Orientador: Ana Elizabete Silva / Banca: Patrícia Matos Biselli Chicote / Banca: Marília de Freitas Calmon Saiki / Resumo: Introdução: A inflamação crônica desencadeada pela bactéria Helicobacter pylori (H. pylori), a qual é considerada o principal fator ambiental relacionado ao câncer gástrico, está associada ao desenvolvimento e progressão de lesões gástricas pré-cancerosas, desencadeando diversas modificações histológicas e moleculares que promovem a transformação maligna do estômago. Para isso, conta com fatores de virulência que promovem alterações superficiais e em vias de sinalização das células epiteliais gástricas. Consequentemente pode levar a alterações no padrão de expressão de genes supressores tumorais e da atividade de enzimas DNA metil transferases (DNMTs), responsáveis pela metilação do DNA e silenciamento gênico. Objetivos: O presente estudo avaliou se a infecção pela bactéria H. pylori, bem como sua erradicação, altera a expressão do RNAm dos genes supressores SOCS1, RPRM, RUNX3 e dos genes de DNMTs (DNMT1, DNMT3A e DNMT3B) em pacientes com gastrite crônica infectados (Hp+) em comparação com indivíduos com gastrite crônica sem infecção (Hp-). Além disso, investigou a ocorrência de correlação negativa entre a expressão do RNAm dos genes supressores tumorais com a dos genes das DNMTs, assim como a associação dos níveis de expressão gênica em relação aos fatores de risco idade, sexo, tabagismo, etilismo e genótipo bacteriano cagA. Material e Métodos: A quantificação relativa (RQ) do RNAm foi realizada por PCR (polymerase chain reaction) quantitativa em tempo real (qPCR) utilizando ensaios TaqMan® em 9 pacientes com gastrite crônica Hp- e 19 Hp+, sendo estes também avaliados três meses depois da terapia de erradicação bacteriana. O diagnóstico molecular e genotipagem do fator de virulência cagA foram realizados por PCR convencional. Resultados: Os resultados mostraram que a infecção pela H. pylori e sua erradicação... / Abstract: Introduction: Chronic inflammation caused by Helicobacter pylori (H. pylori), which is considered the main environmental factor related to gastric cancer, is associated with the development and progression of precancerous gastric lesions, triggering several histological and molecular changes that promote stomach malignant transformation. For this, it has virulence factors promoting superficial and signaling pathways of gastric epithelial cells changes. Consequently, it can lead to alterations in the expression of tumor suppressor genes and DNA enzyme activity methyl transferases (DNMTs), responsible for DNA methylation and gene silencing. Objectives: This study evaluated whether the infection by the bacterium H. pylori and its eradication change the mRNA expression of suppressor genes SOCS1, RPRM, RUNX3 and DNMTs (DNMT1, DNMT3A and DNMT3B) genes in patients with chronic gastritis infected (Hp+) compared to individuals with chronic gastritis without infection (Hp-). In addition, we investigated the occurrence of negative correlation between mRNA expression of tumor suppressor genes with the ones of DNMTs, as well as the association of gene expression levels in relation to the risk factors age, sex, smoking, drinking and bacterial genotype cagA. Methods: The relative quantification (RQ) mRNA was performed by PCR (polymerase chain reaction) quantitative real-time (qPCR) using TaqMan® assays in 9 patients with chronic gastritis Hp- and 19 Hp+, which are also evaluated three months after bacterial eradication therapy. The molecular diagnostics and genotyping of the virulence factors CagA were performed by standard PCR. Results: The results showed that the infection by H. pylori and eradication did not significantly alter the gene expression of SOCS1, RPRM, RUNX3 and DNMTs, which presented, in general, reduced expression (RQ <1.0); on the other hand, higher expression of SOCS1 and ... / Mestre

Orientador: Ana Elizabete Silva / Coorientador: Sonia Maria Oliani / Banca: Henrique César Santejo Silveira / Banca: Patricia Maluf Cury / Banca: Dorotéia Rossi S. Souza / Banca: Debora Ap. Pires de C. Zuccari / Resumo: Apesar do declínio da incidência, o câncer gástrico ocupa ainda a terceira posição em causa de morte por câncer no mundo, tendo como principal fator de risco a bactéria Helicobacter pylori. Esta bactéria pode levar a uma inflamação persistente através da produção de citocinas pró-inflamatórias e de espécies reativas de oxigênio e nitrogênio, estimulando a proliferação celular bem como outros processos envolvidos na carcinogênese. Ainda envolvidos nestes processos tem sido observada a participação de microRNAs, que exercem papel importante na regulação pós-transcricional, influenciando processos fisiológicos normais da célula bem como aqueles ligados às doenças, como por exemplo o câncer gástrico. Alguns miRNAs podem atuar como oncogenes, genes supressores de tumor e biomarcadores para diversas patologias, podendo alvejar genes relacionados com inflamação e câncer como o gene ANXA1 (Anexina-A1). A Anexina-A1 é uma proteína anti-inflamatória e com ação anti-proliferativa, que se liga à receptores do tipo formil peptídeo como por exemplo FPR1 e FPR2, ambos sabidamente relacionados com a progressão de doenças como o câncer. Desta forma o presente trabalho teve como objetivos avaliar a expressão da proteína Anexina A1 e seus receptores FPR1 e FPR2, bem como avaliar a expressão do RNAm da ANXA1 e de miRNAs que possam modular a expressão desse gene (hsa-mir-27a, hsa-mir-196a e hsa-mir-222) em adenocarcinoma gástrico e correlacionar estes resultados com os aspectos clínico-patológicos. Foram avaliadas 31 amostras de adenocarcinoma gástrico, assim como as regiões metaplásica ou normal adjacentes ao tumor. A quantificação relativa (RQ) do RNAm da ANXA1 e miRNAs foi realizada por PCR quantitativo em tempo real utilizando ensaio TaqMan, e a expressão proteica da AnxA1, FPR1 e FPR2 por imuno-histoquímica e análise densitométrica. Em... / Abstract:Gastric cancer still ranks third in cause of cancer death worldwide, despite the decline in its incidence, being Helicobacter pylori the main risk factor for this disease. This bacterium can lead to persistent inflammation via the production of proinflammatory cytokines and reactive oxygen and nitrogen species, stimulating cell proliferation and other processes involved in carcinogenesis. Still involved in this process, it has been observed the participation of miRNAs, which play an important role in post- transcriptional regulation, influencing normal physiological processes of the cell as well as those linked to diseases such as gastric cancer. Some miRNAs can act as oncogenes, tumor suppressor genes and biomarkers for various diseases, can targetting genes linked to inflammation and cancer such as ANXA1 gene (Annexin A1). Annexin A1 is an anti-inflammatory protein with anti-proliferative action that binds to formyl peptide receptors such as, FPR1 and FPR2, both known to be related to the progression of diseases such as cancer. Thus, the present study aimed to evaluate the expression of Annexin A1, FPR1 and FPR2 receptors, and the expression of mRNA ANXA1 and miRNAs (hsa-mir-27a, hsa-mir-196a and hsa-miR 222) in gastric adenocarcinoma, also correlate these results to the clinicopathological features. 31 adenocarcinoma samples were evaluated, as well as normal or metaplastic region adjacent to the tumor. Relative quantification (RQ) of miRNA and mRNA ANXA1 was evaluated by TaqMan assay and protein expression AnxA1, FPR1 and FPR2 by immunohistochemistry and densitometry analysis. Regarding the relative expression the following results were observed, increased expression in tumors of ANXA1 (RQ = 1.374; p < 0.001) and miR- 196a that showed increased expression it he metaplastic tissue (RQ = 4.784; p = 0.0016) and tumor (RQ = 16.99; p < 0.001) compared to normal tissue. The miR- 27a did not appear differentially expressed in different tissues ... / Doutor

Evalúa la presencia de H. pylori en el agua de consumo humano de los pacientes diagnosticados con cáncer gástrico y que son H. pylori positivo. La población de estudio está conformada por 84 muestras de agua que corresponden a 84 pacientes con diagnóstico de cáncer gástrico. Se recolectan las muestras de agua, con un volumen de 2L., de cada domicilio. Se evalúa la presencia de los genes de factores de colonización: ureasa (ureA) y proteína de shock térmico (hspA), mediante la técnica de Reacción en cadena de las polimerasas cuantitativa (qPCR). De la población en estudio se observa que el 47.62 % son mujeres y el 52.38 % son varones. Todos los pacientes en estudio residen en Lima, pero el 70.24% nacieron en provincia y el 29.76% nacieron en Lima. La zona de Lima sur presenta la mayor cantidad de pacientes con diagnóstico de cáncer gástrico con un 28.57%, seguida de Lima norte y Lima este con 21.43% y 20.24% respectivamente. Se documenta que el 84.52% de los pacientes venían residiendo en sus hogares un tiempo mayor a 10 años, y sólo el 8.33% residían en su domicilio actual un tiempo no mayor a 5 años. De las 84 muestras de agua evaluadas, se observa una prevalencia de H. pylori, con al menos un resultado positivo en alguno de los dos factores de colonización evaluados, en el 11.90 % (10/84) del total de las muestras agua evaluadas, ubicándose 6 de ellas en la zona este de Lima. / Tesis

El Helicobater pylori, principal agente causal de la gastritis, condiciona el estrés oxidativo debido a la producción de radicales libres. La Satureja brevicalyx, es usada para tratar problemas gastrointestinales, como la gastritis. El objetivo del presente trabajo fue caracterizar los componentes químicos, la actividad anti-Helicobacter pylori y antioxidante del aceite esencial de Satureja brevicalyx. El aceite esencial reportó un rendimiento (1.80%v/p), rotación óptica (-2 a +4º), densidad (0.9047 g/mL) e índice de refracción (1.475). Mediante CG-SM y CG-FID se elucidó 35 compuestos al 97.1%: monoterpenos oxigenados (74.8%), como componentes mayoritarios la pulegona (27.2%), linalol (20.3%), mentona (11.1%), isomentona (8.3%), cis-isopulegona (2.7%), trans-isopulegona (0.9%), carvacrol (0.6%), timol (0.6%) y α-terpineol (0.5%); hidrocarburos sesquiterpénicos (16%), destacando biciclogermacreno (8.2%), β-cariofileno (6.5%) y bicicloelemeno (0.5%); hidrocarburos monoterpénicos (4.1%), con el p-cimeno (2.0%), limoneno (0.7%) y γ-terpineno (0.6%); y sesquiterpenos oxigenados (1.5%), con el espatulenol (0.8%). Por el método de Artemia salina mostró una bioactividad significativa, con una CL50 de 13.35 μg/mL, y por el método de Dosis Fija, en ratones albinos, presenta una DL50 de 655.26 mg/kg, siendo ligeramente tóxica. Por el método de Difusión de Discos, tiene un efecto antibacteriano frente al H. pylori, con un halo de inhibición de 33.33 % en comparación con la amoxicilina a una concentración de 10 μg/mL; por el método de Difusión en Microplacas, la concentración mínima inhibitoria es de 1.00 μg/mL, y la concentración mínima bactericida de 2.00 μg/mL. Se determinó en 3 modelos la actividad antioxidante a concentraciones de 10, 50 y 100 μg/mL: 1) El aceite inhibió al radical DPPH en 67.76, 75.33 y 86.84%, frente al trolox que inhibió en 80.59, 87.17 y 93.09%; 2) secuestró al radical hidroxilo en 28.95, 51.57 y 76.84%; y 3) inhibió de la peroxidación lipídica en 16.4x10-8, 9.8x10-8 y 8.5x10-8 moles x cm, comparado con el trolox que lo hizo en 14.9x10-8, 8.0x10-8 y 7.5 x10-8 moles x cm, frente a 53.9x10-8 moles x cm para el control negativo. Las estructuras químicas del aceite esencial de la S. brevicalyx justifican su actividad anti-Helicobacter pylori y antioxidante. Palabras Clave: Satureja brevicalyx, gastritis, radicales libres, anti-Helicobacter pylori, antioxidante / --- The Helicobacter pylori is the main causal agent of gastritis, it conditions the oxidative stress due to the fact that it produces free radicals. The Satureja brevicalyx is used to treat gastrointestinal problems as gastritis. The objective of this work was to characterize the chemical components, the anti-Helicobacter pylori activity and the antioxidant of the main oil of Satureja brevicalyx. Te main oil reported a performance (1.80%v/p), optical rotation (- 2 to + 4º), density (0,9047 g/mL) and index of refraction (1.475). By means of CG-SM and CG-FID elucidated 35 compounds at 97,1%: oxygenated monoterpens (74,8%) as majority compound the pulegone (27,2%), linalool (20,3%), menthone (11,1%), isomenthone (8,3%), cis-isopulegone (2,7%), trans-isopulegone (0,9%), carvacrol (0,6%), thymol (0,6%) and α-terpineol (0,5%); sesquiterpenes hydrocarbons (16%), emphasizing biciclogermacrone (8,2%), β-caryophyllene (6,5%) and bicicloelemene (0,5%); monoterpenic hydrocarbons (4,1%), with the p-cymene (2,0%), limonene (0,7%) and γ-terpineno (0,6%); and sesquiterpenes (1,5%), with espatulenol (0,8%). By the method of Artemia salina it showed a significant bioactivity, with a CL50 of 13,35 μg/mL, and by the method of Fixed Dose, in albinae mice shows a DL50 of 655,26 mg/kg being slightly toxic. By the method of the Diffusion of Discs, it has an antibacterial effect of the H. pylori, with an halo of inhibition of 33,33% in comparation with the amoxiciline in a concentration of 10 μg/mL; by the method of Diffusion in Microplacas, the minimum inhibitory concentration is of 1,00 μg/mL, and the minimum bactericide concentration is of 2,00 μg/mL. The antioxidant activity to concentrations was determined in three models the concentrations must be of 10, 50 and 100 μg/mL: 1) The oil inhibited the radical DPPH in 67.76, 75,33 and 86,84%, in relation with trolox which inhibited in 80.59, 87,17 and 93,09%; 2) kidnapped the hidroxile radical in 28.95, 51,57 and 76,84%; and 3) inhibited the lipidic peroxidation in 16.4x10-8, 9.8x10-8 and 8.5x10-8 moles x cm, in relation with trolox which inhibited in 14.9x10-8, 8.0x10-8 and 7.5 x10-8 moles x cm, in relation to a 53.9x10-8 moles x cm for the negative control. The chemical structures of the main oil of the S. brevicalyx justify its activity as an anti-Helicobacter pylori and as an antioxidant. Key Words: Satureja brevicalyx, gastritis, free radicals, anti-Helicobacter pylori, antioxidant / Tesis

H. pylori is one of the most significant discoveries in gastroenterology in the past century. It is associated with a wide range of gastroduodenal pathologies and gastric cancer. Antibiotic resistance in H. pylori has emerged as a significant clinical problem. The body of work contained within this dissertation was carried out to investigate an alternative therapy based on observations of the traditional therapy for gastric disease in the Middle East. One of these traditional therapies centres on plants belonging to the Pistacia genus. This study represents the first reported investigation into the composition and biological activity of the trunk bark exudates of Pistacia atlantica Kurdic (P. a. Kurdica), Pistacia atlantica Mutica (P. a. Mutica) and Pistacia atlantica Cabolica (P. a. Cabolica), resinous gums that have been termed here ‘Kurdica Gum’, ‘Mutica Gum ’ and ‘Cabolica Gum ’ respectively. The antimicrobial screening of the trunk exudates of the genus Pistacia led to the characterization of the most active fraction of the Kurdica gum. This fraction was subsequently subjected to sub-fractionation leading to the discovery of fundamentally new information that went beyond H. pylori, expanding the original parameters of the project. The extent of these findings suggests that new classes of antibiotics might have been discovered. Primary studies on their structure and potential mechanism of action has been undertaken. Thirteen novel antimicrobial agents were identified. Based on the characteristics of these isolated fractions, 50 new compounds were modelled; of which 30 hypothetically have an MIC consistent with contemporary antibiotics and could represent viable lead compounds for commercial development. / Doctor of Philosophy

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.

Ce travail traite de la formulation et de la caractérisation de liposomes porteurs de glycolipides synthétiques, en vue du ciblage d'une bactérie : Helicobacter pylori. Après avoir passé en revue les différents systèmes à temps de résidence gastrique prolongé, il décrit la synthèse et l'utilisation de néoglycolipides de type "ancre-espaceur-sucre ", constitué respectivement du cholestérol, du tétraéthylène glycol et enfin du fucose (ou N-acétylglucosamine). Ont été étudiées dans ce travail l'organisation supramoléculaire des néoglycolipides seuls en fonction de leur état d'hydratation, les altérations de la bicouche liposomale suite à l'incorporation du néoglycolipide, l'accessibilité des sucres à la surface des liposomes, la variation du pH intraliposomal en fonction de pH externes acides, et enfin, l'interaction de quatre formulations de liposomes contenant ou non les néoglycolipides avec 2 souches d'H. pylori

La conversion par les thymidylate synthases du désoxyuridine 5'-monophosphate (dUMP) en désoxythymidine 5'-monophosphate (dTMP) est une des étapes clés de la biosynthèse des désoxyribonucléotides pyrimidiques. Chez les procaryotes (bactéries et archées), il existe deux familles de thymidylate synthases non homologues : ThyA et ThyX. La distribution phylogénétique quasi-exclusive des deux familles est complexe et ne suit pas les relations phylogéniques entre les organismes. Les protéines de ces deux familles ne présentent aucune similarité de séquence, de structure et leurs mécanismes et efficacités catalytiques diffèrent. Les travaux présentés dans cette thèse ont permis de montrer que l'activité de ThyX est essentielle à la survie de la bactérie Rhodobacter capsulatus en absence de thymidine exogène. A partir des résultats de tests de complémentation génétique et de la modélisation mathématique du métabolisme des folates nous avons pu mettre en évidence que seulement une faible activité dihydrofolate réductase était nécessaire à la survie des organismes utilisant ThyX. D'autre part, j'ai pu montrer par des approches génétiques et par l'étude statistique de plus de 400 génomes procaryotes que les organismes utilisant ThyX ont une réplication et un taux de croissance lents, et possèdent majoritairement un petit génome. Je propose un modèle dans lequel la faible activité catalytique de ThyX limite la réplication de l'ADN e! t par conséquent l'expansion du génome. Enfin, j'ai étudié le métabolisme des pyrimidines chez la bactérie pathogène humaine, Helicobacter pylori. J'ai pu en particulier mettre en évidence qu'en absence de voie de récupération de l'uracile, cette bactérie est capable de métaboliser un analogue toxique le 5-fluorouracile (5FU), utilisé comme anticancéreux. J'ai par ailleurs montré que l'orotate phosphoribosyltransférase de H. pylori était capable de restaurer l'auxotrophie pour l'uracile d'une souche d'Escherichia coli indiquant que cette enzyme pourrait être responsable de la sensibilité de H. pylori au 5FU. Les travaux présentés dans cette thèse ont permis une meilleure compréhension des conséquences physiologiques de l'utilisation de la thymidylate synthase ThyX. Ils ont notamment permis d'expliquer la distribution apparemment sporadique des deux familles de thymidylate synthases dans le monde procaryote.

Helicobacter pylori est une bactérie qui infecte l'estomac de la moitié de la population mondiale et est impliquée dans la plupart des maladies gastriques, dont les ulcères et le cancer de l'estomac. La bactérie produit deux toxines, CagA et Tipα, qui sont associées avec le développement du cancer. CagA est injectée dans les cellules et interagit avec de nombreuses protéines des voies de signalisation cellulaire. Tipα est secrétée et internalisée dans les cellules gastriques où elle induit la production de cytokines pro-inflammatoires. Dans ce travail de thèse, nous avons tout d'abord étudié les interactions entre un domaine central de CagA et des protéines de H. pylori. Nous avons ensuite identifié de nouveaux fragments solubles de CagA par une méthodologie à ‘'haut-débit''. L'un d'eux, correspondant à l'extrémité C-terminale de la protéine de 33kDa, CagAC33, a été caractérisé par différentes techniques de biochimie et de biophysique. CagAC33 forme des dimères de dimères en solution et des particules en microscopie électronique. De plus, CagAC33 peut être phosphorylé in vitro par les kinases c-Src et c-Abl mais l'efficacité de la phosphorylation dépend de la kinase utilisée. L'étude de l'interaction entre la phosphatase SHP-2 et CagAC33 montre que CagA est dephosphorylée in vitro. Par ailleurs, les structures de deux formes cristallines de Tipα ont été déterminées par cristallographie aux rayons X. La structure du monomère de Tipα adopte un nouveau repliement, et la protéine forme des dimères différents dans les deux formes cristallines. L'étude de la protéine en solution indique qu'un des dimères est sans doute favorisé et suggère que les ponts disulfures identifiés en N-terminal ont un rôle durant la sécrétion de la protéine.

<p>Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. </p><p>The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. <i>Rickettsia prowazekii</i>, the causative agent of epidemic typhus and <i>Helicobacter pylori</i>, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in <i>Rickettsia</i>. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in <i>H. pylori</i> were studied.</p><p>The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) <i>Rickettsia</i>, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in <i>R. conorii</i> indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the <i>H. pylori </i>carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in <i>H. pylori</i> were studied, of which antigenicity and gene location were identified as the strongest factors. </p><p>In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.</p>