CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A infecÃÃo pelo Helicobacter pylori à uma das infecÃÃes mais prevalentes do mundo. Existem fortes evidÃncias de que a transmissÃo dessa bactÃria ocorre na infÃncia, e estudos epidemiolÃgicos em crianÃas sÃo essenciais para que se entenda os aspectos relacionados com a dinÃmica da infecÃÃo e suas conseqÃÃncias. Previamente pesquisadores vinculados ao LaboratÃrio de Gastroenterologia da Universidade Federal do Cearà avaliaram a prevalÃncia de H.pylori em crianÃas atà quatorze anos de idade em uma comunidade fixa de baixa renda, denominada Parque UniversitÃrio. No presente estudo, foi realizado outro corte transversal aproximadamente oito anos apÃs a anÃlise inicial nesse mesmo grupo de crianÃas, com o objetivo de avaliar a dinÃmica da infecÃÃo e se as crianÃas infectadas sÃo mais susceptÃveis a terem anemia, deficiÃncia de ferrritina, folato e vitamina B12 do que as nÃo infectadas. A infecÃÃo por H. pylori foi avaliada em 133 crianÃas por meio do teste respiratÃrio, previamente validado para a populaÃÃo brasileira. Foram coletadas amostras de sangue em 108 crianÃas para a realizaÃÃo de hemograma e a dosagem de concentraÃÃes sÃricas de ferritina, vitamina B12 e folato por meio da tÃcnica de quimioluminescencia utilizando Immulite  2000. A prevalÃncia da infecÃÃo por H. pylori nas crianÃas foi de 53,4% (71/133) em 2000, enquanto a prevalÃncia de H. pylori nas mesmas crianÃas em 2008 foi de 64,7% (86/133). ApÃs 8 anos, a maioria (88,7%) das crianÃas H. pylori positivas permaneceram infectadas. A taxa de aquisiÃÃo da infecÃÃo por H. pylori apÃs 8 anos da anÃlise inicial foi de 17,3% (23/133) e a taxa de cura espontÃnea da infecÃÃo foi de 6% (8/133). As taxas de aquisiÃÃo foram maiores em crianÃas com idade de 10 anos ou menos, atingindo 30,2% em crianÃas menores de 5 anos. A prevalÃncia da infecÃÃo em crianÃas com menos de 5 anos aumentou de 39.6% em 2000 para 62,2% em 2008 (p=0,020). NÃo houve diferenÃa estatisticamente significante entre anemia, concentraÃÃes sÃricas de ferritina, vitamina B12 e folato em indivÃduos H. pylori positivos comparados aos H. pylori negativos. Em conclusÃo, a infecÃÃo por H. pylori à adquirida precocemente na infÃncia, com altas taxas de aquisiÃÃo em crianÃas com 10 anos ou menos. NÃo foi encontrado associaÃÃo entre a infecÃÃo por H. pylori e anemia, nÃveis sÃricos de vitamina B12, ferritina e folato / A infecÃÃo pelo Helicobacter pylori à uma das infecÃÃes mais prevalentes do mundo. Existem fortes evidÃncias de que a transmissÃo dessa bactÃria ocorre na infÃncia, e estudos epidemiolÃgicos em crianÃas sÃo essenciais para que se entenda os aspectos relacionados com a dinÃmica da infecÃÃo e suas conseqÃÃncias. Previamente pesquisadores vinculados ao LaboratÃrio de Gastroenterologia da Universidade Federal do Cearà avaliaram a prevalÃncia de H.pylori em crianÃas atà quatorze anos de idade em uma comunidade fixa de baixa renda, denominada Parque UniversitÃrio. No presente estudo, foi realizado outro corte transversal aproximadamente oito anos apÃs a anÃlise inicial nesse mesmo grupo de crianÃas, com o objetivo de avaliar a dinÃmica da infecÃÃo e se as crianÃas infectadas sÃo mais susceptÃveis a terem anemia, deficiÃncia de ferrritina, folato e vitamina B12 do que as nÃo infectadas. A infecÃÃo por H. pylori foi avaliada em 133 crianÃas por meio do teste respiratÃrio, previamente validado para a populaÃÃo brasileira. Foram coletadas amostras de sangue em 108 crianÃas para a realizaÃÃo de hemograma e a dosagem de concentraÃÃes sÃricas de ferritina, vitamina B12 e folato por meio da tÃcnica de quimioluminescencia utilizando Immulite  2000. A prevalÃncia da infecÃÃo por H. pylori nas crianÃas foi de 53,4% (71/133) em 2000, enquanto a prevalÃncia de H. pylori nas mesmas crianÃas em 2008 foi de 64,7% (86/133). ApÃs 8 anos, a maioria (88,7%) das crianÃas H. pylori positivas permaneceram infectadas. A taxa de aquisiÃÃo da infecÃÃo por H. pylori apÃs 8 anos da anÃlise inicial foi de 17,3% (23/133) e a taxa de cura espontÃnea da infecÃÃo foi de 6% (8/133). As taxas de aquisiÃÃo foram maiores em crianÃas com idade de 10 anos ou menos, atingindo 30,2% em crianÃas menores de 5 anos. A prevalÃncia da infecÃÃo em crianÃas com menos de 5 anos aumentou de 39.6% em 2000 para 62,2% em 2008 (p=0,020). NÃo houve diferenÃa estatisticamente significante entre anemia, concentraÃÃes sÃricas de ferritina, vitamina B12 e folato em indivÃduos H. pylori positivos comparados aos H. pylori negativos. Em conclusÃo, a infecÃÃo por H. pylori à adquirida precocemente na infÃncia, com altas taxas de aquisiÃÃo em crianÃas com 10 anos ou menos. NÃo foi encontrado associaÃÃo entre a infecÃÃo por H. pylori e anemia, nÃveis sÃricos de vitamina B12, ferritina e folato / Helicobacter pylori infection is one the most prevalent infections in the world. There is a strong evidence that it is acquired in childhood. Therefore epidemiological studies in children are essential to understand the dynamics of this infection and also to evaluate the possible consequences of H. pylori infection in children. Previously it was evaluated the prevalence of H. pylori in children up to fourteen years of age at a low-income community called Parque UniversitÃrio. The present study is a cohort of a group of children eight years after the first study, with the aim of evaluating the dynamics of H. pylori infection and also to check if infected children are more likely to have anemia and ferrritin, folate and vitamin B12 deficiency when compared to non-infected children. The infection was evaluated in 133 individuals through the 13C urea breath test, previously validated for the brazilian population. Blood was sampled from 108 individuals to dose levels of ferritin, vitamin B12 and folate by chemiluminescence using Immulite  2000 and hemoglobin. The prevalence of H. pylori infection in the individuals was 53.4% (71/133) in 2000, while the prevalence of H. pylori in the same group in 2008 was 64.7% (86/133). It was found that after eight years, the majority (88.7%) of H. pylori positive individuals remained positive for infection. The rate of acquisition of infection by H. pylori after 8 years of the initial analysis was 17.3% (23/133) and the rate of loss of infection was 6% (8/133). The acquisition rates were higher in children younger than 10 years, reaching 30.2% in children younger than 5 years. The prevalence of infection in children under 5 years increased from 39.6% in 2000 to 62.2% in 2008 (p = 0.020). There was no statistically significant difference between anemia, serum ferritin, vitamin B12 and folate in H. pylori-positive individuals compared to H. pylori-negative. In conclusion, H. pylori infection is acquired early in childhood, with high rates of acquisition in children under 10 years. There was no association between H. pylori infection and anemia, serum vitamin B12 and folate levels among the individuals studied. / Helicobacter pylori infection is one the most prevalent infections in the world. There is a strong evidence that it is acquired in childhood. Therefore epidemiological studies in children are essential to understand the dynamics of this infection and also to evaluate the possible consequences of H. pylori infection in children. Previously it was evaluated the prevalence of H. pylori in children up to fourteen years of age at a low-income community called Parque UniversitÃrio. The present study is a cohort of a group of children eight years after the first study, with the aim of evaluating the dynamics of H. pylori infection and also to check if infected children are more likely to have anemia and ferrritin, folate and vitamin B12 deficiency when compared to non-infected children. The infection was evaluated in 133 individuals through the 13C urea breath test, previously validated for the brazilian population. Blood was sampled from 108 individuals to dose levels of ferritin, vitamin B12 and folate by chemiluminescence using Immulite  2000 and hemoglobin. The prevalence of H. pylori infection in the individuals was 53.4% (71/133) in 2000, while the prevalence of H. pylori in the same group in 2008 was 64.7% (86/133). It was found that after eight years, the majority (88.7%) of H. pylori positive individuals remained positive for infection. The rate of acquisition of infection by H. pylori after 8 years of the initial analysis was 17.3% (23/133) and the rate of loss of infection was 6% (8/133). The acquisition rates were higher in children younger than 10 years, reaching 30.2% in children younger than 5 years. The prevalence of infection in children under 5 years increased from 39.6% in 2000 to 62.2% in 2008 (p = 0.020). There was no statistically significant difference between anemia, serum ferritin, vitamin B12 and folate in H. pylori-positive individuals compared to H. pylori-negative. In conclusion, H. pylori infection is acquired early in childhood, with high rates of acquisition in children under 10 years. There was no association between H. pylori infection and anemia, serum vitamin B12 and folate levels among the individuals studied.

Caesalpinia pyramidalis Tul. (Fabaceae), known as catingueira , have been used in folk medicine for the treatment of various disorders such as gastritis, heartburn, indigestion, and stomach ache. However, the gastroprotective properties of this species have not yet been studied. The ethanol extract of C. pyramidalis inner bark was used in rats via oral, at the doses of 30, 100, and 300 mg/kg. The antiulcer assays were performed using the ethanol- and nonsteroidal anti-inflammatory drug-induced ulcer protocols. Gastric secretion parameters (volume, pH, and total acidity) were also evaluated by the pylorus ligated model, and the mucus in the gastric content was determined. The anti-Helicobacter pylori activity of the ethanol extract of Caesalpinia pyramidalis was performed using the agar-well diffusion and broth microdilution methods to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values. The ethanol extract (30, 100, and 300 mg/kg) produced dose dependent inhibition (p < 0.01) on the ulcer lesion index in the ethanol-induced ulcer model. The ethanol extract (30, 100, and 300 mg/kg) also reduced (p < 0.001) the ulcer index in the indomethacin-induced ulcer model. In the model ligature pylorus, the treatment with C. pyramidalis ethanol extract failed to significantly change the gastric secretion parameters. However, after treatment with the ethanol extract of C. pyramidalis (30, 100, and 300 mg/kg), there was a significant increase (p < 0.05) in mucus production. The ethanol extract of Caesalpinia pyramidalis had an anti-Helicobacter pylori activity with a MIC of 625 ìg/mL and a MBC of 10.000 ìg/mL. Collectively, the present results suggest that the ethanol extract of C. pyramidalis display gastroprotective actions, supporting the folkloric usage of the plant to treat various gastrointestinal disturbances. / Caesalpinia pyramidalis Tul. (Fabaceae), conhecida popularmente como gcatingueira h, tem sido usada pela medicina popular para tratamento de diversas desordens fisiologicas como gastrite, indigestao e dores estomacais. Entretanto, a acao gastroprotetora dessa especie ainda nao foi estudada. O extrato etanolico da entrecasca (EEE) da Caesalpinia pyramidalis foi administrado em ratos pela via oral, nas doses de 30, 100 e 300 mg/kg. Para avaliacao da atividade gastroprotetora foram utilizados os modelos de ulceras agudas induzidas por etanol e anti-inflamatorio nao esteroidal (AINE). Parametros da secrecao gastrica (volume, pH e acidez total) e producao de muco gastrico tambem foram avaliados pela tecnica de ligadura de piloro. A atividade antimicrobiana do EEE da Caesalpinia pyramidalis foi mensurada atraves da tecnica de difusao em agar e calculados os valores da concentracao inibitoria minima (CIM) e da concentracao bactericida minima (CBM) pela tecnica de microdiluicao em placa. O EEE da Caesalpinia pyramidalis nas doses de 30, 100 e 300 mg/kg inibiu de maneira dose dependente (p < 0,01) o indice de lesoes ulcerativas no modelo de ulceras induzidas por etanol. No modelo de ulceras induzidas por AINE, o extrato em todas as doses utilizadas, tambem reduziu o indice de lesoes ulcerativas (p < 0,001). No modelo de ligadura de piloro, o tratamento com o EEE da Caesalpinia pyramidalis nao foi capaz de modificar os parametros da secrecao gastrica. Entretanto, apos tratamento com o EEE da Caesalpinia pyramidalis (30, 100 e 300 mg/kg) observou-se um aumento significativo (p < 0,05) na producao do muco gastrico. O EEE da Caesalpinia pyramidalis possui atividade antimicrobiana contra o Helicobacter pylori com CIM de 625 Êg/mL e CBM de 10.000 Êg/mL. Os resultados deste estudo sugerem que o EEE da Caesalpinia pyramidalis possui acao gastroprotetora, desta forma validando a utilizacao popular desta planta no tratamento de disturbios gastrointestinais.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / O H. pylori infecta atualmente metade da populaÃÃo mundial e à relacionado com o desenvolvimento das afecÃÃes gÃstricas. O Enteroteste à um mÃtodo minimamente invasivo que pode ser usado para detecÃÃo do H. pylori por meio nÃo endoscÃpico. O objetivo deste estudo foi avaliar a sensibilidade e especificidade do Enteroteste frente ao Teste RespiratÃrio e estudar o perfil genÃtico das cepas de H. pylori em indivÃduos residentes na comunidade Parque UniversitÃrio. O suco gÃstrico foi colhido a partir do Enteroteste, e realizada a cultura e a extraÃÃo do DNA do H. pylori. A genotipagem das cepas foi realizada atravÃs da tÃcnica de PCR e os sÃtios de fosforilaÃÃo EPIYA de sequenciamento Participaram do estudo 50 indivÃduos, 43 positivos e 7 negativos para a infecÃÃo pelo H. pylori atravÃs do Teste RespiratÃrio. A cultura e o PCR a partir do Enteroteste apresentaram sensibilidade de 86% e 77%, respectivamente, com especificidade de 100% em ambos os mÃtodos 33 cepas foram genotipadas como vacA positivas, sendo 39,4% vacA s1m1; 15,2% vacA s1m2; 18,2% vacA s2m2 27,2% com perfil vacA s com ausÃncia do alelo m. Foram cagA positivas 66,6% das cepas sendo 54,5% com perfil EPIYA-ABC, 41,0% EPIYA-ABCC e 4,5% EPIYA-AB. O Enteroteste mostrou-se um mÃtodo confiÃvel com boa sensibilidade e especificidade para identificaÃÃo do H. pylori atravÃs do cultivo e da tÃcnica de PCR. A maioria das cepas expressou o alelo vacA s1, com predomÃnio do subtipo s1b e da combinaÃÃo alÃlica s1m1. Mais da metade das cepas estudadas expressaram o gene cagA e a maioria dessas cepas tinham 3 ou 4 sÃtios de fosforilaÃÃo, com perfil EPIYA-ABC ou EPIYA-ABCC. O perfil genÃtico apresentado por essas cepas à o descrito para a AmÃrica do Sul e diferente do padrÃo AsiÃtico, a maioria das cepas circulantes na comunidade tÃm importante potencial patogÃnico. O Enteroteste à um mÃtodo seguro, sensÃvel e especÃfico para detecÃÃo do H. pylori. / The H. pylori infect currently half of the worldâs population and is related to the development of gastric disorders. The Enterotest is a minimally invasive method that can be used for detection of H. pylori through endoscopic not. The objective of this study was to evaluate the sensitivity and specificity of Enterotest opposite the Respiratory Testing and studying the genetic profile of strains of H. pylori individuals resident in the Community College Park. The gastric juice was collected from Enteroteste, and held the culture and the extraction of DNA from H. pylori. The genotyping of strains was carried out by the PCR technique and phosphorylation sites EPIYA sequencing participated in the study 50 individuals, positive and negative 7 43 for infection H. pylori through Respiratory Test. Culture and PCR from Enteroteste showed sensitivity of 86% and 77%, respectively, with 100% specificity in both methods. 33 strains were positive, genotipadas as Cow being 39.4% Cow s1m1; 15.2% Cow s1m2; 18.2% Cow s2m2 27.2% with Cow s profile with absence of allele m. Were positive 66.6% Bran of strains being profiled 54.5% EPIYA-ABC, 41.0% EPIYA-ABCC and 4.5% EPIYA-AB. The Enterotest proved to be a reliable method with great sensitivity and specificity for identification of H. pylori through cultivation and PCR technique. Most strains expressed alelo Cow s1, with a predominance of subtype s1b and allele combination s1m1. More than half of the studied strains expressed gene Shit and most of these strains were 3 or 4, phosphorylation sites profiled EPIYA-ABC or EPIYA-ABCC. The genetic profile presented by these strains is described for South America and nonstandard Asia, most strains circulating in the community have important potential pathogenic. The Enterotest is a safe method, sensitive and specific detection of H. pylori.

Philosophiae Doctor - PhD / Species of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals; and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42ºC as well as at ambient room temperature (±222ºC). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4ºC for up to 297 days. / South Africa

The stem bark of Peltophorum africanum and Bridelia micrantha are used in South Africa traditional medicine for treatment of intestinal parasites, relieve problems and human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS). The growing problem of antibiotic resistance by Helicobacter pylori the major etiological agent in gastritis, gastric cancer, peptic and gastric ulcer demands the search for novel compounds from plant based sources. This study was aimed to determine the antimicrobial activity of five solvent (ethylacetate, acetone, ethanol, methanol and water) extracts of the stem bark of P. africanum and B. micrantha on clinical strains of H. pylori in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. H. pylori strains were isolated from patients presenting with gastric related morbidities at the Livingstone Hospital, Port Elizabeth for endoscopy and confirmed following standard microbiology procedures. The plant extracts including clarithromycin were tested against 31 clinical strains of H. pylori by the agar well diffusion method. The most potent extract was evaluated by the microdilution method to determine the Minimum Inhibitory Concentration (MIC50&90), followed by the rate of kill. Preliminary phytochemical analysis was carried out. The one way ANOVA test was used to statistically analyse the results. All the extracts demonstrated anti-H. pylori activity with zone diameters of inhibition that ranged from 0 to 23 mm for the extracts and 0 to 35 mm for clarithromycin. Marked susceptibility (100%) was recorded for the ethyl acetate extract of P. africanum (P. afr. EA) and the acetone extract of B. micrantha (B. mic. A), which were statistically significant (P < 0.05) compared to all other extracts and clarithromycin. For B. micrantha ethyl acetate extract, 93.5 percent susceptibility was observed while for the control iv antibiotic, clarithromycin it was 58.1 percent. The MIC50 ranged from 0.0048 to 0.313 mg/mL for P. afr. EA, and from 0.0048 to 0.156 mg/mL for B. mic. EA; MIC90 ranged from 0.156 mg/mL to 0.625 mg/mL and 0.0048 to 2.5 mg/mL for P. afr. EA and B. mic. EA respectively. There was a significant statistical difference observed in potency of both P. afr. EA and B. mic. A compared to the two antibiotics (P < 0.05). One hundred percent killing by P. afr EA was observed at 0.05 mg/mL (½ x MIC) and 0.2 mg/mL (2 x MIC) in 66 h for strain PE466C and PE252C respectively. For B. mic. EA, 100 percent killing effect of both strains (PE430C and PE369C) was observed at 0.1 mg/mL (2 x MIC) in 66 h. Qualitative phytochemical analysis confirmed the presence of alkaloids, flavonoids, steroids, tannins and saponins in the ethyl acetate extracts of both plants, which could be a potential template of lead molecule for the design of new anti- Helicobacter pylori therapies.

Existen estudios que encontraron mayor prevalencia de anticuerpos contra Helicobacter pylori (Hp) en gestantes con hiperémesis gravídica, en comparación con mujeres embarazadas asintomáticas. Objetivos. Determinar la relación entre la seropositividad a Hp y la presencia de náusea y vómitos durante las primeras 20 semanas del embarazo en gestantes de una red hospitalaria, entre marzo y diciembre de 2015. Material y métodos. Estudio de casos y controles no pareado en gestantes hasta las 20 semanas de embarazo. El índice de Rhodes para náusea y vómitos clasificó a las gestantes en casos (9-40 puntos) y en controles (8 puntos). La seropositividad a Hp fue definida como IgG ≥ 1,1 U/mL. La asociación entre Hp y náusea y vómitos del embarazo se determinó con el análisis de regresión logística, controlando por edad, paridad, edad gestacional y nivel socioeconómico. Resultados. Un total de 108 pacientes fueron incluidas, 21 controles y 87 casos. No hubo diferencias significativas en edad (p = 0,916), paridad (p = 0,18) y nivel socioeconómico (p = 0,36). La seropositividad a Hp en los casos fue 78,2% (68/87) y en los controles 61,9% (13/21). En el análisis de regresión logística, los casos presentaron mayor riesgo de seropositividad a Hp que los controles (OR = 3,05; IC 95%: 0,92-10,1; p = 0,068), pero no fue significativa. Conclusiones. Las pacientes con náusea y vómitos en las primeras 20 semanas de gestación tuvieron un mayor riesgo de haber estado expuestas a Hp, aunque esta relación no fue significativa debido al pequeño tamaño de muestra. / There are studies that found a higher prevalence of antibodies against Helicobacter pylori (Hp) in patients with hyperemesis gravidarum, compared to asymptomatic pregnant women. Objective. To determine the relationship between seropositivity to Hp and the presence of nausea and vomiting during the first 20 weeks of gestation in pregnant women of a hospital network, from March to December 2015. Material and methods. Unmatched case-control studies in pregnant women until 20 weeks of gestation. The Rhodes’ index for nausea and vomiting classified pregnant women in cases (9-40 points) and controls (8 points). Hp seropositivity was defined as IgG ≥ 1.1 U/mL. The association between Hp and nausea and vomiting of pregnancy was determined by logistic regression analysis controlling for age, parity, gestational age and socioeconomic status. Results. A total of 108 patients were included, 21 controls and 87 cases. There were no significant differences in age (p = 0.916), parity (p = 0.18) and socioeconomic status (p = 0.36). Hp seropositivity in cases was 78.2% (68/87) and controls 61.9% (13/21). In the logistic regression analysis, cases had higher risk of Hp seropositivity than controls (OR = 3.05; 95% CI: 0.92-10.1; p = 0.068), but was not significant. Conclusions. Patients with nausea and vomiting in the first 20 weeks of gestation had a higher risk of having been exposed to Hp, although this relationship was not significant due to the small sample size.

Helicobacter pylori is one of the most potential and successful human pathogen which colonizes atleast 50% of world population. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by Helicobacter pylori to generate variation, where presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. An important feature of H. pylori biology is the presence of a large number of Restriction-Modification (R-M) systems in its genome. Till date, seven strains have been completely sequenced and each have more than 20 R-M systems. The presence of homopolymeric nucleotide or dinucleotide repeats in many of R-M systems make them an interesting subject for investigation. Here, we show that hp0051 which codes for a C5 cytosine methyltransferase from H. pylori is a hypermutable gene, which undergoes random mutations. In addition it exhibits phase variation due to presence of a dinucleotide (AG) repeat which results in a truncated protein. hp0051 homologs were amplified and sequenced from different clinical isolates of H. pylori. Sequence analysis showed that hp0051 homologs from 23 clinical isolates are different from each other suggesting a hypervariable nature of the sequence. It was observed that when over expressed in E. coli hp0051 undergoes random mutations. These mutations give rise to different variants of HP0051 with different biochemical properties. Different variants of HP0051 were biochemically characterized. All variants recognize 5´-CCTC-3´ and methylate the first cytosine. A few of the isoforms exhibit degeneracy in the recognition site as they recognize 5´- CNCC-3´ (N being any nucleotide) and methylates third cytosine. Molecular modelling studies suggest that HP0051 has two domains, one large domain having catalytic and AdoMet binding motifs and small domain having target recognition domain. DNA sequencing, peptide finger mapping, MALDI MS-MS and CD have been used to establish the differences between the different isoforms of HP0051. Interestingly when a mutant protein which lacks methylation activity was expressed in E.coli random mutations were not observed. To understand the role of methylation in the occurrence of random mutations, microarray analysis was done. Microarray analysis have shown that the overexpression of HP0051 results in the down regulation of deoxyadenine methyltransferase (dam) in E.coli. Microarray data were further authenticated by RT PCR analysis. dam plays a vital role in mismatch repair pathway and down regulation of dam results in enhanced mutation rates. A large number of clinical isolates were analysed for the presence of hp0051 and hp0051 was found to be present in 83% of strains obtained from patients compared to 25 % of strains from healthy volunteers. Single colonies obtained from the same patient were analysed and it was found that variation in hp0051 exists within a patient also. Deletion of an orphan C5 cytosine methyltransferase, hp0051 in H. pylori strains 26695, SS1 and 98.4 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. 98.4∆hp0051 mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. H. pylori strain 26695∆hp0051 is more motile than the wild- type. hp0051 from strain 26695 is able to complement mutant SS1 and 98.4 strains. This study highlights the possible significance of cytosine methylation in the physiology of H. pylori. hp0050 is a N6 DNA adenine methyltransferase which overlaps with the hp0051 ORF .hp0050 was cloned, over expressed and purified to near homogeneity. It recognizes the sequence 5´GRRG 3´ (where R is A or G) and most intriguingly methylates both adenines when R is A (5´GAAG 3´). Kinetic analysis suggest a non processive (repeated hit) mechanism of methylation in which HP0050 methyltransferase methylates one adenine at a time in sequence 5´GAAG 3´. Interestingly, HP0050 homologs from two clinical strains PG227 and 128 methylate only 5´GAGG 3´ compared to 5´GRRG 3´ in strain 26695. HP0050 MTase is highly conserved as it is present in more than 90% of strains. Inactivation of hp0050 in strain PG227 resulted in poor growth suggesting its important role in the physiology of Helicobacter pylori. Collectively, these findings provide impetus for exploring the role(s) of this conserved DNA methyltransferase in the cellular processes of Helicobacter pylori. In one of the clinical isolate it was found that hp0051 and hp0050 can code for a single polypeptide due to an insertion mutation. This mutant ( hp0050 and hp0051 fusion ) was cloned, overexpressed and purified. It was found that fusion protein is able to methylate both adenine and cytosine in the cognate sequence. Similarly, hp1369 - hp1370 is a phase variable type III MTase and it belongs to ɛ group of MTases based on the arrangement of motifs. It has a poly G repeat in its ORF and any change in the number of repeats can result in a functional (full length) or non functional (truncated) protein. Within strain 26695, it has 10 G repeat which results in a truncated protein. Addition of a single nucleotide by site directed mutagenesis in the repeat results in a full length functional protein. HP1369_HP1370 fusion protein recognizes and methylates 5´ TCAGC 3´. DNA methyltransferases are known to play a critical role in gene regulation, cell cycle regulation and pathogenesity in a number of pathogens. H. pylori genome is rich in DNA methyltransferases and this study shows that these methyltransferases exhibit unique features like phase - variation and polymorphism .We propose that high degree of variation that exists in these methyltransferases could play a vital role in enhancing the ability of H. pylori to adapt its host.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T12:19:53Z (GMT). No. of bitstreams: 1 Roesler_BrunaMaria_M.pdf: 3428404 bytes, checksum: ee37171bd6740210c88477c4839317fb (MD5) Previous issue date: 2006 / Resumo: O Helicobacter pylori é uma bactéria gram-negativa que está estritamente relacionada com o desenvolvimento de doenças gástricas, inclusive malignas, sendo classificada como carcinógeno do grupo I pela Agência Internacional para Pesquisa do Câncer. Apesar de grande parte da população mundial estar contaminada pela bactéria, a maioria dos indivíduos permanece assintomática. Muitas abordagens são sugeridas para diferenciar os isolados de H. pylori e diversas técnicas de biologia molecular têm sido aplicadas para caracterizar as amostras clínicas de diferentes pacientes. A análise de restrição (RFLP) usada em conjunto com a PCR tem sido amplamente utilizada para a tipagem de isolados clínicos, utilizando vários genes do genoma do H. pylori como alvos para esse método. Padrões de eletroforese da nested PCR e da PCR para as regiões dos genes urease C e urease B do H. pylori foram obtidos com enzimas de restrição e comparados entre pacientes com úlcera péptica e gastrite crônica. Os produtos da nested PCR para urease C foram digeridos com a enzima Mbo I e os produtos da PCR para urease B com a enzima Hae III, sendo os fragmentos obtidos analisados por eletroforese em gel de agarose. Foram encontrados 7 padrões de genotipagem para urease B e 17 padrões para urease C. O grupo prevalente para gastrite crônica obtido com essa última genotipagem foi o denominado grupo ureC MboI G, com fragmentos de 110, 120, 250 e 310 pares de bases, ocorrendo em 13 pacientes (15,7% dos casos). Em relação aos casos de úlcera péptica, o grupo de genotipagem mais prevalente foi o denominado ureC MboI E, com 110, 180 e 500 pares de bases, ocorrendo em 11 pacientes (24,4% do total de casos). O grupo prevalente obtido com a genotipagem para urease B foi o denominado ureB I, com um fragmento único de 750 pares de bases. Esse grupo foi o prevalente tanto para os casos de úlcera péptica (27), com 60,0%, quanto para os de gastrite crônica (53), com 63,9%. Não foram observadas diferenças significativas entre os padrões encontrados para ambas as doenças. A nested PCR para detecção prévia do DNA do H. pylori também foi realizada, obtendo-se 100% de concordância. Os métodos de tipagem dos produtos obtidos por nested PCR e PCR proporcionam um esquema funcional e de grande reprodutibilidade para estudos epidemiológicos e de caracterização de cepas / Abstract: Helicobacter pylori is a gram-negative bacterium that has been implicated as a major human gastric pathogen responsible for gastritis and peptic ulcer disease. It has been classified as a group I carcinogen by the International Agency for Research on Cancer and is regarded as a primary factor for gastric cancer. A significant proportion of the world population is infected with H. pylori, even though most infections are asymptomatic. To isolate H. pylori, many approaches have been presented and several molecular techniques have been applied to separate clinical isolates from different patients. PCR-RFLP analysis has been widely developed for the typing of clinical isolates, with several genes within H. pylori having been targeted by this method. Eletroforesis patterns of nested PCR and PCR to urease C and urease B gene regions of H. pylori were obtained by restriction fragment length polymorphism and compared among patients with peptic ulcer and chronic gastritis. Nested PCR products for urease C were digested with MboI enzyme and PCR products for urease B with Hae III enzyme, and the obtained fragments were analyzed by eletroforesis in agarosis gel. We have encountered 7 genotyping patterns for urease B and 17 patterns for urease C. The prevalent group for chronic gastritis which was obtained with this last genotyping was named ureC MboI G group, with fragments of 110, 120, 250 and 310 base pairs, occurring in 13 patients (15,7% of the cases). Regarding the peptic ulcer cases, the most prevalent genotyping group was named ureC MboI E group, with 110, 180 and 500 base pairs, occurring in 11 patients (24,4% of the cases). The prevalent group obtained with the genotyping for urease B was named ureB I, with only one fragment of 750 base pairs. This group was the prevalent one both in peptic ulcer cases (27) with 60,0%, and in chronic gastritis case (53), with 63,9%. We haven¿t found meaningful differences among the encountered patterns for both diseases. Nested PCR for previous detection of H. pylori DNA has also been performed, and we have obtained 100% of concordance. The typing methods of the products obtained by nested PCR and PCR show a functional scheme and of great reproduction for epidemiologic studies and of H. pylori strains characterization / Mestrado / Mestre em Farmacologia

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13442号 / 論医博第2241号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 藤田 恭之, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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