Helicobacter pylori ist ein pathogenes Bakterium, das verantwortlich gemacht wird für verschiedene Erkrankungen des Magens und Duodenums, wie beispielsweise chronische Gastritis, peptische Ulzera und maligne Lymphome. Das Bakterium zeichnet sich durch eine hohe Rekombinationsrate aus und besitzt ein hohes Maß an genetischer Allelvielfalt. Im ersten Teil dieser Arbeit wurde die Rekombinationsrate und die Länge der rekombinerten DNA-Importe anhand von sequentiellen Isolaten, die zu definierten Zeitpunkten aus dem selben Patienten isoliert wurden, untersucht. Es wurden zehn Gene, darunter sieben 'housekeeping' Gene und drei virulenzassoziierte Gene, amplifiziert und sequenziert. Die Ergebnisse zeigten eine bis dahin noch nicht für Bakterien beschriebene Fragmentlänge der DNA-Importe von durchschnittlich lediglich 417 Basenpaaren. Die Rekombinationsrate war außergewöhnlich hoch. DNA-Microarray-Analysen konnten zeigen, dass es trotz dieser hohen Rekombinationsrate nur wenige Veränderungen in der genomischen Genausstattung gab. Jedoch hing das Auftreten von Rekombinationsereignissen direkt mit Veränderungen der Genausstattung zusammen. Im zweiten Teil der Arbeit wurde ein neues in vitro-Transformationsmodell entwickelt, das die in vivo ermittelten Resultate nachvollziehen sollte. Das Modell konnte sowohl die in vivo gefundene Rekombinationsrate als auch den Import von kurzen DNA-Fragmenten bestätigen, die zu einem Allelmosaik zwischen DNA-Rezipient und Donor führten. Auffällig war eine stark verminderte Transformierbarkeit mit Donor-DNA aus asiatischen H. pylori-Stämmen. Um eine mögliche Beteiligung des Nukleotid-Excisions-Reparatur (NER) Mechanismus an der Rekombination zu ermitteln, wurden zwei Gene des Mechanismus ausgeschaltet. Die Ergebnisse der NER--Mutanten (uvrA-, uvrD-) zeigten eine starke Verminderung der Transformierbarkeit. Diese Verminderung hatte jedoch keinen Einfluss auf die Länge der rekombinierten DNA-Importe. Das Ausschalten des uvrA-Gens führte zudem zu einer erhöhten Sensibilität gegenüber UV-Licht. Der NER-Mechanismus ist bei H. pylori in einer noch nicht aufgeklärten Weise an der Rekombination beteiligt. In einem Rhesusaffen-Tiermodell wurde die initiale Besiedlung mit H. pylori untersucht. Die Tiere stellen einen natürlichen Wirt dar und zeigen ähnliche Krankheitssymptome wie menschliche Patienten. Die Rhesusaffen wurden experimentell mit zwei klinischen H. pylori-Isolaten infiziert. Die Reisolation zu bestimmten Zeitpunkten zeigte, dass sich nur einer der beiden Stämme im Affenmagen etablieren konnte und der zweite Stamm verdrängt worden war. In einem zweiten Versuchsansatz wurden die persistent infizierten Affen mit vier weiteren H. pylori-Stämmen infiziert, um eine transiente Koinfektion zu simulieren. Diese Stämme verdrängten jedoch den bereits etablierten Stamm, und es konnte keine in vivo-Rekombination festgestellt werden. Dennoch ist dieses Modell das Erste, in dem eine persistierende experimentelle H. pylori-Infektion in Rhesusaffen über einen Zeitraum von mehr als vier Jahren nachgewiesen werden konnte. Die Ergebnisse liefern wichtige Hinweise auf den beim Menschen meist unentdeckten Anfang der H. pylori-Infektion. Die Untersuchungen an weiteren Spezies des Genus Helicobacter zeigten, dass die beschriebene Spezies Heelicobacter nemestrinae keine eigene Spezies darstellt, sondern der Spezies H. pylori zugeordnet werden konnte. Den damit nächsten 'Verwandten' stellt die Spezies H. acinonychis dar, deren Stämme sich untereinander wesentlich weniger stark unterscheiden als H. pylori-Stämme. Die Ergebnisse dieser Arbeit liefern wichtige Daten zum Verständnis der Evolution und Mikroevolution innerhalb eines Wirtes von H. pylori, die zu besseren Strategien in der Bekämpfung dieses pathogenen Bakteriums führen können. / The human pathogen Helicobacter pylori colonizes the gastrointestinal tract and causes a long-term infection leading to several diseases such as gastritis, peptic ulcers and cancer. H. pylori is the most genetically diverse bacterial species known. Population genetic analysis has shown that the diversity is largely due to recombination between different H. pylori strains during mixed infection. To analyse the recombination rate in vivo, sequential isolates, taken from the same patient at different timepoints, were used. Fragments of seven housekeeping genes, the two flagellin genes and the vacuolating cytotoxin gene vacA, were sequenced and pairwisely compared to detect genetic changes that had occurred during chronic colonization. The recombination rate was unexpectedly high and the size of the imported DNA-fragments had an average of only 417 basepairs. DNA-imports of this extraordinarily short length were never found before in other bacterial species so far. A Microarray analysis showed a high stability of the genetic content in the paired isolates. The very few differences in this genetic content were mainly driven by recombination events. A new developed in vitro transformation model was able to measure the recombination frequency and the length of the imported DNA. The model confirmed the unusually high recombination frequency and the very short imported DNA fragments found in vivo in the sequential isolates. Interestingly, by using Asian strains as DNA donor, the recombination frequency was much lower compared to European and African strains. To answer the question whether the nucleotide-excision-repair (NER) mechanism was involved in the recombination process, knock out mutants of two key genes of the mechanism were used (uvrA-, uvrD-). The NER mutants showed a notable decrease in their transformation ability, but the length of the imported fragments was not affected. The NER mechanism seems to be involved in the recombination process, but it is still unknown in how far and in which way. A rhesus monkey model was developed to establish an experimentally persistent H. pylori infection and to investigate the initial infection steps. Macaques are natural hosts of H. pylori and develop similar disease. Two clinical isolates were chosen for infection, but only one strain survived in the stomach of the macaques. The second strain was outcompeted by the first one. In a second trial the macaques were infected with four new strains of H. pylori to simulate a transient co-colonisation. Only two of the new strains survived and the formerly established strain was outcompeted by the two new strains. No recombination events could be detected. Nevertheless, this is the first time that rhesus monkeys were experimentally persistently infected for more than four years with H. pylori. In this model the first steps of an new H. pylori infection can be investigated, which is not possible in humans. Investigations of other members of the genus Helicobacter showed, that the species Helicobacter nemestrinae was not an independent species, but represented a strain of H. pylori. The closest related species then was represented by Helicobacter acinonychis. Genetic analyses revealed a much more clonal genome between different H. acinonychis strains compared to H. pylori.

Flagellen-basierte Motilität und Chemotaxis stellen essentielle Pathogenitätsfaktoren dar, die für die erfolgreiche Kolonisierung der Magenschleimhaut durch H. pylori notwendig sind. Die Mechanismen der Regulation der Flagellensynthese und das Chemotaxis-System von H. pylori weisen trotz einiger Ähnlichkeiten fundamentale Unterschiede zu den Systemen anderer Bakterien auf. In H. pylori ist die Flagellensynthese durch eine komplex regulierte Kaskade kontrolliert, die Regulatorkomponenten wie das Zweikomponentensystem HP244/FlgR, die Sigma Faktoren 54 und 28 und den Sigma Faktor28-Antagonisten FlgM enthält. Das Signal, welches über die Histidinkinase des Zweikomponentensystems HP244/FlgR die Expression der Sigma Faktor54-abhängigen Klasse 2 Flagellengene reguliert, ist bisher noch nicht bekannt. Allerdings konnte mit HP137 ein Protein identifiziert werden, das im „yeast two-hybrid“ System sowohl mit der korrespondierenden Kinase HP244 des Flagellenregulators FlgR, als auch mit der Flagellenkomponente FlgE´ interagiert (Rain et al., 2001). In dieser Arbeit wurde eine mögliche Rolle von HP137 in einem Rückkopplungsmechanismus untersucht, welcher die Aktivität der Histidinkinase in der Flagellenregulation kontrollieren könnte. Obwohl die Deletion des ORF hp137 zu einer unbeweglichen Mutante führte, legen die erfolglosen Komplementations Experimente, sowie die Beobachtung, dass HP137 in vitro keinen bedeutenden Effekt auf die Aktivität der Histidinkinase HP244 hat nahe, dass HP137 weder in H. pylori noch im nahe verwandten C. jejuni direkt an der Flagellenregulation beteiligt ist. Das Chemotaxis-System von H. pylori unterscheidet sich vom gutuntersuchten Chemotaxis-System der Enterobakterien in einigen Aspekten. Zusätzlich zu dem CheY Response Regulator Protein (CheY1) besitzt H. pylori eine weitere CheY-artige Receiver-Domäne (CheY2) welche C-terminal an die Histidinkinase CheA fusioniert ist. Zusätzlich finden sich im Genom von H. pylori Gene, die für drei CheV Proteine kodieren die aus einer N-terminalen Domäne ähnlich CheW und einer C-terminalen Receiver Domäne bestehen, während man keine Orthologen zu den Genen cheB, cheR, and cheZ findet. Um einen Einblick in den Mechanismus zu erhalten, welcher die chemotaktische Reaktion von H. pylori kontrolliert, wurden Phosphotransferreaktionen zwischen den gereinigten Signalmodulen des Zweikomponentensystems in vitro untersucht. Durch in vitro-Phosphorylierungsexperimente wurde eine ATP-abhängige Autophosphorylierung der bifunktionellen Histidinkinase CheAY2 und von CheA´, welches ein verkürztes Derivat von ChAY2 ohne Receiver-Domäne darstellt, nachgewiesen. CheA´ zeigt eine für an der Chemotaxis beteiligte Histidinkinasen typische Phosphorylierungskinetik mit einer ausgeprägten exponentiellen Phase, während die Phosphorylierungskinetik von CheAY2 nur eine kurze exponentielle Phase aufweist, gefolgt von einer Phase in der die Hydrolyse von CheAY2~P überwiegt. Es wurde gezeigt, dass die Anwesenheit einer der CheY2 Domäne die Stabilität der phosphorylierten P1 Domäne im CheA Teil des bifunktionellen Proteins beeinflusst. Außerdem wurde gezeigt, dass sowohl CheY1 als auch CheY2 durch CheAY2 phosphoryliert werden und dass die drei CheV Proteine die Histidinkinase CheA´~P dephosphorylieren, wenn auch mit einer im Vergleich zu CheY1 und CheY2 geringeren Affinität. Außerdem ist CheA´ in der Lage seine Phosphatgruppen auf CheY1 aus C. jejuni und CheY aus E. coli zu übertragen. Retrophosphorylierungsexperimente weisen darauf hin, dass CheY1~P die Phosphatgruppe zurück auf die Histidinkinase CheAY2 übertragen kann und dass die CheY2-Domäne in dem bifunktionellen Protein CheAY2 als „Phosphat Sink“ agiert der den Phosphorylierungszustand und damit die Aktivität des frei diffundierbaren Proteins CheY1 reguliert, das vermutlich es mit dem Flagellenmotor interagiert. Es konnte weiterhin gezeigt werden, dass die unabhängige Funktion der beiden Domänen CheA´ und CheY2 für eine normale chemotaktische Signalgebung in vivo nicht ausreicht. In dieser Arbeit wurden also Hinweise auf eine komplexe Kaskade Phosphatübertragungsreaktionen im chemotaktischen System von H. pylori gefunden, welches Ähnlichkeiten zu dem Syteme-Chemotaxis von S. meliloti aufweist an denen multiple CheY Proteine beteiligt sind. Die Rolle der CheV Proteine bleibt im Moment unklar, jedoch könnte es sein, dass sie an einer weiteren Feinregulierung der Phosphatgruppenübertragungsreaktionen in diesem komplexen chemotaktischen System beteiligt sind / Flagellar motility and chemotaxis are essential virulence traits required for the ability of Helicobacter pylori to colonize the gastric mucosa. The flagellar regulatory network and the complex chemotaxis system of H. pylori are fundamentally different from other bacteria, despite many similarities. In H. pylori expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors 54 and 28 and the anti-sigma 28 factor FlgM. Thus far, the input signal for histidine kinase HP244, which activates the transcriptional regulator FlgR, which triggers sigma factor 54-dependent transcription of the flagellar class 2 genes, is not known. Based on a yeast two-hybrid screen a highly significant protein-protein interaction between the H. pylori protein HP137 and both the histidine kinase HP244 and the flagellar hook protein HP908 (FlgE´) has been reported recently (Rain et al., 2001). So far, no function could be assigned to HP137. Interestingly, the interaction between HP137 and histidine kinase HP244 was observed in the characteristic block N sequence motif of the C-terminal ATP-binding kinase domain. In this work a potential role of HP137 in a feedback regulatory mechanism controlling the activity of histidine kinase HP244 in the flagellar regulation of H. pylori was investigated. Although the substitution of the gene encoding HP137 by a kanamycin cassette resulted in non-motile bacteria, the failure to restore motility by the reintroduction of hp137 in cis into the mutant strain, and the observation that HP137 has no significant effect on the activity of histidine kinase HP244 in vitro indicated that HP137 is not directly involved in flagellar regulation. Therefore, it was demonstrated that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium C. jejuni. Chemotactic signal transduction in H. pylori differs from the enterobacterial paradigm in several respects. In addition to a CheY response regulator protein (CheY1) H. pylori contains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome of H. pylori encodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genes cheB, cheR, and cheZ. To obtain insight into the mechanism controlling the chemotactic response of H. pylori the phosphotransfer reactions between the purified two-component signalling modules were investigated in vitro. Using in vitro phosphorylation assays it was shown that both H. pylori histidine kinases CheAY2 and CheA´ lacking the CheY-like domain (CheY2) act as ATP-dependent autokinases. Similar to other CheA proteins CheA´ shows a kinetic of phosphorylation represented by an exponential time course, while the kinetics of phosphorylation of CheAY2 is characterized by a short exponential time course followed by the hydrolysis of CheAY2~P. Therefore, it was demonstrated that the presence of the CheY2-like receiver domain influences the stability of the phosphorylated P1 domain of the CheA part of the bifunctional protein. Furthermore, it was proven that both CheY1 and CheY2 are phosphorylated by CheAY2 and CheA´~P and that the three CheV proteins mediate the dephosphorylation of CheA´~P, although with a clearly reduced efficiency as compared to CheY1 and CheY2. Moreover, CheA´ is capable of donating its phospho group to the CheY1 protein from C. jejuni and to CheY protein from E. coli. Retrophosphorylation experiments indicated that CheY1~P is able to transfer the phosphate group back to the HK CheAY2 and the receiver domain present in the bifunctional CheAY2 protein acts as a phosphate sink fine tuning the activity of the freely diffusible CheY1 protein, which is thought to interact with the flagellar motor. Hence, in this work evidence of a complex phosphorelay in the chemotaxis system was obtained which has similarities to other systems with multiple CheY proteins. The role of the CheV proteins remain unclear at the moment, but they might be engaged in a further fine regulation of the phosphate flow in this complex chemotaxis system and the independent function of the two domains CheA´ and CheY2 is not sufficient for normal chemotactic signalling in vivo.

Helicobacter pylori is a human pathogen that colonizes the stomach and increases the risk of diseases such as ulcers and gastric cancer. The distribution of H. pylori within the stomach is associated with different disease outcomes, with more dispersed colonization correlated with gastric cancer. The focus of this research is to study the chemotactic responses that promote dispersal of H. pylori within the stomach. We have shown previously that H. pylori perceive the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report that H. pylori chemorepulsion from endogenous AI-2 influences the proportion and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS) or are defective for chemotaxis (∆cheA) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wildtype biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified and characterized two novel periplasmic binding proteins, AibA and AibB, that independently both bind AI-2 and are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenously administered AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal and may play an important role in H. pylori colonization of the stomach by promoting dispersal away from areas of high cell density, thereby modulating the disease spectrum of the host. This dissertation contains previously unpublished co-authored material.

Tesis para optar al título de Doctor en Farmacología / Helicobacter pylori (H. pylori) es un bacilo Gram negativo que infecta en algún momento de la vida a la mitad de la población mundial. Se ha demostrado que la interacción de H. pylori con las células del epitelio gástrico provocan cambios en la expresión de genes. Sin embargo, sólo se han realizado estudios en poblaciones adultas que presentan una infección persistente y sintomática por H. pylori o un cáncer gástrico definido. Estudios realizados en nuestro laboratorio permitieron la identificación de genes diferencialmente expresados en niños infectados con H. pylori comparado con niños no infectados, involucrados en la regulación del ciclo celular, apoptosis, migración celular o la activación del sistema inmune, destacando dentro de ellos el gen SLC5A8. Este es un gen caracterizado como un supresor de cáncer, debido a esta cualidad nuestro trabajo se focalizó en la caracterización de este gen. SLC5A8 es expresado en la membrana apical de las células del epitelio gástrico y es un transportador de ácidos grasos de cadena corta como butirato, permitiendo el ingreso de este sustrato dentro de la célula y favoreciendo su rol como supresor tumoral. La hipótesis planteada en esta tesis es la siguiente “La infección persistente con H. pylori se relaciona con una reducción de expresión de SLC5A8 asociada a hipermetilación en la región promotora de SLC5A8”. En primer lugar, se demostró mediante ensayos de qPCR en muestras de sangre y tejido epitelial gástrico, que la infección por H. pylori se asoció a una expresión disminuida de SLC5A8 en niños infectados cuando se comparó con niños no infectados. (2) Para identificar una posible hipermetilación de la región promotora de SLC5A8, que diera cuenta de la reducción de expresión, en tejido epitelial gástrico se realizaron experimentos de secuenciación con bisulfito. Los resultados obtenidos mostraron una tendencia a la hipermetilación del gen en niños infectados, pero estos resultados no son aún concluyentes. (3) Por otro lado, se comprobó una menor expresión del mRNA SLC5A8 en líneas celulares gástricas infectadas con H. pylori comparado con células no infectadas. (4) Finalmente se evaluó el efecto de la infección de H. pylori en efectores de SLC5A8 como: p53, p21, survivina, y COX2, comprobándose un aumento de COX2, pero una disminución de p21. En conclusión, se demostró que la infección por H. pylori se asocia con una disminución de expresión del gen SLC5A8, tanto in vivo en niños sintomáticos y asintomáticos, como in vitro en líneas celulares. El mecanismo epigenético podría involucrar la hipermetilación de la región promotora de este gen, aunque más experimentos son necesarios para comprobarlo con mayor certeza / Helicobacter pylori (H. pylori) is a gram-negative bacillus that infects half of the population worldwide at some point in life. Several studies have shown that the interaction with H. pylori causes many changes in gene expression in gastric epithelial cells. However, to date all these studies have been performed in adult populations with persistent, symptomatic H. pylori infection or gastric cancer. Research in our laboratory lead to the identification of differential expression of genes in children infected with H. pylori, compared to non-infected children, involved in cell cycle regulation, apoptosis, cell migration and/or activation of the immune system. Of the genes identified, we selected SLC5A8 gene as our focus of attention for this thesis project because of its predicted role as a tumor suppressor and potential connection with cancer genesis upon expression reduction. This transporter is present in the apical membrane of gastric epithelial cells, where it facilitates uptake of short-chain fatty acids, such as butyrate, modulating histone deacetylase function, the potential mechanism for tumor suppression. The hypothesis of this work is “The SLC5A8 expression is diminished during H. pylori infection in children due to hyper-methylation in the promoter region of SLC5A8”. Our main results are (1) H. pylori infection in children is associated with a decrease in SLC5A8 expression, as assessed by qPCR analysis of blood and tissue samples, compared to uninfected children. (2) We observed a trend in the association between reduced expression and hyper-methylation of the promoter region in gastric epithelial tissue. However, more experiments are required to confirm these results. (3) We verified in vitro, in gastric cell lines that SLC5A8 expression is decreased upon infection with H. pylori. (4) Finally, we evaluated in these cells the effect of H. pylori infection on the expression of SLC5A8 effector genes such: p53, p21, COX2, and surviving, showing increased expression for COX2, but an decreased expression in p21. In conclusion, we demonstrated an association between H. pylori infection and decrease expression of SLC5A8 gene, both in vivo, among symptomatic and asymptomatic children, and in vitro, in gastric cell lines. The epigenetic mechanism involved can be a hyper-methylation in the gene promoter of SLC5A8 although more experiments are needed to demonstrate this for sure / Conicyt; Fondecyt; Senescyt

Submitted by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2010-11-22T17:15:34Z No. of bitstreams: 1 AnaPaulaTaschetto.pdf: 1307988 bytes, checksum: b742ca1361331070731bafa9b6cc4162 (MD5) / Made available in DSpace on 2010-11-22T17:15:34Z (GMT). No. of bitstreams: 1 AnaPaulaTaschetto.pdf: 1307988 bytes, checksum: b742ca1361331070731bafa9b6cc4162 (MD5) / O gênero Baccharis é largamente utilizado na medicina popular, na forma de infusões, para controle e/ou tratamento de várias doenças. Estudos já comprovaram a atividade antimicrobiana de Baccharis frente a vários microorganismos. Porém, existem poucos relatos da ação de plantas deste gênero, sobre o microorganismo Helicobacter pylori. Esta bactéria Gram-negativa, microaerófila, está associada às úlceras pépticas duodenais ou gástricas, e ao desenvolvimento de adenocarcinoma gástrico. Desta forma, com a realização deste trabalho buscou-se avaliar in vitro, a atividade antimicrobiana de extratos aquosos e etanólicos de quatro amostras da espécie Baccharis trimera, coletadas nas cidades de Marques de Souza (RS), Mata (RS), Santa Clara do Sul (RS) e Westfália (RS), e de duas amostras da espécie Baccharis articulata, coletadas nas cidades Mata (RS) e Santa Clara do Sul (RS). As atividades foram determinadas através da obtenção das Concentrações Inibitórias Mínimas (CIM), e das Concentrações Bactericidas Mínimas (CBM) dos extratos. Todos os extratos etanólicos, tanto de B. trimera, como de B. articulata apresentaram atividades antimicrobianas, porém apenas um, dos seis extratos aquosos, apresentou atividade (CIM 2500 μg/mL). Três das quatro amostras de B. trimera apresentaram CIM 312,5 μg/mL, mesmo coletadas em locais diferentes. As duas amostras de B. articulata, também coletadas em locais diferentes, apresentaram CIM 625 μg/mL. Os valores das CBM foram idênticos aos encontrados para as CIM em cinco das seis amostras. Os resultados demonstram que os extratos etanólicos de B. trimera apresentaram atividade antimicrobiana ligeiramente superior a B. articulata. Ainda, observou-se que a atividade antimicrobiana foi pouco influenciada pelo local de coleta.

Background – A associação da infecção pela bactéria Helicobacter pylori com algumas doenças gástricas, como por exemplo, a úlcera gástrica e o câncer gástrico, é bem definida. No entanto, a associação da bactéria com outras doenças, especialmente extragástricas não está estabelecida. Métodos – Foi realizada avaliação da ansiedade e depressão e pesquisa do Helicobacter pylori com endoscopia em participantes de um estudo que avaliou os efeitos da erradicação do Helicobacter pylori sobre a dispepsia funcional. Pacientes com infecção por Helicobacter pylori dispepsia funcional foram randomizados para receber omeprazol, amoxicilina e claritromicina (grupo antibiótico) ou omeprazol mais placebo (grupo controle) por 10 dias. Os pacientes foram avaliados na baseline e 12 meses após. O principal desfecho do estudo foi avaliar a mudança no status de ansiedade e depressão após 12 meses nos dois grupos. Resultados – Foram incluídos 234 pacientes, 115 no grupo antibiótico e 119 no grupo controle. As informações do Hospital Anxiety and Depression Scale e pesquisa da bactéria foram obtidas de 213 pacientes (91,03%) na visita de 12 meses. Com respeito à avaliação da ansiedade após 12 meses os resultados foram: no grupo antibiótico 66,3%(69/104) não modificaram o status da ansiedade, 13,5% (14/104) que não apresentavam passaram a apresentar ansiedade, 20,2%(21/104) que apresentavam ansiedade passaram a não apresentar; no grupo controle 70,6% (77/109), 7,3% (8/109) e 22,0% (24/109) respectivamente. Resultando em um valor de P de 0,340. Quanto a depressão após 12 meses os resultados foram: no grupo antibiótico 71,2%(74/104) não modificaram o status da depressão, 18,3% (19/104) que não apresentavam passaram a apresentar depressão, 10,6%(11/104) que apresentavam depressão passaram a não apresentar; no grupo controle 69,7% (76/109), 10,1% (11/109) e 20,2% (22/109) respectivamente. Resultando em um valor de P de 0,057. Conclusão – Não foi observada relação estatística significativa entre o tratamento do Helicobacter pylori e a mudança de status de ansiedade e depressão nos sujeitos. / Background – The association of infection due to Helicobacter pylori bacteria with some gastric diseases, such as gastric ulcer and gastric cancer, is well defined. However, the association of the bacterium with other diseases, especially extra gastric ones, is not. Methods – We evaluated anxiety and depression and the presence of Helicobacter pylori through endoscopy, in subjects who participated in a study assessing the effects of Helicobacter pylori eradication, in functional dyspepsia. Subjects with Helicobacter pylori infection, suffering functional dyspepsia, were randomized to receive omeprazol, amoxicillin and claritromicin (antibiotic group) or omeprazol plus placebos (control group) during 10 days. Subjects were evaluated at baseline and after 12 months. The main outcome of the study was to assess the anxiety and depression change of status, after 12 months, in both groups. Results – We included 234 subjects: 115 in the antibiotic group and 119 in the control group. We obtained HADS information and bacteria research from 213 subjects (91.03%) at the 12 months visit. Regarding the 12-month anxiety assessment the results were: 66.3% (69/104) did not modify the anxiety status, 13.5% (14/104) who did not present previous signs of anxiety began showing signs of anxiety and 20.2% (21/104) who presented anxiety, ceased to present the condition in the antibiotic group; in the control group the results was 70.6% (77/109), 7.3% (8/109) and 22.0% (24/109) respectively, resulting in a P value of 0,340. As for depression, after 12 months, the results were: 71.2% (74/104) did not modify the depression status, 18.3% (19/104) who did not present previous signs of anxiety began showing signs of depression and 10.6% (11/104) who presented depression, ceased to present the condition in the antibiotic group; in the control group the results was 69.7% (76/109), 10.1% (11/109) and 20.2% (22/109) respectively, resulting in a P value of 0,057. Conclusion – No significant statistical value was observed between the treatment for Helicobacter pylori and the changing status of anxiety and depression in the subjects.

La bactérie Helicobacter pylori est responsable de l’infection la plus répandue dans la population mondiale. Cette bactérie est considérée comme le principal agent étiologique de la gastrite chronique, de l’ulcère duodénal et du cancer gastrique non-héréditaire. La thérapeutique prescrite pour l’éradication de cette infection est inefficace pour un nombre de plus en plus élevé de patients, dû à l’induction constante de souches résistantes aux antibiotiques habituellement prescrits.H. pylori a aussi d’autres façons d’assurer sa survie dans le milieu gastrique que l’induction de résistances aux antibiotiques. En particulier en intéragissant avec le cholestérol des cellules épithéliales gastriques. De ce fait, l’utilisation de molécules inhibitrices de la croissance de H. pylori, autres que les antibiotiques classiques, est une stratégie importante pour combattre cette infection.L’objectif majeur de ce travail a été de mettre en évidence de nouvelles molécules inhibitrices de la croissance et de la viabilité de H. pylori, permettant ainsi le développement de solutions alternatives à la thérapeutique classiquement utilisée. Les acides gras polyinsaturés et l’acide docosahexaenoic (DHA) se mettre en lumière pour réunir incontestable propriété anti-inflammatoires et anti-tumoral. Nous avons émis l’hypothèse que le DHA influence la survie de H. pylori, et peut moduler la disponibilité des acides gras et du cholestérol cellulaires.Nos résultats montrent que le DHA inhibe la croissance de H. pylori in vitro et affecte sa capacité à coloniser la muqueuse gastrique dans le modèle souris. Dans ces conditions, le DHA diminue la réponse inflammatoire gastrique induite par l’infection. Au niveau des cellules épithéliales gastriques, des modifications du profil des acides gras et du cholestérol avec des conséquences sur le métabolisme et la signalisation cellulaire sont observées. De plus, un traitement antibiotique classique combiné à une administration de DHA aux souris infectées diminue de façon drastique la récidive de l’infection. En conclusion, cette étude démontre un effet inhibiteur du DHA sur l’infection par H. pylori et sa récidive. Ces résultats justifient la proposition du DHA comme coadjuvant thérapeutique, constituant ainsi une stratégie prophylactique alternative de l’éradication de l’infection par H. pylori. / H. pylori infection is extremely common worldwide and is recognized as a major etiological factor in chronic active gastritis, gastric duodenal ulcers and gastric cancer development. H. pylori eradication treatment has not changed to a large extent in the last decades and can raise some concern mainly due to recurrence of infection, and most importantly, acquired resistance to classically used antibiotics. In this context, the use of compounds other than antibiotics that could decrease H. pylori infection in a safe way could provide an alternative to tackle this problem. It is known that H. pylori extracts cholesterol from host cell-membrane rafts, modifies it into an α-glycosylated form, and uses this mechanism to increase its survival. The main aim of this thesis work was to explore the role of different non-antibiotic molecules in inhibiting H. pylori growth. Among molecules known to affect in vitro H. pylori growth and viability are certain polyunsaturated fatty acids (PUFAs). Within the many molecules available, we concentrated our efforts on the study of docosahexaenoic acid (DHA). We also pursued the hypothesis that DHA affects survival of H. pylori by modulating the host epithelial cell levels of fatty acids and cholesterol availability.Our results show that DHA inhibits H. pylori growth both in vitro and in vivo, and attenuates the host inflammatory response. Additionally, we demonstrate that DHA induces morphological and cell wall protein composition changes that altogether decrease bacteria-gastric epithelial cell adherence, inflammation and survival. Also, we demonstrated that DHA alters cholesterol levels in epithelial cells, thereby influencing H. pylori ability to uptake and use epithelial cholesterol. This will ultimately impair H. pylori survival. Importantly, the combination of DHA and antibiotic standard treatment decreased the recurrence of H. pylori infection in a mouse model. Our results have gathered important evidence to pave the way for DHA use in the clinical setting and in prophylactic/preventive strategies against H. pylori infection.

by Enders Kwok Wai Ng. / Thesis (M.D.)--Chinese University of Hong Kong, [1999]. / Includes bibliographical references (p. 178-203). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Introdução: A reinfecção pelo Helicobacter pylori em países em desenvolvimento parece ser maior do que nos países desenvolvidos. O retratamento da bactéria e o controle periódico de cura são necessários, quando elevadas taxas de reinfecção são verificadas. O objetivo do trabalho foi determinar a taxa anual de reinfecção, em pacientes brasileiros com úlcera péptica, num seguimento de cinco anos. Métodos: Pacientes com úlcera péptica, diagnosticada por endoscopia digestiva alta e infecção pelo Helicobacter pylori documentada por histologia, teste da urease, reação em cadeia da polimerase e teste respiratório, foram tratados para erradicação da bactéria. A cura da infecção foi verificada com os mesmos exames, três meses após o término do tratamento. Avaliação clínica e teste respiratório foram realizados aos seis e nove meses. Com um ano de seguimento, endoscopia, exame histológico, teste da urease, reação em cadeia da polimerase e teste respiratório foram novamente realizados. Até o quinto ano de seguimento, foram feitas consultas semestrais e testes respiratórios anuais. Na inclusão dos pacientes e nos reinfectados foram estudados 15 diferentes genes da bactéria. Resultados; Cento e quarenta e sete pacientes foram seguidos: 19 por um ano, oito por dois anos, quatro por três anos, cinco por quatro anos e 98 por cinco anos, num total de 557 pacientes/ano. Não ocorreu reinfecção no primeiro ano. No segundo ano, dois pacientes se reinfectaram, no terceiro quatro pacientes, no quarto três pacientes e no quinto um paciente, num total de 10 reinfecções. A taxa anual de reinfecção por pacientes/ano foi de 1,8%. Conclusão: O Brasil, um país em desenvolvimento com alta prevalência da infecção pelo Helicobacter pylori, apresenta uma taxa de reinfecção semelhante à dos países desenvolvidos / Introduction: The Helicobacter pylori reinfection rate seems to be higher in developing countries, than in developed ones. If a high reinfection rate is verified, periodical exams and bacterial retreatment would be necessary. The objective of this study was to determine the annual reinfection rate of Helicobacter pylori, in Brazilian patients with peptic ulcer disease, in a five year follow-up. Methods: Patients, with peptic ulcer disease diagnosed by upper digestive endoscopy and Helicobacter pylori infection verified by histological examination, rapid urease test, polymerase chain reaction and urea breath test, were treated for bacterial eradication. The infection cure was determined by the same proceedings, three months after the treatments end. Clinical evaluation and urea breath test were performed at sixth and ninth months of the follow-up. At one year of the follow-up, upper digestive endoscopy, histological examination, rapid urea test, polymerase chain reaction and urea breath test were repeated. Up to the fifth year of follow-up semester clinical evaluation and annual urea breath test were performed. All the patients included in the study and all the reinfected patients were tested for fifteen different genes of the Helicobacter pylori. Results: One hundred and forty-seven patients were followed: nineteen patients for one year, eight patients for two years, four patients for three years, five patients for four years and ninety-eight patients for five years. The total of patients/years follow-up was 557. Reinfection did not occur in the first year of the follow-up. In the second year, two patients became reinfected; in the third year, four patients; in the fourth, three and in the fifth, one patient. The total of reinfected patients was 10. The annual reinfection rate was 1,8%. Conclusion: Brazil, a developing country, presents a high prevalence of Helicobacter pylori infection. Nonetheless, the reinfection rate is similar to the developed countries.

by Chan Chi Wai, Michael. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 238-272). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.