Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-91). Also available for download via the World Wide Web; free to University of Oregon users.

Contexte : Amélioration de la prise en charge de l’infection à H. pylori. Matériels et Méthodes : Détection de H. pylori, la résistance à la clarithromycine, la tétracycline, la lévofloxacine, et la détermination des gènes de pathogénicité ont été réalisées par PCR en temps réel, du gène de l’ARNr 23 S, de l’ARNr 16S, PCR classique et séquençage. L’évaluation de la stabilité du mutant résistant par rapport à l’isolat sensible était obtenue par compétition en culture sur cellules gastriques AGS sur une longue période, suivie du séquençage du génome entier. L’évaluation de l’effet de l’extrait de Ceiba pentandra sur H. pylori était réalisée par la détermination de la concentration minimale inhibitrice. Résultats : Prévalence de l’infection à H. pylori : 75.52%, résistance à la clarithromycine et tétracycline : 4,2% et 1,2%, résistance à la lévofloxacine : 57%. Gène CagA : 92,2%. Gène Vac As1m1 : 82%. Absence de stabilité du mutant résistant dans le couple de souches 3695 R/S (ratio R / S 0,1), à 30 jours de la co-culture (p <0.05) ; ce mutant présentait la mutation A2142G, conférant la résistance à la clarithromycine. On notait la stabilité du mutant résistant dans l’autre couple de souches 3657R/S (ratio R / S ratio : 1,7) à 40 jours de la co-culture (p <0.05), avec développement des mutations compensatoires ; ce mutant présentait la mutation A2143G. L’activité modérée à faible était notée avec les extraits hydroéthanolique et butanolique de Ceiba pentandra, avec une concentration minimale inhibitrice de 50 à 80 μg / ml.Conclusion : il est possible de traiter l’infection à H. pylori avec une thérapie à base de clarithromycine au Congo. L’absence d’une activité forte ne permet pas de recommander Ceiba pentandra dans le traitement de l’infection à H. pylori . La réversion de la résistance dans le cas de H. pylori peut être envisagée. / Context: The objective of this work was to improve Helicobacter pylori infection management. Materials and methods: H. pylori detection, it’s resistance to clarithromycine, tetracyclin, levofloxacin, and determination pathogenic genes were done by real-time PCR on 23 S rRNA, on 16 S rRNA gene, classic PCR, sequencing. Evaluation of the resistant mutant stability to its sensitive isolate was carried out by competing them over a long period in culture on AGS gastric cells and whole sequencing genome. The evaluation of Ceiba pentandra extract effect on H. pylori was carried out by determining the minimum inhibitory concentration. Results: Prevalence of H. pylori infection: 75.52%, resistance to clarithromycin and tetracycline: 4.2% and 1.2%, levofloxacin resistance: 57%. CagA gene: 92.2%. Vac As1m1 gene: 82%. Lack of stability of the resistant mutant in a 3695 R/S pair of isolates (R/S ratio 0.1), at the 30 day of the co-culture (p <0.05); this mutant had an A2142G mutation conferring resistance to clarithromycin. Stability of the resistant mutant in the other 3657 pair of isolates (R/S ratio of 1.7) at the 40 day of the co-culture (p <0.05), with development of compensatory mutations; this mutant had an A2143G mutation conferring resistance to clarithromycin. The moderate to low activity was noted with the hydroethanol extract and the butanol extract: minimum inhibitory concentration: 50 to 80 μg / ml. Conclusion: It’s possible to treat H. pylori infection with therapy based on clarithromycin in Congo. The absence of a strong activity does not make it possible to recommend Ceiba pentandra in the treatment of H. pylori infection. Reversion resistance is possible with H. pylori.

Introdução: Helicobacter pylori é uma bactéria que infecta aproximadamente metade da população mundial e é considerada uma importante causa de câncer gástrico. A terapia de erradicação nem sempre é eficaz, pois pode ocorrer a resistência aos antimicrobianos. Objetivo: Determinar o perfil de sensibilidade de isolados de H. pylori frente aos antibióticos amoxicilina, claritromicina e ciprofloxacina na população do Rio Grande do Sul empregando distintas padronizações. Material e Métodos: Estudo transversal. Avaliaram-se 54 amostras de H. pylori obtidas através de cultivo de biópsias gástricas em Agar Belo Horizonte e incubação a 37°C em microaerofilia, durante cinco dias. A sensibilidade aos antibióticos foi determinada segundo as orientações das padronizações britânica (BSAC) e australiana (CDS Method) (quantitativas), além da francesa (CA-SFM) (qualitativa). Resultados e discussão: Sete (13%) isolados de H. pylori foram resistentes à claritromicina, um (1,9%) à amoxicilina e três (5,5%) à ciprofloxacina. Estes índices de resistência são considerados satisfatórios e demonstram que todos esses antibióticos podem ser utilizados na terapia empírica na população local, sobretudo a amoxicilina e a claritromicina como primeira linha de tratamento. As metodologias quantitativas BSAC e CDS Method revelaram concordância muito semelhante nos resultados de sensibilidade, sendo a interpretação mais facilitada na técnica CDS Method. A padronização CA-SFM parece ser mais atrativa sob o aspecto econômico, mas fornece resultados apenas qualitativos. Conclusão: Os antibióticos amoxicilina e claritromicina ainda são uma boa opção no tratamento anti-H. pylori na população do Rio Grande do Sul. / Introduction: Helicobacter pylori is a bacteria which infects nearly half the world population and it is considered an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. Objective: To determine the susceptibility profile of H. pylori isolates to the antibiotics amoxicillin, clarithromycin and ciprofloxacin in the population of Rio Grande do Sul using different standardizations. Material and Methods: Transversal study. Were evaluated 54 samples of H. pylori obtained by gastric biopsies which were cultured on Belo Horizonte agar and incubated at 37°C in a microaerophilic environment for five days. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy (BSAC), the Australian (CDS Method) (quantitative) and the French (CA-SFM) (qualitative). Results and discussion: Seven (13%) H. pylori isolates were resistant to clarithromycin, one (1,9%) to amoxicillin and three (5,5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy of the local population, especially amoxicillin and clarithromycin as a first line treatment. The quantitative methodologies BSAC and CDS Method revealed very similar agreement in the susceptibility results. Moreover, the CDS method had an easier interpretation technique. The CA-SFM standards seem to be more attractive on the economic aspect but they only provide qualitative results. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for anti-H. pylori treatment in the population of Rio Grande do Sul.

Introdução: Helicobacter pylori é uma bactéria que infecta aproximadamente metade da população mundial e é considerada uma importante causa de câncer gástrico. A terapia de erradicação nem sempre é eficaz, pois pode ocorrer a resistência aos antimicrobianos. Objetivo: Determinar o perfil de sensibilidade de isolados de H. pylori frente aos antibióticos amoxicilina, claritromicina e ciprofloxacina na população do Rio Grande do Sul empregando distintas padronizações. Material e Métodos: Estudo transversal. Avaliaram-se 54 amostras de H. pylori obtidas através de cultivo de biópsias gástricas em Agar Belo Horizonte e incubação a 37°C em microaerofilia, durante cinco dias. A sensibilidade aos antibióticos foi determinada segundo as orientações das padronizações britânica (BSAC) e australiana (CDS Method) (quantitativas), além da francesa (CA-SFM) (qualitativa). Resultados e discussão: Sete (13%) isolados de H. pylori foram resistentes à claritromicina, um (1,9%) à amoxicilina e três (5,5%) à ciprofloxacina. Estes índices de resistência são considerados satisfatórios e demonstram que todos esses antibióticos podem ser utilizados na terapia empírica na população local, sobretudo a amoxicilina e a claritromicina como primeira linha de tratamento. As metodologias quantitativas BSAC e CDS Method revelaram concordância muito semelhante nos resultados de sensibilidade, sendo a interpretação mais facilitada na técnica CDS Method. A padronização CA-SFM parece ser mais atrativa sob o aspecto econômico, mas fornece resultados apenas qualitativos. Conclusão: Os antibióticos amoxicilina e claritromicina ainda são uma boa opção no tratamento anti-H. pylori na população do Rio Grande do Sul. / Introduction: Helicobacter pylori is a bacteria which infects nearly half the world population and it is considered an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. Objective: To determine the susceptibility profile of H. pylori isolates to the antibiotics amoxicillin, clarithromycin and ciprofloxacin in the population of Rio Grande do Sul using different standardizations. Material and Methods: Transversal study. Were evaluated 54 samples of H. pylori obtained by gastric biopsies which were cultured on Belo Horizonte agar and incubated at 37°C in a microaerophilic environment for five days. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy (BSAC), the Australian (CDS Method) (quantitative) and the French (CA-SFM) (qualitative). Results and discussion: Seven (13%) H. pylori isolates were resistant to clarithromycin, one (1,9%) to amoxicillin and three (5,5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy of the local population, especially amoxicillin and clarithromycin as a first line treatment. The quantitative methodologies BSAC and CDS Method revealed very similar agreement in the susceptibility results. Moreover, the CDS method had an easier interpretation technique. The CA-SFM standards seem to be more attractive on the economic aspect but they only provide qualitative results. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for anti-H. pylori treatment in the population of Rio Grande do Sul.

Introdução: Helicobacter pylori é uma bactéria que infecta aproximadamente metade da população mundial e é considerada uma importante causa de câncer gástrico. A terapia de erradicação nem sempre é eficaz, pois pode ocorrer a resistência aos antimicrobianos. Objetivo: Determinar o perfil de sensibilidade de isolados de H. pylori frente aos antibióticos amoxicilina, claritromicina e ciprofloxacina na população do Rio Grande do Sul empregando distintas padronizações. Material e Métodos: Estudo transversal. Avaliaram-se 54 amostras de H. pylori obtidas através de cultivo de biópsias gástricas em Agar Belo Horizonte e incubação a 37°C em microaerofilia, durante cinco dias. A sensibilidade aos antibióticos foi determinada segundo as orientações das padronizações britânica (BSAC) e australiana (CDS Method) (quantitativas), além da francesa (CA-SFM) (qualitativa). Resultados e discussão: Sete (13%) isolados de H. pylori foram resistentes à claritromicina, um (1,9%) à amoxicilina e três (5,5%) à ciprofloxacina. Estes índices de resistência são considerados satisfatórios e demonstram que todos esses antibióticos podem ser utilizados na terapia empírica na população local, sobretudo a amoxicilina e a claritromicina como primeira linha de tratamento. As metodologias quantitativas BSAC e CDS Method revelaram concordância muito semelhante nos resultados de sensibilidade, sendo a interpretação mais facilitada na técnica CDS Method. A padronização CA-SFM parece ser mais atrativa sob o aspecto econômico, mas fornece resultados apenas qualitativos. Conclusão: Os antibióticos amoxicilina e claritromicina ainda são uma boa opção no tratamento anti-H. pylori na população do Rio Grande do Sul. / Introduction: Helicobacter pylori is a bacteria which infects nearly half the world population and it is considered an important cause of gastric cancer. The eradication therapy is not always effective because resistance to antimicrobials may occur. Objective: To determine the susceptibility profile of H. pylori isolates to the antibiotics amoxicillin, clarithromycin and ciprofloxacin in the population of Rio Grande do Sul using different standardizations. Material and Methods: Transversal study. Were evaluated 54 samples of H. pylori obtained by gastric biopsies which were cultured on Belo Horizonte agar and incubated at 37°C in a microaerophilic environment for five days. The antibiotics susceptibility was determined according to the guidelines of the British Society for Antimicrobial Chemotherapy (BSAC), the Australian (CDS Method) (quantitative) and the French (CA-SFM) (qualitative). Results and discussion: Seven (13%) H. pylori isolates were resistant to clarithromycin, one (1,9%) to amoxicillin and three (5,5%) to ciprofloxacin. These indices of resistance are considered satisfactory and show that all of these antibiotics can be used in the empirical therapy of the local population, especially amoxicillin and clarithromycin as a first line treatment. The quantitative methodologies BSAC and CDS Method revealed very similar agreement in the susceptibility results. Moreover, the CDS method had an easier interpretation technique. The CA-SFM standards seem to be more attractive on the economic aspect but they only provide qualitative results. Conclusion: The antibiotics amoxicillin and clarithromycin are still a good option for anti-H. pylori treatment in the population of Rio Grande do Sul.

Les systèmes Toxine Antitoxine (TA) sont présents chez la plupart des génomes bactériens. Nous rapportons dans cette étude la présence de tels systèmes chez la bactérie pathogène Helicobacter pylori. Ce nouveau système TA de type I, de la même manière que les autres systèmes de type I décrits, est composé d’une toxine peptidique membranaire (AapA) dont la traduction est inhibée par un ARNnc (IsoA) suivant une interaction côté 5’ non traduit de l’ARNm. La structuration particuliere de l’ARNm a permit l’identification d’orthologue de ce système dans les chromosomes des genres Helicobacter et Campylobacter mais aussi sur leur patrimoine génétique mobiles, tels les plasmides. Ceci impliquant leur potentielle acquisition dans le génome par transfert génétique horizontal. La deuxième partie de l’étude se focalise sur la résolution par RMN du liquide de la structure atomique de la toxine AapA1 dans un environnement pseudo-membranaire. Une approche par mutation révèle les determinants structuraux de la toxicité, pointant la présence d’une empreinte de charge dans la partie helicoidale transmenbranaire de la toxine présente aussi chez d’autres toxines de Type I. La dernière partie de l’étude se centre sur l’expression et la purification de la toxine afin d’en étudier la structure en environnement membranaire complexe par RMN du solide. Les résultats prometteurs ouvrent la voie à une caractérisation de la toxine par l’expérience de PISEMA. / Toxin-antitoxin (TA) systems are present in almost all bacterial genomes. Here we report that the genome of the major human gastric pathogen, Helicobacter pylori, is hosting several copies of a new family of type I TA systems. Similarly to other type I TA systems, the toxin (AapA) is a small membrane protein whose expression is controlled by a small antisense RNA (IsoA, antitoxin) that binds to the 5’ untranslated region (UTR) of the mRNA. In addition we used the strong conservation of the mRNA folding to identify homologs of this TA system not only in other Helicobacter and Campylobacter chromosomes but also on plasmids, indicating that this new TA system might have been spread over different genomes via horizontal gene transfer.The second part of the study take account of the AapA toxin itself. We acutely determine the structure of AapA1 by liquid state NMR in membrane mimicking environment. We then probe first structural insight on atomic structural determinants of its toxicity following a mutation studies.These results reveal a particular charge pattern on the transmembrane α helix domain of AapA toxin similary to other Type I toxin. A third part of the studies is based on expression and purification of the toxin in order to determine the structure in complex membrane environment by solid state NMR. The promosisng result open the way to characterize the toxin by PISEMA experiment.

Les microARN sont de petits ARN non codant régulant post-transcriptionnellement l’expression de certains gènes. Du fait de leur fort potentiel régulateur, une modification de leur expression peut conduire à l’apparition de pathologies telles que le cancer ou l’inhibition des mécanismes de défense contre des pathogènes. Notre objectif est de caractériser le rôle de certains miARN dans la formation de cancer gastrique dû à Helicobacter pylori. En effet, cette bactérie peut conduire à l’apparition d’adénocarcinome gastrique et de lymphome du MALT. Sa virulence est essentiellement due à la protéine CagA, injectée dans les cellules de la muqueuse gastrique. Par séquençage à haut débit du contenu en miARN d’une lignée épithéliale gastrique humaine, co-cultivée ou non avec H. pylori, nous avons observé que les niveaux de miR-200b/c sont augmentés par l’infection. Ces miARN sont des inhibiteurs puissants de la transition épithélio-mésenchymateurse (TEM), modification morphologique promotrice d’invasion. Ils ciblent les facteurs de transcription ZEB1/2 avec lesquels ils sont impliqués dans une boucle de rétro-action mutuellement répressive. Le niveau basal élevé de miR-200b/c dans ces cellules réprime totalement ZEB1, tandis que l’infection par H. pylori, sous la dépendance de CagA, promeut une TEM en induisant ZEB1. Paradoxalement, les miR-200b/c sont aussi augmentés lors de l’infection transcriptionnellement. Nous avons pu démontrer que l’augmentation des miR-200b/c dans les cellules infectées a pour rôle de modérer l’induction de ZEB1 via l’activation de NF-kB, constituant ainsi un mécanisme de défense des cellules hôte contre la perte de leur identité épithéliale. / MicroRNA are small noncoding RNA that post-transcriptionally regulate gene expression. Due to their high regulator potential, a change in their expression may lead to the emergence of diseases such as cancer or inhibition of defense mechanisms against pathogens. Our aim is to characterize the role of miRNA in the response of gastric eptithelial cells to Helicobacter pylori (H. pylori). Indeed, H. pylori promote gastric adenocarcinoma and MALT lymphoma. Its virulence is essentially mediated by CagA, injected into cells of the gastric mucosa. Thanks to high throughput sequencing of miRNA content of a gastric epithelial cell line, infected or not with H. pylori: miR-200b and -200c appeared up-regulated upon infection. These miRNA are potent inhibitors of the “epithelial-to-mesenchymal transition” (EMT), a process that drastically alters cell morphology and promotes cell invasion. MiR-200b/c target the transcription factors ZEB1 and ZEB2, with which they are involved in a mutually repressive feedback loop. In basal conditions, the high levels miR-200b/c in gastric epithelial cells totally silence ZEB1 mRNA whereas H. pylori promotes EMT via ZEB1 expression, on the dependence of CagA translocation into host cells. But, paradoxically, miR-200b/c levels were also up-regulated upon infection. The increased miR-200b/c levels in infected cells moderate ZEB1 induction thanks to NF-kB activation and constitute a self-defense mechanism to thwart the loss of their epithelial phenotype upon infection.

Orientadores: José Murilo Robilotta Zeitune, Sandra Cecília Botelho Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T12:37:01Z (GMT). No. of bitstreams: 1 Roesler_BrunaMaria_D.pdf: 2549394 bytes, checksum: 8534c8dd497e83d0a6b58932dcf0c4cf (MD5) Previous issue date: 2011 / Resumo: O entendimento acerca da carcinogênese gástrica, que é um processo multifatorial, avançou consideravelmente nas últimas décadas, especialmente no que diz respeito ao papel do Helicobacter pylori no desenvolvimento das lesões pré-cancerosas e na neoplasia propriamente dita, tanto em seu estágio precoce quanto avançado. O H. pylori é uma bactéria gram-negativa, classificada como carcinógeno tipo I pela IARC e que possui um alto grau de diversidade genética, assim como importantes fatores de virulência, o que pode ser verificado através das diferentes cepas encontradas nas doenças do trato gastrointestinal. A determinação, portanto, das cepas mais virulentas, responsáveis, pelo menos teoricamente, pelo desenvolvimento das doenças gástricas mais graves, é de vital importância tanto para estudos epidemiológicos quanto para a determinação de quais são os pacientes com maior probabilidade de desenvolvimento do câncer gástrico, que permanece como um grave problema de saúde pública. No presente estudo foram comparadas cepas da bactéria prevalentes em amostras de tecido gástrico provenientes de pacientes com diagnóstico de adenocarcinoma gástrico distal do tipo intestinal precoce e avançado através do uso de técnicas de biologia molecular para avaliar diferentes regiões genômicas da bactéria: genes ureaseC, vacA (regiões s e m), cagA, cagT e dupA (regiões jhp0917 e jhp0918). Os resultados obtidos foram semelhantes tanto para os casos precoces quanto avançados da doença, porém, uma relação estatisticamente significativa foi encontrada entre o gene cagA e o adenocarcinoma avançado. Em geral, as cepas foram classificadas como vacA s1m1, cagA positivas, cagT positivas e dupA negativas, concluindo-se que cepas com o genótipo vacA s1m1 cagA positivo e cagT positivas podem ser consideradas mais virulentas. Além disso, conclui-se que o gene dupA, apesar de pouco presente nas amostras de câncer gástrico estudadas, não pode ser considerado um marcador exclusivo da úlcera duodenal, como descrito inicialmente / Abstract: The understanding of gastric carcinogenesis, that is a multifactorial process, has advanced considerably in recent decades, especially with regard to the role of Helicobacter pylori in the development of precancerous lesions and cancer, both in its early or advanced stage. H. pylori is a gram-negative bacteria, classified as a group I carcinogen by the IARC and it has a high degree of genetic diversity, as well as important virulence factors, which can be verified through the different strains present in gastrointestinal diseases. Therefore, determination of the most virulent strains, responsible, at least theoretically, for the development of the most severe diseases, as gastric adenocarcinoma, is of fundamental importance for epidemiological studies and for determination of the patients with high probability for development of the disease, which remains as a serious public health problem. In the present study they were compared strains of the bacteria prevalent in early and advanced gastric distal type intestinal adenocarcinoma through molecular biology techniques to evaluate different regions of bacterium genoma: urease C, vacA (regions s and m), cagA, cagT and dupA (jhp0917 and jhp0918 regions) genes. The results were similar for both early and advanced cases of the disease, however, a statistically significant relationship was found between the cagA gene and advanced adenocarcinoma. In general, the strains were classified as vacA s1m1, cagA positive, cagT positive and dupA negative, conducting that strains with the genotype vacA s1m1, cagA positive and cagT positive may be considered more virulent. Moreover, it is concluded that the dupA gene, although found in a little proportion of the gastric cancer H. pylori strains, can not be considered an exclusive marker for duodenal ulcer, as described earlier / Doutorado / Ciencias Basicas / Doutor em Clínica Médica