolarity thus requiring homeostatic adjustments for proper cell function. In a hypoosmotic shock the central players are the mechanosensitive channels (MS channels), considered the major routes for the release of cytoplasmic solutes to achieve rapid reduction of turgor pressure. In the present study a combination of in vivo and in vitro techniques were used to investigate three of the seven MS channels present in the genome of E. coli, these channels being MscL, MscS and MscK. The first chapter focused on three residues (R46, R59 and K60) in the transmembrane helices TM1 and TM2 of MscS. It has been hypothesized that these residues act as part of the tension sensor mechanism, interacting either with membrane phospholipids or ions within the pore of the channel. In addition to MscS, the second chapter included MscL and MscK and analyzed the precise localization and diffusion rate of these proteins in the cytoplasmic membrane by the use of a super resolution fluorescence microscopy technique. The same technique was used in the third chapter to quantify the two main MS channels, MscL and MscS. As well as providing a single cell census these analyses provided a correlation between abundance of channels and cells survival during a downshock, the situation in which these channels gate and protect cells. Quantification revealed a high abundance of these proteins, thus MS channels were also investigated for an alternative role, namely the release of solutes during hyperosmotic shocks. This last chapter focused specifically on glutamate excretion, which is part of the hyperosmotic response. In summary, the results presented in this thesis substantially increased our knowledge of MS channels, both from a functional and a quantitative perspective.

The cytochrome o and cytochrome d oxidase complexes provide twin termini for the branched respiratory chain of aerobically grown Escherichia coli. Combined use of mutant strains, modulated growth conditions and high resolution analytical techniques enabled cytochromes to be resolved, identified and partially characterized. The cytochrome complement of everted membrane vesicles and detergent extracts fractionated by liquid chromatography is more complex than previously recognised. Multiple type-b cytochromes were resolved by potentiometry and by high resolution spectrophotometry in membrane vesicles from mutant strains lacking the cytochrome d oxidase complex and grown under conditions minimising respiratory chain diversity. Cytochrome o was identified with Em = +235 mV (vs. NHE) as were low potential cytochromes associated with dehydrogenases. Spectrally distinct components of the cytochrome d complex yielded Em values of +125 mV (cytochrome 6595) and +187 mV (cytochrome d). The latter displayed atypical redox behaviour with extreme hysteresis during potentiometric titrations. Several cytochromes b₅₅₆ displaying single, symmetrical redox α-bands at 77 K were resolved from detergent extracts of vesicles. Mutant strains identified one with Mr = 52500 (gel filtration) and Em = +20 mV as the sdhC gene product, a component of succinate dehydrogenase. DL-lactate induced another while a hydroperoxidase, Mr = 386000 (gel filtration) with twin Em values of -2mV and -121 mV and a split Soret absorption band at 77 K (λ[symbol omitted]max= 426.0 nm + 434.0 nm) was produced under limited oxygen tension. The Triton-solubilized and purified cytochrome 0 complex exhibited Mr = 516000 (gel filtration) with five component peptides of Mr= 55000, 32000, 31000, 21000 and 16000 (SDS-PAGE). It displayed mid-point potentials of -58 mV, +127 mV and +260 mV and three a-absorption maxima at 77 K : 554.5 nm, 557.0 nm and 563.5 nm. These components were reduced equivalently during poised-potential low temperature spectrophotometric analyses. Carbon monoxide binding changed the complex's redox α-absorption spectrum minimally but shifted the high potential Em to approximately +420 mV. Quinone analogues inhibited both reduction and reoxidation of the complex. Cytochrome o complex prepared from cloned sources contained a significantly greater proportion of the component with mid range electrochemical potential absorbing at 554.0 nm. These results are discussed in relation to possible structures of the complex, its respiratory interactions and the identity of cytochrome o itself. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The acetohydroxy acid synthetase levels in streptomycin-sensitive, -dependent and -resistant mutants have been studied in four different strains of Escherichia coli. The activity of the ∝-acetolactate-forming system was found to be greater both at pH 6.0 and at pH 8.0 in streptomycin-dependent mutants than in the corresponding streptomycin-sensitive cultures. In general, streptomycin-resistant mutants demonstrated enzyme activities within the range found for streptomycin-sensitive organisms regardless of whether they were grown in the presence or absence of antibiotic. The acetohydroxy acid synthetase activity of streptomycin-sensitive and -resistant revertants was observed to be lower than that of the dependent Escherichia coli culture from which they were derived by back-mutation. Mutation to streptomycin-resistance or -dependence had no effect on glucokinase and glutamic dehydrogenase activities. The addition of the coenzyme flavin adenine dinucleotide to the incubation mixtures markedly stimulated the activities of all the extracts. This enhancement of acetohydroxy acid synthetase activity had little or no effect on the ratio of activities of this enzyme in the dependent and sensitive Escherichia coli strains investigated. ∝-Acetohydroxybutyrate formation was found to be greater in extracts from the streptomycin-dependent organism than in extracts prepared from the same strain of sensitive and resistant Escherichia coli. The degree of elevation of ∝-acetohydroxybutyrate paralleled that of ∝-acetolactate formation in the dependent mutant. It was concluded from these observations that excretion of L-valine by streptomycin-dependent Escherichia coli was a consequence of the elevated acetohydroxy acid synthetase activity of these mutants. In the dependent organism, it was postulated that streptomycin functioned as a wde-repressorw of acetohydroxy acid synthetase thus permitting the biosynthetic pathway leading to L-valine to serve as an important route of pyruvate dissimilation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The compound α-acetolactate has been prepared by the chemical method of Krampltz (1948) and by an enzymatic procedure. The products obtained by each method were characterized chromatographically and spectrophotometrically by conversion to their 2,4—dinitrophenylhydrazone derivatives as well as by conversion to acetoin. The Identity of the hydrazone of enzymatically prepared α-acetolactate with hydrazones of the chemical product was established by comparison of R[subscript: F] values, the identical absorption maxima, and by the infrared spectra of these compounds. The α-acetolactate so obtained was used as substrate in a comparison of the activity in streptomycin mutants of the reductoisomerase enzyme which catalyzes the rearrangement and reduction of α-acetolactate to α,β-dihydroxyisovaleric acid. This reaction is the second step in valine biosynthesis. Streptomycin-dependent mutants of Escherichia coli previously has been shown to be derepressed in acetohydroxy acid synthetase, the enzyme which initiates valine biosynthesis. In addition, it had been reported previously that in streptomycin-dependent E. coli K-12, threonine dehydratase, the enzyme which initiates biosynthesis of isoleucine, also is derepressed. In contrast, reductoisomerase, which is common to both the valine and isoleucine pathway, has been found in this work to be normal (i.e., not derepressed) in streptomycin mutants. An additional enzyme of the common pathway, transaminase B, was found to be about 2 to 3 times higher in streptomycin-dependent mutants than in sensitive or resistant strains. The structural genes for both transaminase B and threonine dehydratase of E. coli K-12 have been shown by genetic studies (Ramakrishnan and Adelberg, 1965b) to be coordinately regulated (i.e., on the same operon). The observations made in this study with streptomycin-dependent E. coli K-12 support the observations of these workers. That is, transaminase B of streptomycin-dependent E. coli K-12 is derepressed coordinately with threonine dehydratase. However, the degree of derepression of transaminase B (about 2 to 3 fold) was much less than that of threonine dehydratase (about 9 fold, according to Desai and Polglase, 1966). It may be concluded from these studies that the type of derepression of certain enzymes which has been observed in streptomycin-dependent E. coli has contrasting features to the type of derepression which would be expected on the basis of the Jacob and Monod model (1961) from a nonfunctional regulatory gene or product thereof. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate