Berlin, Humboldt-Universiẗat, Diss., 2004.

Aquatic microalgae produce a variety of toxic secondary metabolites, which are a concern for public health and seafood industries, while also presenting a source of pharmacologically valuable compounds. The present study deals with the physiology and molecular genetics of saxitoxin (STX), a cyanobacterial neurotoxic alkaloid. Ecological and chemical parameters have been investigated for their effects on growth and STX production in the cyanobacterium Cylindrospermopsis raciborskii T3, in order to better understand the physiological responses of this cyanobacterium to the anthropogenic eutrophication of water bodies. The results indicated that phosphate, in particular, had an incremental effect on STX production, as well as promoting the up-regulation of transcription of the STX biosynthetic gene cluster (sxt). High temperature was found to negatively affect growth and STX production in this organism. The effects of the plant hormone, jasmonic acid, were also tested, since it has previously been shown to affect plant alkaloid production. The hypothesised similarity between cyanobacterial and plant secondary metabolism in response to this plant hormone was confirmed in the neurotoxic cyanobacterium, C. raciborskii T3, as well as the non-toxic Anabaena sp. PCC7120. Furthermore, investigation of the sxt gene cluster transcriptional map in C. raciborskii T3 was carried out, with identification of three main polycistronic and one monocistronic transcripts. Promoter regions putatively involved in the regulation of STX production in C. raciborskii T3 were also identified. Transcription factor consensus motifs, the pho boxes, were identified in the main promoter region. These conserved motifs are the binding regions for the transcriptional regulator, PhoB, to the pho regulon genes, involved in phosphate uptake during conditions of its depletion in the environment. Moreover, a genomic region adjacent to the sxt gene cluster in C. raciborskii T3 was identified and characterised, putatively encoding a regulatory two-component system. This system appears to be involved in the sensing of environmental signals, in particular depleted phosphate, while activating the transcription of genes involved in its uptake and transport. The results of this study lead to a greater understanding of the complex factors associated with the regulation of STX biosynthesis and bloom-formation, by the cyanobacterium C. raciborskii T3.

Aquatic microalgae produce a variety of toxic secondary metabolites, which are a concern for public health and seafood industries, while also presenting a source of pharmacologically valuable compounds. The present study deals with the physiology and molecular genetics of saxitoxin (STX), a cyanobacterial neurotoxic alkaloid. Ecological and chemical parameters have been investigated for their effects on growth and STX production in the cyanobacterium Cylindrospermopsis raciborskii T3, in order to better understand the physiological responses of this cyanobacterium to the anthropogenic eutrophication of water bodies. The results indicated that phosphate, in particular, had an incremental effect on STX production, as well as promoting the up-regulation of transcription of the STX biosynthetic gene cluster (sxt). High temperature was found to negatively affect growth and STX production in this organism. The effects of the plant hormone, jasmonic acid, were also tested, since it has previously been shown to affect plant alkaloid production. The hypothesised similarity between cyanobacterial and plant secondary metabolism in response to this plant hormone was confirmed in the neurotoxic cyanobacterium, C. raciborskii T3, as well as the non-toxic Anabaena sp. PCC7120. Furthermore, investigation of the sxt gene cluster transcriptional map in C. raciborskii T3 was carried out, with identification of three main polycistronic and one monocistronic transcripts. Promoter regions putatively involved in the regulation of STX production in C. raciborskii T3 were also identified. Transcription factor consensus motifs, the pho boxes, were identified in the main promoter region. These conserved motifs are the binding regions for the transcriptional regulator, PhoB, to the pho regulon genes, involved in phosphate uptake during conditions of its depletion in the environment. Moreover, a genomic region adjacent to the sxt gene cluster in C. raciborskii T3 was identified and characterised, putatively encoding a regulatory two-component system. This system appears to be involved in the sensing of environmental signals, in particular depleted phosphate, while activating the transcription of genes involved in its uptake and transport. The results of this study lead to a greater understanding of the complex factors associated with the regulation of STX biosynthesis and bloom-formation, by the cyanobacterium C. raciborskii T3.

Le travail de thèse a eu pour but de contribuer à une meilleure connaissance de la dangerosité pour l’homme des souches STEC d’origine bovine, en explorant la corrélation pouvant exister entre la présence du gène stx chez de telles souches et la réalité de son expression. La quantification de l’expression du gène stx2 présent chez 46 souches STEC bovines a été réalisée à l’aide d’un test ELISA commercial détectant spécifiquement les shiga toxines, le test ProSpecT® Shiga toxin (OXOID). L’ensemble des résultats de validation préalable obtenus pour ce test a permis de considérer qu’il pouvait être valablement appliqué à l’étude du panel de souches d’E. coli O157 :H7 bovines collectées au laboratoire, tout en déterminant les limites méthodologiques, et donc d’interprétation. Utilisé comme outil de quantification de la production de Stx2 par les souches du panel choisi, et dans deux conditions expérimentales différentes (présence ou absence d’induction par l’enrofloxacine), ce test a permis de mettre en évidence que seulement 15,2% des souches d’E.coli O157:H7/H- étudiés produisent des quantités significatives de Stx2 détectables sans induction, et ce à des niveaux variables. En revanche, la majorité de ces isolats, bien que n'exprimant pas la protéine Stx2 de manière constitutive, produit des quantités significatives de Stx2 en présence de concentrations subinhibitrices d'enrofloxacine, antibiotique de la famille des fluoroquinolones et utilisé en médecine vétérinaire. Enfin, des mutants résistants à l'enrofloxacine sélectionnés à partir de certaines souches d’E. coli O157:H7, produisent, après induction par l'enrofloxacine, 3 fois plus de toxine Stx2 que les souches sauvages. Les mutants sont également inductibles en utilisant des doses d'enrofloxacine 100 fois supérieures à celles utilisables pour les souches sauvages. L’ensemble de ces résultats montre (i) la corrélation, ou non, qui peut exister entre la présence du gène stx2 et son expression, (ii) que la proportion inductible des souches STEC bovines est potentiellement importante, (iii) que l’enrofloxacine induit fortement l’expression du gène stx2 chez les souches STEC bovines et que (iv) l’induction par l’enrofloxacine conduit à des taux d’expression du gène stx2 supérieurs chez des souches résistantes aux fluoroquinolones que chez les souches sensibles. Au final, cette étude contribue à documenter la variabilité des niveaux d’expression des gènes stx et illustre le risque que des STEC issus de bovins puissent devenir plus fréquemment pathogènes pour l'homme suite à l'usage croissant des fluoroquinolones vétérinaires. / The present study contributed to a better knowledge of the pathogenicity of STEC for humans by quantifying the expression of the stx2 gene from a panel of 46 cattle STEC isolates by ELISA. Succesful validation experiments of the ProSpecT® Shiga toxin ELISA (OXOID) first concluded to its capability to be used for a valuable quantification of the Stx2 protein. Stx2 expression was tested in presence and absence of subtherapeutic concentrations of enrofloxacin, an antibiotic of the fluoroquinolones family used in veterinary medicine. Whereas only 15.2% of the strains displayed significant amounts of detectable Stx2 in absence of induction, most of them were shown to be inducible, and at various levels, in presence of subtherapeutic concentrations of enrofloxacin. Also, enrofloxacin-resistant mutants of Stx2-producing E. coli O157:H7 were selected and produced 3-fold higher Stx2 levels than native strains after induction with enrofloxacin. Mutants were also inducible using hundred-fold higher enrofloxacin concentrations than the useful ones for native strains. At the end, these results show (i) the inconstant and variable expression of the stx2 gene from cattle STEC isolates in native conditions, (ii) the potentially high number of inducible STEC isolates in cattle, (iii) that enrofloxacin is a strong inducer of the stx2 expression in cattle STEC isolates and (iv) that the stx2 gene is stronger induced in isolates resistant to fluoroquinolones compared to susceptible ones. Finally, this all study documents the variable expression of the stx2 gene and also suggests that E. coli O157:H7 from cattle may become more frequently pathogenic to humans as a side-effect of the increasing use of veterinary fluoroquinolones.

Die Auszeichnungssprache XML definiert eine einfache Syntax für strukturierte Daten, die sich so applikationsübergreifend einsetzen lassen. Eine der wichtigsten Voraussetzungen für den Austausch solcher XML-Daten ist die Möglichkeit ihrerTransformation. Unter den derzeit verfügbaren Transformationsmethoden für XML hat die Sprache XSLT als W3C-Standard die größte Verbreitung gefunden. Allerdings skaliert XSLT nicht für große Datenmengen, da hier eine Gesamtsicht auf das XML-Dokument vorausgesetzt wird. Andere existierende Transformationsmethoden besitzen entweder die gleiche Eigenschaft oder erfordern die Programmierung auf der XML-fernen Ebene einer Programmiersprache. In dieser Arbeit wird mit STX eine Transformationssprache für XML entwickelt, die diese Lücke füllt. STX orientiert sich sehr stark an XSLT, verarbeitet jedoch ein XML-Dokument als Datenstrom. STX kann so prinzipiell beliebig große Dokumente transformieren. Die aus der Sprache XPath 2.0 des W3C abgeleitete STX-Pfadsprache (STXPath) trägt dabei der eingeschränkten Sicht auf die zu transformierenden Daten Rechnung, indem sie nur den Zugriff auf die Vorfahren des jeweiligen Kontextknotens ermöglicht. Zu den neuartigen Konzepten in STX zählen neben prozeduralen Eigenschaften vor allem Gruppen, Schnittstellen zu externen Transformationsprozessen, die komplexe Transformation von Zeichenketten sowie Sprachmittel zur Fehlerbehandlung. Diese Arbeit stellt Entwurfsmuster für die wichtigsten Transformationstypen in STX vor und demonstriert an drei Fallbeispielen den Einsatz in realen Projekten. Der dazu verwendete STX-Prozessor Joost verfügt zudem über standardisierteJava-Schnittstellen, die dessen Integration in bestehende Java-Applikationen erleichtern. / The markup language XML defines a simple syntax for structured data that can be used across application boundaries. One of the most important prerequisites for the interchange of such XML data is the possibility of its transformation. Among the currently available transformation approaches for XML, the W3C standard XSLT has gained the biggest popularity. However, XSLT doesn''t scale for huge amounts of data because it requires an overall view to an XML document. Other existing transformation approaches either have the same character or require low-level programming using a general programming language. This PhD thesis introduces STX, an XML transformation language that fills this gap. STX is strongly geared to XSLT, though it processes an XML document as a stream. Therefore, STX is able to transform documents of any size. The STX path language (STXPath), derived from the W3C standard XPath 2.0, considers the restricted view to the input data and enables the access only to the ancestors of the current context node. The new concepts in STX include besides its procedural behaviour mainly groups, interfaces to external transformation processes, complex transformations of strings, as well as language means for error handling. This work introduces design patterns for the most important transformation types in STX and demonstrates three real-life scenarios. The STX processor Joost used for this purpose provides in addition standardized Java interfaces that facilitate its integration into existing Java applications.

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-05Bitstream added on 2014-06-13T20:35:49Z : No. of bitstreams: 1 cardoso_pa_me_jabo.pdf: 283666 bytes, checksum: 9d4635249701354d18b4a667c65f5861 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi investigar a ocorrência de cepas de Escherichia coli produtoras de Shiga toxina (STEC) em queijos mussarela produzidos artesanalmente. Foram analisadas 59 amostras de queijo, produzidas no Vale do Jequitinhonha (Nordeste de Minas Gerais, Brasil). Isolando-se 147 cepas de E. coli e através da técnica de PCR, foram investigadas a presença dos genes da Shiga toxina (stx 1 e stx 2) e da intimina (eae). Dezesseis cepas bacterianas (10,8%) apresentaram o gene stx ( todas portavam o gene stx 1) e 13 delas também se mostraram eae positivas. Os isolados de E. coli foram também examinados para a detecção dos genes codificadores de adesinas (pap, sfa e afa). Não foram identificados nenhum desses genes.Todas as cepas STEC isoladas foram pesquisadas para resistência a 12 agentes antimicrobianos. As resistências predominantes detectadas foram de 37,5% para estreptomicina, 37,5% para a tetraciclina, 31,2% para a ampicilina e 31,2% para a amicacina. A resistência a múltiplas drogas foi encontrada em 5 cepas (31,2%). A presença dos genes codificadores dos fatores de virulência indica que o queijo mussarela produzido artesanalmente pode representar um risco à saúde dos consumidores. / The aim of the present study was to investigate the occurrence of Escherichia coli strains presenting the Shiga toxin gene (probably STEC strains) in mussarela cheese produced by artesanal method in the Jequitinhonha Valey (Northeast of Minas Gerais State, Brazil). Fifty-nine cheese samples were analyzed and a hundred forty seven strains of E. coli were isolated. Using the PCR method the strains were screened for the Shiga toxin (stx 1 and stx 2) and the intimin (eae) genes. Sixteen isolates (10,8%) carried the stx gene (all of them showed the stx 1 gene ) and thirteen also presented the eae gene. Using the same method the strains were screened for the presence of pap, afa, and sfa genes, adhesin genes caracteristics of the E. coli extraintestinal pathogenic strains (ExPEC). None of them showed these adhesin genes and could not be classified as an ExPEC strains. The susceptibility of the probably STEC strains to twelve antimicrobial drugs were evaluated. The most important resistance was detected to the streptomycin (37,5%), tetracycline (37,5%), ampicillin (31,2%) and amikacin (31,2%). The multidrug resistance was detected in 5 isolates (31,2%). The presence of coding genes for virulence factors in the E. coli isolates recovered from mussarela cheese produced by artesanal method could represent a risk for the human health.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le travail de thèse a eu pour but de contribuer à une meilleure connaissance de la dangerosité pour l'homme des souches STEC d'origine bovine, en explorant la corrélation pouvant exister entre la présence du gène stx chez de telles souches et la réalité de son expression. La quantification de l'expression du gène stx2 présent chez 46 souches STEC bovines a été réalisée à l'aide d'un test ELISA commercial détectant spécifiquement les shiga toxines, le test ProSpecT® Shiga toxin (OXOID). L'ensemble des résultats de validation préalable obtenus pour ce test a permis de considérer qu'il pouvait être valablement appliqué à l'étude du panel de souches d'E. coli O157 :H7 bovines collectées au laboratoire, tout en déterminant les limites méthodologiques, et donc d'interprétation. Utilisé comme outil de quantification de la production de Stx2 par les souches du panel choisi, et dans deux conditions expérimentales différentes (présence ou absence d'induction par l'enrofloxacine), ce test a permis de mettre en évidence que seulement 15,2% des souches d'E.coli O157:H7/H- étudiés produisent des quantités significatives de Stx2 détectables sans induction, et ce à des niveaux variables. En revanche, la majorité de ces isolats, bien que n'exprimant pas la protéine Stx2 de manière constitutive, produit des quantités significatives de Stx2 en présence de concentrations subinhibitrices d'enrofloxacine, antibiotique de la famille des fluoroquinolones et utilisé en médecine vétérinaire. Enfin, des mutants résistants à l'enrofloxacine sélectionnés à partir de certaines souches d'E. coli O157:H7, produisent, après induction par l'enrofloxacine, 3 fois plus de toxine Stx2 que les souches sauvages. Les mutants sont également inductibles en utilisant des doses d'enrofloxacine 100 fois supérieures à celles utilisables pour les souches sauvages. L'ensemble de ces résultats montre (i) la corrélation, ou non, qui peut exister entre la présence du gène stx2 et son expression, (ii) que la proportion inductible des souches STEC bovines est potentiellement importante, (iii) que l'enrofloxacine induit fortement l'expression du gène stx2 chez les souches STEC bovines et que (iv) l'induction par l'enrofloxacine conduit à des taux d'expression du gène stx2 supérieurs chez des souches résistantes aux fluoroquinolones que chez les souches sensibles. Au final, cette étude contribue à documenter la variabilité des niveaux d'expression des gènes stx et illustre le risque que des STEC issus de bovins puissent devenir plus fréquemment pathogènes pour l'homme suite à l'usage croissant des fluoroquinolones vétérinaires.

No description available.

Orientador: José Moacir Marin / Banca: Everlon Cid Rigobelo / Banca: Maria Cristina Monteiro de Souza-Gugelmin / Resumo: O objetivo deste estudo foi investigar a ocorrência de cepas de Escherichia coli produtoras de Shiga toxina (STEC) em queijos mussarela produzidos artesanalmente. Foram analisadas 59 amostras de queijo, produzidas no Vale do Jequitinhonha (Nordeste de Minas Gerais, Brasil). Isolando-se 147 cepas de E. coli e através da técnica de PCR, foram investigadas a presença dos genes da Shiga toxina (stx 1 e stx 2) e da intimina (eae). Dezesseis cepas bacterianas (10,8%) apresentaram o gene stx ( todas portavam o gene stx 1) e 13 delas também se mostraram eae positivas. Os isolados de E. coli foram também examinados para a detecção dos genes codificadores de adesinas (pap, sfa e afa). Não foram identificados nenhum desses genes.Todas as cepas STEC isoladas foram pesquisadas para resistência a 12 agentes antimicrobianos. As resistências predominantes detectadas foram de 37,5% para estreptomicina, 37,5% para a tetraciclina, 31,2% para a ampicilina e 31,2% para a amicacina. A resistência a múltiplas drogas foi encontrada em 5 cepas (31,2%). A presença dos genes codificadores dos fatores de virulência indica que o queijo mussarela produzido artesanalmente pode representar um risco à saúde dos consumidores. / Abstract: The aim of the present study was to investigate the occurrence of Escherichia coli strains presenting the Shiga toxin gene (probably STEC strains) in mussarela cheese produced by artesanal method in the Jequitinhonha Valey (Northeast of Minas Gerais State, Brazil). Fifty-nine cheese samples were analyzed and a hundred forty seven strains of E. coli were isolated. Using the PCR method the strains were screened for the Shiga toxin (stx 1 and stx 2) and the intimin (eae) genes. Sixteen isolates (10,8%) carried the stx gene (all of them showed the stx 1 gene ) and thirteen also presented the eae gene. Using the same method the strains were screened for the presence of pap, afa, and sfa genes, adhesin genes caracteristics of the E. coli extraintestinal pathogenic strains (ExPEC). None of them showed these adhesin genes and could not be classified as an ExPEC strains. The susceptibility of the probably STEC strains to twelve antimicrobial drugs were evaluated. The most important resistance was detected to the streptomycin (37,5%), tetracycline (37,5%), ampicillin (31,2%) and amikacin (31,2%). The multidrug resistance was detected in 5 isolates (31,2%). The presence of coding genes for virulence factors in the E. coli isolates recovered from mussarela cheese produced by artesanal method could represent a risk for the human health. / Mestre