Made available in DSpace on 2014-12-17T14:13:45Z (GMT). No. of bitstreams: 1 SilvaEL_DISSERT.pdf: 2857936 bytes, checksum: 9a0ed46e2b6c06e351eff5810e5f24d8 (MD5) Previous issue date: 2010-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Magnetic targeting is being investigated as a means of local delivery of drugs, combining precision, minimal surgical intervention, and satisfactory concentration of the drug in the target region. In view of these advantages, it is a promising strategy for improving the pharmacological response. Magnetic particles are attracted by a magnetic field gradient, and drugs bound to them can be driven to their site of action by means of the selective application of magnetic field on the desired area. Helicobacter pylori is the commonest chronic bacterial infection. The treatment of choice has commonly been based upon a triple therapy combining two antibiotics and an anti-secretory agent. Furthermore, an extended-release profile is of utmost importance for these formulations. The aim of this work was to develop a magnetic system containing the antibiotic amoxicillin for oral magnetic drug targeting. First, magnetic particles were produced by coprecipitation of iron salts in alkaline medium. The second step was coating the particles and amoxicillin with Eudragit? S-100 by spray-drying technique. The system obtained demonstrated through the characterization studies carried out a possible oral drug delivery system, consisting in magnetite microparticles and amoxicillin, coated with a polymer acid resistant. This system can be used to deliver drugs to the stomach for treatment of infections in this organ. Another important finding in this work is that it opens new prospects to coat magnetic microparticles by the technique of spray-drying. / A vetoriza??o magn?tica tem sido investigada como uma forma de entrega local de f?rmacos combinando precis?o, m?nima interven??o cir?rgica e concentra??o satisfat?ria do f?rmaco na regi?o de interesse. Part?culas magn?ticas s?o atra?das a partir da aplica??o de um campo magn?tico e f?rmacos associados a essas part?culas podem ser direcionados ao seu s?tio de a??o atrav?s de uma aplica??o seletiva do campo na regi?o de interesse. Helicobacter pylori ? a mais comum causa de infec??o bacteriana cr?nica no est?mago. O tratamento padr?o ? a tripla terapia oral contendo dois antibi?ticos e um inibidor da bomba de pr?tons. Sendo assim, um perfil de libera??o prolongada ? de suma import?ncia para essas formula??es. O objetivo deste trabalho foi desenvolver um sistema magn?tico com potencial emprego na vetoriza??o de antibi?tico por via oral. Inicialmente, part?culas magn?ticas foram produzidas por co-precipita??o de sais de ferro em meio alcalino. Em seguida, as part?culas foram revestidas a partir da dispers?o da suspens?o magn?tica em uma solu??o contendo o pol?mero dissolvido e a amoxicilina, e ent?o submetido ? secagem por aspers?o (spray-drying). Atrav?s das caracteriza??es realizadas p?de-se verificar a obten??o de um potencial sistema para vetoriza??o de f?rmacos por via oral contendo micropart?culas de magnetita e amoxicilina revestidos por um pol?mero gastro-resistente. Adicionalmente, um importante aspecto nesse trabalho ? a abertura de novas perspectivas para o revestimento de micropart?culas magn?ticas atrav?s da t?cnica de spray-drying.

Made available in DSpace on 2014-12-17T14:16:32Z (GMT). No. of bitstreams: 1 DalianaMBOS_DISSERT.pdf: 1178965 bytes, checksum: a8c57235d95835138c537bc774c7af70 (MD5) Previous issue date: 2012-02-27 / The expression of human leukocyte antigen G (HLA-G) and human leukocyte antigen E (HLA-E) in physiological and pathological processes remains unknown, it is believed that these molecules play a fundamental role in the establishment and maintenance of immune tolerance by inhibiting the functions of immunocompetent cells. In literature we found no published study involving the bacterium Helicobacter pylori (H. pylori) with expression of HLA-G and HLA-E. The objective this study is investigated the expression of this protein in gastric biopsies of patients with the bacterium H. pylori. Sixty-four biopsies of the patients with diagnosis of infection by H. pylori were evaluated to expression of HLA-G and HLA-E. The samples were stratified according to the presence of carcinoma or peptic ulcers. Patients without H. pylori were used to control. To investigate the expression of this protein were used immunohistochemistry technique with monoclonal antibody anti-HLA-G and anti-HLA-E. Other criteria such as analysis of the inflammatory infiltrate (hematoxylin-eosin) and identification of H. pylori (Giemsa) were analyzed. We detected HLA-G and HLA-E molecules in the most samples containing ulcer and gastric carcinoma. In negative control group was not detected the presence of HLA-G and HLA-E. The presence of H. pylori seems modulate the expression of HLA-G and HLA-E, favoring the evolution of infection, giving different degrees of gastric lesion in epithelium of these patients / A express?o do ant?geno leucocit?rio humano G (HLA-G) e do ant?geno leucocit?rio humano E (HLA-E) em processos fisiol?gicos e patol?gicos permanece pouco conhecida. Acredita-se que essas mol?culas desempenham papel fundamental no estabelecimento e manuten??o da toler?ncia imunol?gica, inibindo as fun??es das c?lulas imunocompetentes. Na literatura internacional, at? o momento, n?o foi encontrado nenhum estudo publicado correlacionando Helicobacter pylori (H. pylori) com express?o de HLA-G e HLA-E. O presente trabalho tem como objetivo correlacionar a express?o dessas prote?nas em bi?psias g?stricas de pacientes acometidos com H. pylori. Sessenta e quatro esp?cimes g?stricas de pacientes acometidos com H. pylori foram avaliados para express?o de HLA-G e HLA-E. As amostras foram estratificadas de acordo com a presen?a de carcinoma ou de ?lcera p?ptica. Como controle foram analisados esp?cimes g?stricas de pacientes vivos sem H. pylori. Para detectar a express?o dessas mol?culas utilizou-se a t?cnica de imunohistoqu?mica com os anticorpos monoclonais anti-HLA-G e anti-HLA-E. Outros crit?rios como an?lise do infiltrado inflamat?rio (hematoxilina-eosina) e identifica??o do H. pylori (Giemsa) foram analisados. As mol?culas de HLA-G e HLA-E foram detectadas em grande parte das amostras contendo ?lcera e carcinoma g?strico. No grupo controle n?o foi detectada a presen?a de HLA-G e HLA-E, indicando que a bact?ria H. pylori modula a express?o dessas mol?culas no grupo dos pacientes que apresentaram ?lcera ou carcinoma g?strico. A presen?a da bact?ria H. pylori parece modular a express?o do HLA-G e do HLA-E, favorecendo assim a evolu??o da infec??o, o que confere diferentes graus de les?o do epit?lio g?strico desses pacientes

Orientador: Helena Zerlotti Wolf Grotto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T08:24:50Z (GMT). No. of bitstreams: 1 Alvarenga_ElianadaCosta.pdf: 3519502 bytes, checksum: cc024f9bd1176e4c4f56d989bba5a487 (MD5) Previous issue date: 2009 / Resumo: A deficiência de ferro é provavelmente o distúrbio nutricional mais freqüente no mundo. O ferro é um componente essencial da molécula de hemoglobina, da mioglobina e de diversas enzimas. Tem papel fundamental no transporte de oxigênio, na transferência de elétrons e atua como cofator em muitos processos enzimáticos, incluindo a síntese de DNA. Diversos estudos têm mostrado a contribuição da infecção pelo Helicobacter pylori (H. pylori) no desenvolvimento da anemia ferropriva e a associação entre o H. pylori e a diminuição do estoque de ferro. O objetivo do presente trabalho foi verificar a possível associação entre a infecção pelo H. pylori e a deficiência de ferro em um grupo de pacientes adultos. Desse modo pretendeu-se conhecer melhor as alterações hematológicas presentes nos pacientes infectados pelo H. pylori, principalmente as relacionadas ao metabolismo do ferro. Foram estudados 156 pacientes adultos de ambos os sexos que foram submetidos à endoscopia digestiva alta para esclarecimento diagnóstico. Desses 156 pacientes, 125 apresentaram alterações à endoscopia, que justificaram a realização da biópsia gástrica. A avaliação da presença de anemia foi feita pelos dados hematimétricos e pelo conteúdo de hemoglobina dos reticulócitos (Ret-He) e o estado do ferro foi avaliado pelas dosagens de ferro sérico, capacidade de ligação do ferro à transferrina e ferritina sérica. A possível participação da atividade inflamatória na eritropoiese foi verificada pela correlação entre os parâmetros hematimétricos e do estado do ferro com os níveis de proteína C reativa (PC-R). O diagnóstico do H. pylori foi realizado através da análise histológica, teste da urease e teste sorológico (IgG anti- H. pylori). Os 125 pacientes foram separados em H. pylori positivo (n= 75) e negativo (n= 50). Não houve diferença significativa nos valores dos parâmetros hematológicos e bioquímicos, tendo sido diferentes somente os valores de IgG anti-H. pylori e de gastrina sérica, significativamente superiores no grupo H. pylori positivo. Foi observada uma correlação inversa fraca, mas significativa entre os níveis de PC-R e Ret-He e entre gastrina sérica e Ret-He no grupo H. pylori positivo. De acordo com os critérios laboratoriais, dos 125 pacientes apenas 17 (13,6%) mostraram níveis de Hb indicativos de anemia, sendo que desses, 7 (5,6%) mostraram ser positivos para a infecção pelo H. pylori. Não foi observada diferença na freqüência da deficiência de ferro entre os grupos de pacientes infectados pelo H. pylori e os não infectados pelo H. pylori. Dentre os pacientes estudados a gastrite foi confirmada em 110 pacientes, sendo que 74 (67,2%) eram H. pylori positivo. A gastrite foi classificada de acordo com o sistema de Sidney em: moderada/intensa (36.63%), leve (63,37%) e inespecífica (8,1%). Quando os pacientes com gastrite moderada/intensa foram comparados com os com gastrite leve, não houve diferença entre os parâmetros estudados, exceto os valores da titulação de IgG, que foram significativamente superiores no grupo com gastrite moderada/intensa. Portanto, podemos concluir que no grupo de indivíduos estudados a infecção pelo H. pylori e a intensidade da gastrite não foram fatores determinantes ao desenvolvimento da deficiência de ferro. / Abstract: Iron deficiency is probably the most common nutritional disorder in the world. Iron is an essential component of the molecule of hemoglobin, myoglobin and various enzymes. It has a fundamental role in oxygen transport, in electrons transference and acts as a cofactor in many enzymatic processes, including synthesis of DNA. Several studies have shown the contribution of Helicobacter pylori (H. pylori) infection in the development of anemia and the association between H. pylori and reduction of iron store. The objective of this study was to investigate the association between H. pylori infection and iron deficiency in a group of adult patients. Thus we intend to study the hematological alterations in H. pylori infected patients, mainly those related to iron metabolism. We studied 156 adult patients of both sex undergoing routine upper endoscopy. Of these 156 patients, 125 had gastric biopsies stained for H. pylori identification. The evaluation of the presence of anemia was performed by erythrocyte indeces and reticulocyte hemoglobin content (RET-He) and the iron status was assessed by measurements of serum iron, iron binding capacity of the transferrin and serum ferritin levels. Inflammatory activity influence on erythropoiesis was verified by the correlation among erythrocyte parameters and the state of the iron with C-reactive protein (CR-P) levels. The diagnosis of H. pylori was performed by histological, urease and serological (IgG anti-H. pylori) methods. Patients were divided into H. pylori positive (n = 75) and negative (n = 50). There was no significant difference in hematological and biochemical parameters, except for IgG anti-H. pylori values and serum gastrin, significantly higher in H. pylori positive group. There was a weak but significant inverse correlation between CR-P and Ret-He levels and between serum gastrin and Ret-He in H. pylori positive group. According to laboratory criteria, only 17 of 125 patients (13.6%) showed levels of hemoglobin indicative of anemia, 7 of 17 patients were positive for H. pylori infection. There was no difference in the frequency of iron deficiency anemia between H. pylori positive and negative groups. Gastritis was confirmed in 110 patients, of these 74 (67.2%) were H. pylori positive. The gastritis was classified according to the Sydney system as: moderate / severe (36.63%), mild (63.37%) and nonspecific (8.1%). There was no difference between groups with moderate/ severe gastritis and mild gastritis concerning hematologic and biochemical parameters, except the values of the titers of IgG, which were significantly higher in the group with moderate / severe gastritis. Therefore, we conclude that infection by H. pylori and the intensity of gastritis were not determining factors for the development of iron deficiency in the studied group. / Universidade Estadual de Campi / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T23:14:22Z (GMT). No. of bitstreams: 1 Oliveira_TelmaBarbosade_M.pdf: 1175004 bytes, checksum: 5912fb5d3dc25609414bb097190091e2 (MD5) Previous issue date: 2008 / Resumo: O He/icobacter pylori é uma bactéria gram-negativa que está relacionada ao desenvolvimento de doenças gástricas e extragástricas. Dentre as doenças gástricas incluem-se o câncer gástrico, a gastrite crônica, a úlcera péptica e linfoma tipo MALT. Com relação às doenças extragástricas essa bactéria recentemente foi relacionada com a anemia por deficiência de ferro e com algumas doenças autoimunes, como a artrite reumatóide e a púrpura trombocitopênica idiopática. A PCR tem sido uma importante ferramenta para a análise de pequenos fragmentos de DNA, os quais podem, inclusive, ser armazenados por um tempo maior em amostras emblocadas em parafina ou obtidas a fresco à temperatura de -80°C. Dessa maneira, foram estudados 33 pacientes com púrpura trombocitopênica idiopática (PTI) e H.pylori positivo. No que diz respeito à detecção da bactéria pela nested PCR, foi obtido um resultado de 100% de concordância em comparação aos resultados de histologia e teste da urease, usados rotineiramente. Além dos primers para detecção do He/icobacter pylori foram utilizados primers para as regiões do gene urease C e do gene cagA, sendo os fragmentos obtidos analisados em gel de 2% agarose observados sob luz ultravioleta. Foi obtida também uma concordância de 100% para a região do gene urease C. Em relação ao gene cagA, 30,3% desses pacientes apresentaram esse. Dos 33 pacientes positivos para H pylori, após tratamento específico, 27,7% tiveram remissão completa do quadro clínico, 33% remissão parcial e 40% não tiveram remissão. Para o gene cagA positivo, 9,0% dos pacientes com PTI tiveram remissão completa e em 21,2% não houve remissão. Em relação ao gene cagA negativo, 21,2% tiveram remissão completa e em 48,4% não houve remissão. Foram aplicados testes estatísticos para observar a relação do gene cagA com a PTI, (teste exato de Fisher, Box-plot média e desvio padrão da contagem das plaquetas e Mann-Withney). Os resultadoS' obtidos não foram estatisticamente significantes neste grupo estudado. Portanto o gene cagA não está relacionado com o desenvolvimento da PTI / Abstract: Helicobacter pylori is a gram-negative bacterium that is related to the development of gastric and extragastric diseases. Gastric diseases include gastric cancer, chronic gastritis, peptic ulcer and MALT lymphoma. Extragastric diseases include iron deficiency anemia and some auto-immune conditions such as reumathoid arthritis and idiopathic thrombocytopenic purpura. PCR has been an important tool for the analysis of small fragments of DNA that may be stored for a longer time inserted in paraffin blocks or fresh tissue at -80ºC. Here we studied 45 patients with ITP and 33 of these ones had positive tests, with agreement of 100% of histological and rapid urease test. Besides the primers for H. pylori detection, primers for urease C region and cagA gene were used and the fragments obtained were analysed in agarose gel by ultraviolet radiation. We obtained 100% of agreement for urease C region and for detection of H. pylori and 30% of agreement for cagA gene. In these 33 patients which were H. pylori positive, 27,7% had complete remission, 33,0% partial remission and 40,0% had no remission. In positive patients for cagA gene, 9,0% had complete remission and 21,2% had no remission. In negative patients for cagA gene, 21,2% had complete remission and 48,4% had no remission. We used statistical analysis (Exact Fishers test, Box-plot and Mann-Withney) to relation cagA gene and thrombocytopenic idiopathic purpura Our results don't suggest or correlate the presence of cagA gene with idiopathic thrombocytopenic purpura development / Mestrado / Mestre em Farmacologia

INTRODUÇÃO: A prevalência da úlcera péptica e outras afecções esofagogastroduodenais associadas à infecção pelo H. pylori foram alteradas em decorrência da erradicação desta infecção e uso de inibidores de secreção gástrica ácida. OBJETIVO: Determinar a prevalência da infecção pelo Helicobacter pylori associada às afecções diagnosticadas pela endoscopia digestiva alta e analisar fatores de risco. MÉTODOS: Foram analisados dados de 1478 pacientes, e as informações dos achados endoscópicos foram correlacionadas com resultado de teste de urease, faixa etária e gênero. Os pacientes com exame endoscópico normal foram considerados como grupo controle para análise estatística dos fatores de risco, perfazendo um total de 272 indivíduos. RESULTADOS: A prevalência da infecção por H. pylori foi de 53% (n=786), e maior na faixa etária entre 31 e 40 anos. Os achados endoscópicos mais frequentes foram gastrites (n=810; 54,8%), úlceras pépticas duodenais e gástricas (n=494; 33,4%), duodenites (n=287; 19,4%) e esofagites (n=217; 14,7%). Apenas a gastrite nodular e úlcera péptica foram associadas com infecção por H. pylori (p<0,05). Gastrite erosiva no antro (n=644, 78,5%) predominou em relação à pangastrite (n=166; 20,2%) e aquelas no corpo (n=19; 2,3%). Entre os casos de úlcera péptica, 103 (7%) foram gástricas, 343 (23,2%) foram duodenais e 48 (3,2%) foram gástrica e duodenal. A esofagite geralmente foi leve (grau A; 63,1%), 23,5% foram moderada (grau B) e 13,3% foram intensa (graus C e D). Infecção por H. pylori aumentou o risco de úlceras gástrica e duodenal em 1,9 e 1,6 vezes, respectivamente. Gênero masculino e maior idade foram riscos de todas as outras afecções. CONCLUSÃO: Infecção pelo H. pylori associada com maior idade e gênero masculino foram determinantes importantes para evolução de afecções gastrintestinais / Introduction: Peptic ulcer prevalence and other esophageal and gastroduodenal disorders associated with H. pylori infection changed as a consequence of its eradication and the use of gastric acid secretor inhibitors. Purpose: To establish H. pylori infection prevalence associated with clinical disorders diagnosed by upper gastrointestinal endoscopy, and determine the risk factors. Methods: Data from 1478 patients were analyzed, and the endoscopic findings were correlated with the urease test results, age and gender. Patients with normal endoscopy were considered control group for statistical analysis of the risk factors, comprising a total of 272 individuals. Results: The overall prevalence of H. pylori infection was 53% (n=786), being higher between 31 and 40 years old. The most frequent endoscopic findings were gastritis (n=810, 54.8%), peptic ulcer (n=494, 33.4%), duodenitis (n=287, 19.4%) and esophagitis (n=217, 14.7%). Only nodular gastritis and peptic ulcer were associated with H. pylori infection (p<0.05). Erosive gastritis (70%) in the antrum (n=644; 78.5%) predominated in relation to pangastritis (n=166, 20.2%) and the ones in the corpus (n=19, 2.3%). Among peptic ulcer cases, 103 (7%) were gastric, 343 (23.2%) were duodenal and 48 (3.2%) were gastric and duodenal. Esophagitis usually was mild (grade A in 63.1%), 23.5% were moderate (grade B) and 13.3% were intense (grades C and D). H. pylori infection increased the risk of gastric and duodenal ulcers by 1.9 and 1.6-fold, respectively. Male gender and being older were risks of all the other conditions. Conclusion: H. pylori infection associated with older age and male gender were important determinants to gastrointestinal diseases outcome

Durant la réplication du chromosome bactérien, le désappariement du double brin d'ADN est réalisé par l'hélicase hexamérique DnaB. Chez Escherichia coli, le positionnement de l'hexamère de DnaB sur l'ADN simple brin dans le sens 5'-3'est permis par le facteur de chargement. La primase DnaG interagit ensuite avec l'hélicase pour former le primosome. Chez Helicobacter pylori, aucun facteur de chargement n'a été identifié, ce qui est également le cas pour la majorité des espèces bactériennes. De plus, DnaB d'H. pylori (HpDnaB) peut complémenter des souches mutantes d'E.coli DnaBts et DnaCts suggérant que HpDnaB peut jouer le rôle des deux protéines. Pour mieux comprendre le mode d'action de HpDnaB, nous avons résolu sa structure cristallographique à une résolution de 6.7 Å. Celle-ci révèle que la protéine s'assemble en dodécamère, formé par deux hexamères interagissant par leurs domaines N-terminaux (NTD). Nos expériences en diffusion des rayons X aux petits angles (SAXS) montrent que le dodécamère de HpDnaB adopte une conformation modifiée et dynamique en solution. Nous avons ensuite étudié la structure de HpDnaB après interaction avec HpDnaGHBD et/ou l'ADN simple brin par chromatographie d'exclusion stérique couplée à la diffusion de la lumière multi-angles (SEC-MALS) et par SAXS. Ces expériences suggèrent qu'après interaction avec HpDnaGHBD, le double hexamère est dissocié en simples hexamères formant un complexe avec HpDnaGHBD. De plus, HpDnaB forme des hexamères avec l'ADN simple brin en présence d'AMP-PNP. L'ensemble de nos résultats suggère que la formation du primosome d'H. pylori conduit à la dissociation du dodécamère en deux complexes HpDnaB6•HpDnaG3 / During bacterial chromosomal replication, unwinding of double stranded DNA is performed by the hexameric helicase DnaB. In Escherichia coli, the positioning of DnaB hexamers onto replication forks in the 5’to 3’ direction is dedicated by helicase loader. DnaB then interacts with the DnaG primase helicase binding domain (DnaGHBD) to form the primosome. Helicobacter pylori does not encode for a DnaC homologue, which is also the case of most bacterial species. Moreover, H. pylori DnaB (HpDnaB) could complement two temperature–sensitive mutants of E. coli dnaBts and dnaCts, suggesting that the HpDnaB was able to bypass DnaC in these cells. To gain insights into HpDnaB mode of activation, we have solved the crystal structure of HpDnaB at 6.7Å resolution. The structure reveals a novel dodecameric organisation where HpDnaB assembles as planar stack-twisted double hexamers via N-terminal domain (NTD)-rings interactions. Small angle X-ray scattering analysis (SAXS) demonstrates that HpDnaB adopts a modified and dynamic structure in solution but maintains dodecameric architecture. We have then investigated the structure of HpDnaB upon interaction with HpDnaGHBD and/or ssDNA using size exclusion chromatography coupled to multiangle light scattering and SAXS. These experiments show that upon interaction with HpDnaGHBD, HpDnaB double hexamer dissociates into single hexamers to form a complex with HpDnaGHBD. Moreover, we found that HpDnaB also forms hexamers in complex with ssDNA in the presence of AMP-PNP. Collectively, these data suggest that primosome assembly in H. pylori results in the dissociation of the double hexamer into two HpDnaB6•HpDnaG3 sister primosomes

H. pylori est une bactérie à Gram négatif qui infecte l'estomac de près de 50% de la population mondiale. L'infection, en général asymptomatique, peut évoluer vers l'ulcère gastrique (15% des cas) ou le cancer de l'estomac (1% des cas). L'infection à H. pylori est traitée par antibiothérapie mais ces dernières années ont vu une augmentation du nombre de souches résistantes. Cette augmentation et la forte prévalence d'H. pylori sont probablement dues à son importante variabilité génétique qui a pour origine un fort taux de mutagénèse spontanée, associée à une recombinaison efficace et un important transfert horizontal de gènes. H. pylori est en effet naturellement compétente pour la transformation qui est le processus biologique permettant la capture, l'internalisation et l'intégration d'ADN exogène dans le génome de la bactérie. Ce processus favorise la diversité génétique au sein d'une population et peut permettre son adaptation rapide aux changements environnementaux. Durant ma thèse, j'ai participé au développement d'une méthode permettant de visualiser la transformation d'ADN fluorescent dans des cellules de H. pylori vivante. Cette méthode nous a permis, pour la première fois, de visualiser directement l'entrée d'un ADN transformant dans le cytoplasme d'une bactérie compétente. Elle nous a également permis de confirmer le rôle de la protéine ComEC dans l'internalisation de l'ADN dans le cytoplasme. Le travail que j'ai réalisé a également permis de mettre en évidence que le niveau de transformation de H. pylori est déterminé par le niveau d'expression du complexe membranaire d'internalisation. La quantité d'ADN capturée serait alors un facteur limitant pour la transformation. / H. pylori is a Gram negative flagellar bacterium that colonizes nearly 50% of the world population. Infection is generally asymptotic but can evolve to ulcerous gastritis (15% of the cases) or stomach cancer (1% of the cases). H. pylori infection is usually treated with antibiotic but the last years saw a dramatic increase in the number of resistant strains. This increase, and the high prevalence of H. pylori, are probably caused by its huge genetic variability likely due to a strong mutagenesis rate associated with efficient recombination and horizontal gene transfer. H. pylori is indeed naturally competent for transformation which is the biological process allowing capture, internalization and integration of exogenous DNA in the genome of a bacterium. This process promotes genetic diversity in a population and could permit rapid adaptation to environmental changes. During my thesis, I participated to the development of a method to visualize transformation in H. pylori living cells. Using fluorescently labelled DNA, this method allowed us for the first time to follow directly the entry of a transforming DNA into the cytoplasm of competent bacteria. It also allowed us to confirm the role of the ComEC protein in the internalization of the DNA in the cytoplasm. The work I performed also allowed to show that the level of expression of the uptake complex determines the transformation efficiency of H. pylori. The amount of captured DNA would then be a limiting factor for the transformation in this bacterium. Finally, I initiated the biochemical and genetic characterization of the NucT protein, a nuclease associated to the membrane and implicated in the transformation.

Medicinal plants have been used as traditional medicine in the treatment of numerous human diseases for thousands of years in many parts of the world. In the developing world, especially in rural areas, herbal remedies continue to be a primary source of medicine. Scientifically, medicinal plants have proven to be an abundant source of biologically active compounds, many of which have already been formulated into useful therapeutic substances or have provided a basis for the development of new lead molecules for pharmaceuticals. Antibiotic resistance, undesireable side effects and expences associated with the use of combination therapy in the treatment of Helicobacter pylori infections have generated a considerable interest in the study of medicinal plants as potential sources of new drugs against this organism. The high complexicity of bioactive compounds accumulated in plants coupled with their broad antimicrobial activity may make it difficult for pathogenic organisms, including H. pylori to acquire resistance during treatment. This study therefore evaluates the antimicrobial potential of selected South African medicinal plants employed in the treatment of H. pylori-related infections, and the subsequent isolation of the plant active principles. An ethnobotanical survey of plants used in the treatment of H. pylori-related infections was conducted in the study area. Crude extracts of Combretum molle, Sclerocarya birrea, Garcinia kola, Alepidea amatymbica and 2 Strychnos species were screened against 30 clinical strains of H. pylori and 2 standard control strains (NCTC 11638 and ATCC 43526). In the preliminary stages of this study, ethyl acetate, acetone, ethanol, methanol and water extracts of the plants were tested against H. pylori by agar well diffusion and micro broth dilution methods. The plant crude extracts that exhibited anti-H. pylori activity with a iv percentage susceptibility of 50 percent and above were considered for the rate of kill assays and the most active crude extracts selected for bio-assay guided isolation of the active ingredient. Preliminary fractionation of the crude extract was achieved by thin layer chromatography (TLC) using different solvent combinations; hexane/diethylether (HDE), ethyl acetate/methanol/water (EMW) and chloroform/ethyl acetate/formic acid (CEF) in order to determine the most suitable combination for column chromatography (CC) and subsequent testing by indirect bioautography. The extract was then fractionated in a silica gel column using previously determined solvent combinations as eluent. Active fractions obtained from column chromatography separations were further fractionated and the compounds identified by gas chromatography/mass spectrometry (GC/MS) analysis. All the plants exhibited antimicrobial activity against H. pylori with zone of inhibition diameters ranging from 0 - 38 mm and minimum inhibitory concentration (MIC) values ranging from 0.06 - 5.0 mg/mL. The most active plant extracts were the acetone extract of C. molle with a percentage susceptibility of 87.1 percent, acetone and aqueous extracts of S. birrea (71 percent each) and the ethanolic extracts of G. kola (53.3 percent). Except for the aqueous extract, these extracts also exhibited a strong bactericidal activity against H. pylori at different concentrations. TLC analysis revealed the presence of 9 components in the acetone extract of S. birrea with the EMW solvent system as opposed to 5 and 8 with HDE and CEF respectively. Bioassay-guided isolation led to the identification of 52 compounds from the acetone extract of S. birrea with n-octacosane being the most abundant (41.68 percent). This was followed by pyrrolidine (38.91 percent), terpinen-4-ol (38.3 percent), n-eicosane (24.98 percent), cyclopentane (16.76 percent), n-triacontane (16.28 percent), aromadendrene (13.63 percent) and α-gujunene (8.77 percent). Terpinen-4-ol and pyrrolidine demonstrated strong antimicrobial activity against H. pylori at all concentrations tested. These results may serve as preliminary scientific validation of the ethnomedicinal uses of the above mentioned plants in the treatment of H. pylori-related infections in South Africa. Terpinen-4-ol and pyrrolidine could be considered for further evaluation as therapeutic or prophylactic agents in the treatment of H. pylori-related infections. However, further investigations would be necessary to determine their toxicological properties, in-vivo potencies and mechanism of action against H.pylori

Helicobacter pylori is one of the most genetically diverse bacterial species that successfully colonizes at least 50% of the world population. It has been associated with humans for thousands of years and most probably evolved from ancestral gastric Helicobacter species in early mammals. 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 H. pylori to generate variation. The 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. Interestingly, R-M genes comprise a significant percentage of H. pylori strain-specific genes and are more prevalent in H. pylori than in other bacterial species whose genomes have been fully sequenced. R-M systems in H. pylori have been identified on the basis of sequence similarity to known restriction endonucleases and methyltransferases, genetic organization, and specific enzyme isolation and characterization. Analysis of genome sequences of H. pylori strains 26695, J99, HPAGI and 26 others has revealed the presence of more than 20 R-M systems in each stain, which are far more than detected in any other bacterial genome sequence till date. hp1369 and hp1370 are two ORFs in stain 26695 coding for hypothetical proteins. hp 1369 has a stretch of poly-G repeats, thus making hp1369-hp1370, a candidate of phase variation. hpag1_1313 is homolog of hp1369-hp1370 which got up-regulated, in a person suffering from acute gastritis, thus making these genes an interesting subject of investigation. This study was therefore initiated with the following objectives: 1. Cloning, over-expression and purification of Type III MTase (ORF- hp1369- hp1370) and its cognate restriction enzyme (hp1371). 2. Biochemical characterization of MTase (HP1369-HP1370): Determination of oligomeric status, kinetic properties, binding affinities for AdoMet and DNA. Sequence analysis shows the presence of a poly-G track (10 Gs) at 3’-end of hp1369 which is a signature sequence for phase variation. Addition of a single nucleotide can place both hp1369 and hp1370 in-frame, which could code for a single polypeptide. hp1369 and hp1370 in H. pylori strain 26695 alone do not code for any functional protein but with the fusion of hp1369 and hp1370 can code for a protein with all the nine motifs of a DNA MTase. Interestingly, on the basis of arrangement of Motifs, it is probably the first example of ε type of methyltransferase. By site-directed mutagenesis a single G nucleotide was inserted in the poly-G track and both the ORFs (hp1369 and hp1370 ) became in-frame, coding for fully functional HP1369-HP1370 MTase. Kinetic parameters for functional HP1369-HP1370 MTase were determined, and has shown that there was substrate inhibition in methylation reaction at higher concentrations of AdoMets. When preincubation studies were done, enzyme-DNA complex was found to be more competent than enzyme-AdoMet complex. HP1369-HP1370 MTase exists as dimer in solution, having affinity for duplex DNA and does not bind to single-stranded DNA. Binding affinity for ligand (AdoMet) was determined by Isothermal Titration Calorimetry method. H. pylori has evolving restriction-modification systems. It is capable of taking new R-M systems from the environment in the form of DNA released from other bacteria or other Helicobacter strains. H. pylori genome is dynamic with high mutation rates. Random mutations in R-M genes can result in a non-functional R-M systems or R-M systems with new properties. The dynamics of R-M system plays a vital role in shaping up the genome.

L’infection par Helicobacter pylori touche environ la moitié de la population mondiale et est responsable de plusieurs pathologies gastrointestinales incluant l’adénocarcinome gastrique. Le développement récent d’un modèle d’étude chez l’animal a permis d’identifier la nature des cellules à l’origine de la transformation cancéreuse en réponse à l’infection chronique par Hélicobacter. Les cellules souches mésenchymateuses de la moelle osseuse (MSC) seraient recrutées au niveau de la muqueuse gastrique afin de reconstituer, par un mécanisme de différenciation épithéliale, les glandes lésées par l’infection. L’adénocarcinome gastrique se développerait à partir des glandes reconstituées par les MSC. Les objectifs de ces travaux ont été d’analyser, in vitro par une approche séquentielle, les différentes étapes responsables de l’initiation tumorale incluant le recrutement des MSC au niveau gastrique, leur différenciation en cellules épithéliales et leur transformation tumorale lors de l’infection par H. pylori. Ces travaux ont tout d’abord démontré que les cellules épithéliales infectées par H. pylori peuvent recruter les MSC après sécrétion de certaines chimiokines. Nous avons ensuite montré que les MSC sont capables de fusionner avec les cellules épithéliales gastriques aboutissant à l’obtention de cellules épithéliales dérivées des MSC. Finalement, les interactions entre H. pylori et les MSC ont été étudiées et ont fourni des premiers éléments de compréhension des mécanismes de l’initiation tumorale. Nos résultats permettent de mieux comprendre le mécanisme physiopathologique de l’adénocarcinome gastrique et seront utiles à la compréhension d’autres cancers dans lesquels le rôle des MSC comme cellules initiatrices de tumeur est suggéré. / Helicobacter pylori infection is found in about half of the world population and can induce several gastrointestinal pathologies including gastric adenocarcinoma. An animal model recently led to the hypothesis of a cellular origin for the cancer initiating cells after Helicobacter infection. Bone marrow-derived mesenchymal stem cells (MSC) are believed to be recruited in the gastric mucosa in order to repair the damages due to infection, by an epithelial differentiation. Gastric carcinoma may rise from MSC reconstituted gastric glands. This study aimed to analyze, by sequential in vitro approaches, the different steps allowing tumor initiation including MSC recruitment by infected epithelial cells, epithelial differentiation of MSC, and cancer marker appearance after H. pylori infection. We first demonstrated that H. pylori infected epithelial cells may recruit MSC by a secretion of cytokines. We then showed that MSC fuse with gastric epithelial cells leading to MSC-derived epithelial cells. Finally, we studied the interaction between H. pylori and epithelial cells providing a preliminary explanation for cancer initiation. Our results allow a better understanding of gastric adenocarcinoma pathophysiology and will be helpful for the understanding of other cancers in which the role of MSC as cancer initiating cells is suspected.