A bactéria Gram negativa Helicobacter pylori, além de estar associada ao câncer gástrico e duodenal, está relacionada a patologias extra gástricas. Entre essas estão as doenças cardiovasculares. Os mecanismos pelos quais o H. pylori pode causar, ou agravar essas doenças, ainda não são claros. A urease de H. pylori (HPU) é considerada um fator de virulência, visto que sua atividade catalítica cria um microambiente de pH mais elevado, possibilitando a sobrevivência do patógeno no estômago. A HPU é capaz de ativar plaquetas de coelho através da indução da secreção de seus grânulos densos com liberação de ADP, culminando na agregação plaquetária. Esse fenômeno ocorre via 12- lipoxigenase, rota de sinalização também utilizada pelo colágeno, um importante agonista intrínseco desse sistema. Demonstramos previamente que também as duas subunidades da HPU, HpUreB e HpUreA, interagem com membranas de plaquetas de coelho, sendo que a HpUreB contém o domínio da holoenzima responsável pela agregação plaquetária. Essa interação entre HPU e plaquetas pode ser mediada por GPVI, o principal receptor de colágeno dessas células No presente trabalho, estudamos a interação da HPU e suas subunidades com plaquetas humanas, através de citometria de fluxo. Demostramos que HPU ativa plaquetas humanas sem exposição significativa de P-selectina. O bloqueio com anticorpos específicos para o receptor de membrana GPIIbIIIa, mas não para GPVI, interfere na ativação plaquetária induzida por HPU. Em plaquetas ativadas por HPU ocorrem modificações do processamento pré-mRNA de proteínas pró-inflamatórias, aumentando os níveis de mRNAs que codificam IL-1 e CD14, indicando que plaquetas passam a apresentar um fenótipo pró-inflamatório após exposição à urease. No conjunto, nossos dados sugerem que a HPU, além de permitir a sobrevivência bacteriana na mucosa gástrica, pode ter um papel importante, e até agora negligenciado, nos estados inflamatórios associados com a infecção por H. pylori. / The Gram negative bacterium Helicobacter pylori, besides its association with gastric and duodenal cancer, correlates positively to several extragastric diseases suchas cardiovascular pathologies. However, the mechanisms by which H. pylori can cause or aggravate these diseases are still unclear. H. pylori urease (HPU) is considered a virulence factor, since its catalytic activity creates a microenvironment, of higher pH, that allows survival of the pathogen in the stomach. HPU is able to activate rabbit platelets by inducing the secretion of their dense granules with release of ADP, culminating in platelet aggregation. This phenomenon occurs with activation of the 12-lipoxygenase pathway, which is also used by collagen, an important intrinsic agonist of this system. We have previously demonstrated that both subunits of HPU, HpUreB and HpUreA, interact with rabbit platelet membranes, and that HpUreB contains the domain responsible for platelet aggregation This interaction of HPU and platelets could be mediated by GPVI, the main collagen receptor in these cells. In this work, by using flow cytometry assay, we have studied the interaction of HPU and of its subunits with human platelets. HPU was shown to activate human platelets without significant P-selectin exposure. Blockage with antibodies against the membrane receptor GPIIbIIIa, but not against GPVI, interfered on platelet activation induced by HPU. The processing of pre-mRNA of proinflammatory proteins was evaluated in HPU-activated platelets and increased levels of mRNAs encoding IL-1 and CD14 were found, indicating that platelets acquire a proinflammatory phenotype when exposed to the urease. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked role in the inflammation associated to the infection by H. pylori.

Objetivo: Evaluar la asociación entre la presencia de H. pylori y sangrado en los primeros 30 días post operatorios de gastrectomía en manga. Diseño: Cohorte retrospectiva basada en un análisis de datos secundarios.

BACKGROUND: For the last 15 years, infection from Helicobacter pylori (H. pylori) has been recognized in gastritis pathogenesis, and is known to trigger an important inflammatory response in these patients. AIM: To determine the association between follicular gastritis and the infection of H. pylori in children seen at a public hospital in Peru. METHODOLOGY: An analytic, cross-sectional study was conducted on all the children treated at the Hospital Nacional Docente Madre "Niño San Bartolomé" in Lima, Peru, within the time frame of 2011-2012. All the personal data from the patients' medical histories and endoscopic procedures were collected. The crude prevalence ratios (PR) were obtained and adjusted (aPR) with their 95% confidence intervals (95%CI), using generalized linear models with the binomial family and log link function. RESULTS: A total of 123 children met the study criteria. Forty-eight (39%) of the study sample were girls and the mean age of the children was 12 years. H. pylori was present in 44% of the sample and 9% presented with more than 100 bacteria per field (classified as +++). Thirty-five percent of the children had esophagitis due to concomitant reflux. The presence of H. pylori was associated with follicular gastritis (P<.01; PRa: 2.3; 95% CI:1.49-3.49), adjusted by the children's age. CONCLUSIONS: Based on the data analyzed, it was concluded that the children with follicular gastritis had a greater likelihood of having H. pylori than those that did not present with gastritis. These results can be extrapolated to other similar populations and should be evaluated in each setting so that this does not become a public health problem within the next few years. / Peer review

Helicobacter pylori (HP), known to cause active chronic gastritis, has primarily been found in gastric-type mucosa. Even in the duodenum, the organism was detected in islands of metaplastic gastric mucosa. HP has also been found in gastric metaplasia of Barrett's esophagus in 15-50%. The aim of our study was to determine: (1) the frequency with which HP is found on histopathological sections of heterotopic gastric mucosa (HGM) patch(es) at the upper esophagus, as compared to that of the stomach proper, and (2) the histopathological significance of infection in the HGM patches. From 63 patients with HGM patches at the upper esophagus, 48 patients were found to have concurrent adequate specimen from the stomach for modified Steiner's stain. In 22 patients (45.8%), pair sections from HGM and stomach were negative for HP. Of 26 patients (54.1%) HP-positive on sections from the antrum and/or body (both in 21 cases) nine patients (18.7%) demonstrated HP in the HGM patches. Whereas focal acute inflammatory changes on the HandE section of HGM was present in six patients, HP was detected in HGM only in one. Chronic inflammatory cell infiltration was detected in all nine HP-positive HGM patches and in 37 of 39 HP-negative patches. A mixed acute and chronic inflammatory cell infiltration was found in five of these 37 patients. Our data demonstrate that HP infection of HGM patches at the upper esophagus is part of the HP gastritis and an independent colonization of HGM patches without gastric infection does not occur. No correlation was found between the presence of acute and chronic inflammatory changes in HandE-stained section and positivity of HP in modified Steiner's section of HGM.

幽門螺桿菌感染在世界範圍內普遍存在，超過50%的世界人口都曾被感染。幽門螺桿菌與胃炎，胃潰瘍，胃癌和其他胃內疾病的發生密切相關。盡管目前已有多種有效除菌的抗生素，但耐藥菌的出現仍然不可忽視。防止幽門螺桿菌感染能有效的減緩疾病進程及其相關疾病引發的死亡率。因此，新藥物或者新的藥物劑型的研發十分重要。 / Cathelicidin是一種宿主免疫防禦系統用於抵抗不同種類病原微生物感染的生物肽。然而，其在幽門螺桿菌感染引發的炎癥中的作用仍未被揭示。本研究旨在發現Cathelicidin在幽門螺桿菌體內及體外感染中的可能抗菌作用及其機制。為了研究不同劑量Cathelicidin對幽門螺桿菌的直接抗菌作用，我們主要觀察了細菌生長，生物膜形成及細菌形態的改變。實驗結果表明，Cathelicidin可有效的抑制幽門螺桿菌的生長，破壞其生物膜形成，及在細菌胞膜形成孔狀結構，以改變其正常的超微形態。 / 在宿主抵禦幽門螺桿菌感染的機制中，自噬不僅具有抗菌活性，同時在清除胃上皮細胞內病原體的方面發揮著重要作用。然而，幽門螺桿菌則可能得益於自噬通路，並掌控自噬這一工具，進而幫助其自身的存活及感染。 / 研究發現，維生素D於體內的活性形式1,25 - 二羥維生素D3（1,25D3）可促進Cathelicidin的合成及激活自噬通路，從而發揮自身免疫來殺死在胃上皮細胞內定植的幽門螺桿菌。此外，通過RNAi沈默技術，與對照基因沈默的細胞相比，LL-37在人胃上皮細胞（HEF-145）中表達被抑制後，細胞內幽門螺桿菌的存活數量顯著上升。同樣的結果也被發現於動物模型中，在急性及慢性幽門螺桿菌感染的小鼠模型中，CRAMP基因敲除小鼠胃內的幽門螺桿菌數量比野生型小鼠胃內更多。 / 為了進一步研究Cathelicidin是否具有潛在的治療幽門螺桿菌感染的作用，本研究采用生物工程的方法將CRAMP轉入乳酸球菌中，再將這種分泌CRAMP型及對照組乳酸球菌餵給被幽門螺桿菌感染的小鼠。預防性和治療性的研究結果表明，這種能夠分泌CRAMP的益生菌可在胃黏膜表面存活和定植。更多的幽門螺桿菌能夠定植在CRAMP基因敲除小鼠的胃內，同時其胃內的促炎癥因子，IL-6，IL-1β及ICAM表達也高於野生型小鼠。此外，幽門螺桿菌感染上調了野生型小鼠胃上皮型來源的CRAMP表達，這一結果可部分解釋為什麽在野生型小鼠胃內只有少量幽門螺桿菌及輕度炎癥反應的原因。 / 重要的是，預防性及治療性的實驗顯著的提高了兩種小鼠胃黏膜中抗菌肽的水平，並降低了幽門螺桿菌感染及促炎癥因子mRNA的表達。值得注意的是，預防性的給藥還促進了胃粘液層的合成及防止表皮細胞雕亡，從而加強胃黏膜屏障的保護作用。 / 總結而言，本研究結果揭示Cathelicidin作為一種天然抗生素，在清除幽門螺桿菌及治療其引發的慢性胃炎中發揮重要的作用。分泌Cathelicidin型食用益生菌和幫助Cathelicidin內源性表達的1,25D3則有望發展成為新型的生物制劑用於防治動物和人體幽門螺桿菌感染及其引發的相關性胃炎。 / Helicobacter pylori (H. pylori) infection is one of the most prevalent infectious diseases, affecting more than 50% of the world’s population and responsible for gastritis, peptic ulcer, gastric cancer and other stomach disorders. / Although there are antibiotics which are effective to eradicate the bacteria, the worldwide appearance of drug resistance to H. pylori is common. It is therefore needed to search for new therapeutic agents or establish a new form of drug delivery system to prevent H. pylori infection at the early stage in order to reduce the disease progression and its associated morbidities. / Cathelicidin, a host defense antibacterial peptide in humans can eradicate different kinds of microbial infection. However, its roles in H. pylori infection and inflammation remain unexplored. This study sought to elucidate the possible actions and mechanisms for cathelicidin to protect against H. pylori infection and its associated inflammation both in vitro and in vivo. / To examine the direct antimicrobial action of cathelicidin, H. pylori survival, biofilm formation and morphology change were determined after exposure to different doses of cathelicidin in vitro. Results showed that exogenous cathelicidin could affect H. pylori growth, destroy bacteria biofilm and cause pore formation in H. pylori membranes. With respect to the host defense against H. pylori infection, autophagy plays a crucial role in antimicrobial activity, and contributes to clearance of intracellular pathogens in gastric cells. In this regard, H. pylori might benefit from autophagy pathway, and subvert the autophagy machinery in favor of its survival and infectious process. / The active form of vitamin D3, 1, 25-dihydroxyvitamin D3 (1, 25D3) activated LL-37, a human cathelicidin antimicrobial peptide and produced autophagy, which could contribute to host immune responses against intracellular survival of H. pylori in gastric cells. Additionally, we transfected gastric epithelial cells (HFE-145) with siRNA specific for LL-37 (siLL-37) to knockdown the expression of LL-37 in HFE-145 cells, which markedly increased the number of intracellular H. pylori when compared to cells transfected with a scrambled control siRNA (Csi). Consistent with these findings, cathelicidin knockout (Cnlp⁻/⁻) mice exhibited stronger H. pylori colonization in stomachs with acute and chronic H. pylori infection when compared to the stomachs in cathelicidin wild-type (Cnlp⁺/⁺) mice. / To further examine whether cathelicidin could be used as a therapeutic agent for H. pylori infection, we replenished exogenous CRAMP in H. pylori infected Cnlp⁺/⁺ and Cnlp⁻/⁻ mice with a bioengineered CRAMP-secreting strain of Lactococcus lactis (L. lactis) in a cost-effective manner. To this end, Cnlp⁺/⁺and Cnlp⁻/⁻ mice were pre-treated or post-treated with the control plasmid-encoded L. lactis or CRAMP-encoded L. lactis in H. pylori infected mice. They were then assessed for H. pylori infection and inflammatory responses in stomachs. Results showed that the probiotic L. lactis could survive in the gastric mucosa. In the absence of CRAMP, Cnlp⁻/⁻ mice exhibited more H. pylori harboring in the stomach together with marked expressions of IL-6, IL-1β and ICAM in the gastric mucosa when compared to wild type mice. Furthermore, in Cnlp⁺/⁺ mice, H. pylori infection stimulated gastric epithelium-derived CRAMP production but not in the Cnlp⁻/⁻ mice. These findings could partially explain why there were less bacterial infection and inflammatory responses in the wild type animals. Importantly, pre-treatment and post-treatment with CRAMP-encoded L. lactis significantly increased mucosal CRAMP level in both types of animals and reduced H. pylori infection and also pro-inflammatory cytokines mRNA expressions in these stomachs. It was noteworthy that delivering CRAMP intragastrically before H. pylori challenge strengthened the mucosal barrier by stimulating mucus layer synthesis and preventing epithelial cell apoptosis. / Collectively, these findings indicate that cathelicidin plays a significant role as a potential natural antibiotic for H. pylori clearance and a therapeutic agent for chronic gastritis. The increase of cathelicidin expression in the gastric mucosa either by the food-grade probiotic encoded with cathelicidin or the active form of vitamin D, could be promising biological preparations for the treatment of H. pylori infection and its associated gastritis in animals and perhaps also in humans. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 144-170). / Abstract also in Chinese. / ABSTRACT --- p.I / 中文摘要 --- p.V / DECLARATION --- p.VII / ACKNOWLEDGEMENTS --- p.VIII / PUBLICATIONS --- p.XIV / LIST OF ILLUSTRATIONS --- p.XIX / INTRODUCTION --- p.1 / Chapter 1.1 --- Helicobacter pylori --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Epidemiology of H. pylori infection --- p.2 / Chapter 1.1.3 --- Diagnosis and treatment strategies for H. pylori-induced diseases --- p.2 / Chapter 1.1.4 --- Bacteria autophagy: restriction or persistence of infection? --- p.3 / Chapter 1.1.5 --- Virulence factors of H. pylori related to autophagy --- p.8 / Chapter 1.1.6 --- Future research directions concerning H. pylori and autophagy --- p.11 / Chapter 1.2 --- Cathelicidins --- p.11 / Chapter 1.2.1 --- Overview --- p.11 / Chapter 1.2.2 --- Cathelicidin and its antimicrobial action and possible mechanisms --- p.12 / Chapter 1.2.3 --- Mouse cathelicidin deficient model --- p.16 / Chapter 1.2.4 --- Multiple receptors enable diversified activities of cathelicidins --- p.17 / Chapter 1.2.5 --- Endogenous cathelicidin induction --- p.23 / Chapter 1.2.5 --- New uses for old drugs --- p.26 / METHODS --- p.29 / Chapter 2.1 --- General Materials --- p.29 / Chapter 2.1.1 --- Chemicals and reagents --- p.29 / Chapter 2.1.2 --- Antibodies --- p.33 / Chapter 2.1.3 --- Commercial Kits --- p.34 / Chapter 2.1.4 --- Bacteria and culture conditions --- p.35 / Chapter 2.1.5 --- Animals --- p.36 / Chapter 2.1.6 --- Cell Line --- p.36 / Chapter 2.2 --- Experimental Designs --- p.37 / Chapter 2.2.1 --- In vitro studies --- p.37 / Chapter 2.2.2 --- In vivo studies --- p.44 / Chapter 2.3 --- Statistical analysis --- p.52 / RESULTS AND DISCUSSION --- p.53 / Chapter 3.1 --- Antimicrobial activity of cathelicidin on H. pylori in vitro --- p.53 / Chapter 3.2 --- Anti-biofilm formation activity of cathelicidin on H. pylori in vitro --- p.58 / Chapter 3.3 --- Manipulation of autophagy by H. pylori for their survival --- p.62 / Chapter 3.3.1 --- H. pylori stimulated dysfunctional autophagy --- p.62 / Chapter 3.3.2 --- H. pylori compromised the autophagic flux in cells and thereby promoting self-multiplication --- p.68 / Chapter 3.3.3 --- Autophagy is a host defense process in controlling intracellular survival of H. pylori --- p.71 / Chapter 3.4 --- Vitamin D3 inhibited H. pylori infection through the induction of autophagy --- p.76 / Chapter 3.4.1 --- 1,25D3 triggered the formation of autophagosomes and autophagolysosomes in gastric epithelial cells --- p.76 / Chapter 3.4.2 --- 1,25D3 treatment inhibited intracellular H. pylori survival through induction of autophagy by cathelicidin --- p.79 / Chapter 3.5 --- Discussion --- p.86 / Chapter 3.6 --- Elucidation of the role of endogenous and exogenous cathelicidin in H. pylori colonization and the associated gastritis --- p.94 / Chapter 3.6.1 --- H. pylori SS1 colonized in Cnlp⁺/⁺ and Cnlp⁻/⁻ mouse gastric epithelium --- p.94 / Chapter 3.6.2 --- Endogenous cathelicidin protects against H. pylori SS1 colonization in vitro and in vivo --- p.96 / Chapter 3.6.3 --- Endogenous cathelicidin protects against drug-resistant H. pylori 10783 colonization --- p.100 / Chapter 3.6.4 --- The bioengineered L. lactis encoded with CRAMP could localize in mouse stomachs and express CRAMP mRNA --- p.104 / Chapter 3.6.5 --- Effects of CRAMP secreting bioengineered L. lactis on H. pylori growth in vitro --- p.106 / Chapter 3.6.6 --- Post-treatment of CRAMP-encoded L.lactis on H. pylori colonization and its associated gastritis --- p.108 / Chapter 3.6.7 --- Pre-treatment of CRAMP-encoded L.lactis on H. pylori colonization and its associated gastritis --- p.118 / Chapter 3.7 --- Discussion --- p.129 / SUMMARY AND FUTURE PERSPECTIVES --- p.140 / REFERENCES --- p.144

預激活綜合體的形成對於脲酶的成熟是必需的。所以作為預激活綜合體一部份，UreF/UreG/UreG綜合體的形成也是脲酶成熟的關鍵之一。從幽門螺桿菌UreF/UreH的晶體結構顯示出是一個由異源二聚體形成的二聚體，這UreF/UreH二聚體和幽門螺桿菌的脲酶都有擁有個獨特的二重對稱性。而UreF/UreH二聚體的長度和幽門螺桿菌脲酶獨特的二次軸上那兩個催化中心的距離很接近。這讓我們聯想到UreF/UreH二聚體的二聚化是否與脲酶的活性有關。所以跟據UreF/UreH的晶體結構，計計了三個証實可以破壞UreF二聚化的突變體(F33D/Q37A, R179A/Y183D and F33D/Q37A/R179A/Y183D)。而這些突變體與UreH的結合體都保留了和脲酶結舍的能力卻失去了和UreG結合的能力，所以都不可以結合成完整的預激活綜合體來熟化脲酶。為了UreF/UreH二聚面的虛擬篩選，AutoDock Vina和Dock6.5,這兩個篩選程式用了DUD去做了一些基準。而基於一個百分比的富集值和首個已知配體的百分比值， Dock6.5比AutoDock Vina優勝，所以會用Dock6.5來篩選可以綁定UreF的二聚分介面的分子。最後，分析Dock6.5前1排名的分子，這些分子可以跟據它們和UreF殘基的接觸分類。 / The formation of the pre-activation complex is essential for the urease maturation. Being part of the pre-activation complex, the formation of theUreF/UreG/UreH complex is crucial for the formation of the complete preactivation complex. The crystal structures of Helicobacter pylor iUreF/UreH had been determined showing a dimer of heterodimer formation. The structure of UreF/UreH complex and H. pylori urease shared a unique two-fold symmetry. Moreover, the length of the UreF/UreH complex is similar to the distance of the two catalytic centres on the two-fold symmetry axis. This brought to the question: whether the dimerization of the UreF in the UreF/UreH complex has an effect on the H. pylori urease activity. According to the UreF/UreH crystal structure, three UreF mutants (F33D/Q37A, R179A/Y183D and F33D/Q37A/R179A/ Y183D) were designed and all were able to break the dimerization of UreF. These mutants were not able to interact with UreG, hence the complete pre-activation complex could not be formed and the maturation of urease was inhibited. Working towards to the virtual screening of the UreF/UreH complex dimerization surface, two docking programs, AutoDock Vina and Dock 6.5 were benchmarked using the DUD set. Dock 6.5 out performed AutoDock Vina by comparing the EF1 (Enrichment Factor of the top1% ranked ligands) and the percentage ranking of the first true hit. Using Dock 6.5, UreF residues that make the most contacts with the ligands had been identified using the top 1% of the ranked ligands. / Detailed summary in vernacular field only. / Yuen, Man Hon Nicholas. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 72-74). / Abstracts also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iii / Table of Content --- p.iv / Figures List --- p.vi / Tables List --- p.vi / Chapter Chapter 1 --- introduction --- p.1 / Chapter Introduction --- p.1 / Chapter 1.1 --- What is urease? --- p.1 / Chapter 1.2 --- Role of urease in H. pylori --- p.3 / Chapter 1.3 --- Structure of urease --- p.4 / Chapter 1.4 --- The active site of urease --- p.6 / Chapter 1.5 --- Accessory proteins are needed for urease maturation --- p.8 / Chapter 1.6 --- Crystal structure of H. pylori UreF/UreH complex --- p.12 / Chapter 1.7 --- Objective --- p.14 / Chapter Chapter 2: --- Material and Methods --- p.15 / Chapter 2.1 --- General Techniques --- p.15 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.15 / Chapter 2.1.2 --- Agarose gel electrophoresis of DNA --- p.16 / Chapter 2.1.3 --- Polymerase Chain reaction, PCR --- p.17 / Chapter 2.1.3.1 --- Basic protocol --- p.17 / Chapter 2.1.3.2 --- Generation of HisGST-UreF mutants --- p.18 / Chapter 2.1.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.1.5 --- SDS-polyacryamide gel electrophoresis, SDS-PAGE --- p.19 / Chapter 2.1.6 --- Staining of polyacrylamide gel --- p.20 / Chapter 2.2 --- Expression and Purification of Recombinant Proteins --- p.21 / Chapter 2.2.1 --- General bacterial culturing, harvesting and lysis procedures --- p.21 / Chapter 2.2.2 --- Purification of wild type HisGST-UreF and mutants with UreH --- p.22 / Chapter 2.2.3 --- Purification of Urease (UreAC) --- p.23 / Chapter 2.2.4 --- Purification of His-SUMO-UreG --- p.24 / Chapter 2.3 --- Static light scattering, SLS --- p.25 / Chapter 2.4 --- In vitor Urease Activity --- p.26 / Chapter 2.5 --- In vitor Urease Activity --- p.27 / Chapter 2.6 --- Virtual Screening --- p.28 / Chapter 2.6.1 --- Docking with Dock 6.5 --- p.28 / Chapter 2.6.2 --- Docking with AutoDock Vina --- p.29 / Chapter 2.6.3 --- Enrichment factor calculation --- p.29 / Chapter 2.7 --- Reagents and Buffers --- p.30 / Chapter 2.7.1 --- Buffers for competent cells preparation --- p.30 / Chapter 2.7.2 --- Nucleic acid electrophoresis buffers --- p.30 / Chapter 2.7.3 --- Media fr bacterial culture --- p.30 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.31 / Chapter 2.7.5 --- Reagents and Buffers for in vitro Urease Activity Assay --- p.32 / Chapter 2.7.6 --- Reagents and Buffers for in vitro Urease Activity Assay --- p.32 / Chapter Chapter 3 --- Dimerization of UreF is Essential for Urease Maturation --- p.33 / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Results --- p.34 / Chapter 3.2.1 --- Mutant design --- p.34 / Chapter 3.2.2 --- When expressed alone, the UreF mutants were found in the inclusion Body --- p.36 / Chapter 3.2.3 --- Co-expressing UreFmutants with UreH would solublize UreF mutants and the interactions between UreF mutants and UreH were retained --- p.36 / Chapter 3.2.4 --- UreF oligomerizationstate determination by size exclusion chromatography / static light scattering (SEC/LS) --- p.39 / Chapter 3.2.5 --- UreF dimerization is necessary for the interaction between the UreF/UreH complex and UreG --- p.41 / Chapter 3.2.6 --- UreF dimerization is not involved in the interaction between the UreF/UreH complex and Urease(UreA/UreC) --- p.43 / Chapter 3.2.7 --- UreF dimerization is essential for in vitro Urase Maturation --- p.45 / Chapter 3.2.8 --- UreF dimerization is essential for in vivo Urase Maturation --- p.47 / Chapter Chapter 4 --- Benchmarking Virtual Screening Performance of AUTODOCK VINA and DOCK 6.5 - Towards Virtual Screening of Inhibitors for Uref/UreH Complex Dimerization --- p.53 / Chapter 4.1 --- Introduction --- p.53 / Chapter 4.2 --- Benchmarking AutoDock Vina and Dock 6.5 --- p.54 / Chapter 4.2.1 --- Description of the Directory of Useful Decoys (DUD) set --- p.54 / Chapter 4.2.2 --- Benchmarking AutoDock Vina and Dock 6.5 shoewing Dock 6.5 has a better overall EF1 --- p.57 / Chapter 4.2.3 --- Dock 6.5 has a higher first hit percentile --- p.60 / Chapter 4.2.4 --- Analysis of the binding site for the top 1% ranked ligand for UreF Dimerization surface --- p.63 / Chapter 4.3 --- Discussion --- p.68 / Chapter Chapter 5 --- Conclusion --- p.71 / References --- p.72

La cancérogenèse gastrique est un processus d’origine multifactorielle, incluant des facteurs génétiques de l’hôte, mais aussi d’origine bactérienne et de l’environnent. Les populations humaines peuvent être exposées directement ou indirectement à des composés toxiques/génotoxiques présents dans les plantes, comme la fougère Pteridium aquilinum. Cette plante comprend une toxine, le ptaquiloside associée à des maladies graves et le développement de cancer chez les animaux. Des études épidémiologiques ont démontré une association entre l'exposition à ces fougères et l’incidence du cancer gastrique dans les populations humaines. Cependant, un autre facteur de risque majeur dans le développement du cancer gastrique est la bactérie Helicobacter pylori qui colonise l'estomac humain et induit une réponse génotoxique. Cette étude vise à caractériser l'implication biologique et moléculaire de Pteridium aquilinum et de sa toxine le ptaquiloside dans le processus de cancérogenèse gastrique et d'explorer un effet de synergie potentiel avec l'infection par H. pylori.Nous avons montré que le traitement avec des extraits de Pteridium aquilinum et le ptaquiloside diminue la viabilité cellulaire et favorise l'apoptose des cellules épithéliales gastriques. L'induction de cassures de l'ADN a été observée, exacerbée en présence de l’infection par H. pylori. Dans les cellules traitées, la protéine p53 est induite et associée à l'activation de la voie de signalisation ATR-Chk1. Cette augmentation de p53 est aussi détectée en présence des souches virulentes de H. pylori. L’induction de lésions à l’ADN par le ptaquiloside est en accord avec la dérégulation observée de l’expression d’un certain nombre de gènes impliqués dans la régulation du cycle cellulaire et la réparation de l'ADN. De plus, des souris exposées à Pteridium aquilinum, montrent des modifications histomorphologiques de la muqueuse gastrique ainsi qu’une augmentation de la prolifération cellulaire et l'induction de mutations dans le gène p53 après 7 semaines de traitement. Toutefois, bien qu’une exacerbation de la prolifération cellulaire et des lésions histologiques soient induites par un traitement chronique en association avec l'infection à H. pylori pendant 12 mois, aucune différence significative dans l'expression du gène p53 a été mise en évidence. Cependant, dans ces conditions, une modification du schéma glycophenotypique a été induite dans la muqueuse gastrique des souris. Différentes glycosyltransférases impliquées dans la biosynthèse des antigènes simples de mucines et terminaux antigènes Lewis ont été différentiellement exprimées chez les souris non-infectées et infectées, respectivement. Ces résultats sont également validés par une augmentation de l’expression de Sialyl-LewisX.De plus, des modifications des glycosyltransférases impliquées dans les étapes initiales de O-glycosylation ont été observées. Le ppGalNAcT6 a présenté une expression altérée dans un carcinome gastrique, associée à la présence de l'invasion veineuse.En conclusion, nos données confirment l’activité génotoxique de Pteridium aquilinum et du ptaquiloside sur les cellules gastriques, supportant leur rôle fondamental dans la promotion de la cancérogenèse gastrique. Cette activité est exacerbée en présence de l’infection par H. pylori, soulignant l'importance de l'interaction de ces deux facteurs de risque dans ce processus. / The multifactorial gastric carcinogenesis process encompasses host genetic susceptibility, bacterial and environmental factors. Humans can directly consume or be indirectly exposed to toxic compounds present in plants, such as the bracken fern Pteridium aquilinum. This plant has a carcinogenic toxin, ptaquiloside, and is known to cause severe health problems in animals, including cancer. Epidemiological evidence also demonstrated an association between bracken exposure and gastric cancer development in Humans. Additionally, another major etiological agent is Helicobacter pylori, a bacterium that colonizes the stomach inducing a genotoxic response in gastric cells. This study aimed to characterize the biological and molecular involvement of Pteridium aquilinum and its ptaquiloside toxin in the gastric carcinogenesis process and to evaluate the potential synergistic effect of H. pylori infection.We observed that treatment with Pteridium aquilinum extracts and ptaquiloside toxin decreased cell viability and promoted cell apoptosis in gastric epithelial cells. A genotoxic effect with induction of DNA strand breaks was noted and it was exacerbated in the presence of H. pylori infection. We further demonstrated that in treated cells a p53 accumulation occurs, controlled by the activation of the ATR-Chk1 DNA damage signalling pathway. An increased level of p53 was also detected in the presence of a H. pylori virulent strain. The contribution of ptaquiloside to this genotoxic activity was supported by the deregulation of other genes involved in DNA cell cycle regulation and DNA repair. In addition, using a mouse model exposed to Pteridium aquilinum, we detected histomorphological alterations with increased cell proliferation and induction of frameshift events in the p53 gene. However, a concomitant chronic treatment with Pteridium aquilinum and H. pylori infection did not produce significant differences in p53 gene expression.Moreover, an altered glycophenotypic pattern was induced in the gastric mucosa of mice upon Pteridium aquilinum treatment in the presence of H. pylori infection. Several glycosyltransferases involved in the biosynthesis of simple mucin-type carbohydrate antigens and terminal Lewis antigens were differently expressed in the absence and presence of H. pylori, respectively. These results were also validated by an increased expression of Sialyl-LewisX.Further alterations in glycosyltransferases involved in the initial steps of O-glycosylation were observed. The ppGalNAcT6 was shown to have a heterogeneous expression in human gastric carcinoma, associated with the presence of venous invasion.Overall, our data supports the notion that cell exposure to the genotoxic and carcinogenic Pteridium aquilinum and ptaquiloside has a fundamental role in the promotion of gastric carcinogenesis. The synergistic environment associated to H. pylori infection underlines the importance of risk factor interplay in this process. / A carcinogénese gástrica é um processo multifatorial que engloba fatores genéticos, bacterianos e ambientais. O Homem pode consumir diretamente ou ser exposto de forma indireta a compostos tóxicos presentes em plantas, como é o caso do feto vulgar Pteridium aquilinum. Esta planta tem uma toxina carcinogénica, o ptaquilosídeo, sendo conhecida a sua capacidade natural para induzir lesões neoplásicas em animais. Estudos epidemiológicos também demonstraram a existência de uma associação entre a exposição ao feto e o desenvolvimento de cancro gástrico em humanos. Outro fator etiológico importante é a Helicobacter pylori, uma bactéria que coloniza o estômago, induzindo nas células gástricas uma resposta genotóxica. Este estudo tem por objetivos caracterizar o envolvimento biológico e molecular do Pteridium aquilinum e da sua toxina, ptaquilosídeo, no processo de carcinogénese gástrica e avaliar o potencial efeito sinergístico da infeção por H. pylori.Observámos em células epiteliais gástricas que o tratamento com extratos de Pteridium aquilinum e com a toxina ptaquilosídeo, diminui a viabilidade celular e promove a apoptose. Foi demonstrado um efeito genotóxico com indução de quebras na cadeia de ADN, exacerbado pela presença da infeção por H. pylori. Demonstrámos ainda que, em células tratadas, ocorria uma acumulação de p53, controlada pela ativação da via de sinalização ATR-Chk1. Um aumento nos níveis de p53 foi igualmente detetado na presença de estirpes virulentas de H. pylori. A contribuição do ptaquilosídeo para esta atividade genotóxica foi também apoiada pela desregulação de outros genes envolvidos na regulação do ciclo celular e na reparação do ADN. Adicionalmente, usando um modelo de ratinho exposto ao Pteridium aquilinum, foram detetadas alterações histomorfológicas, bem como um aumento da proliferação celular e indução de mutações no gene p53. Contudo, um tratamento crónico com Pteridium aquilinum e infeção concomitante por H. pylori não produziu diferenças significativas na expressão do gene p53.Um padrão glicofenotípico alterado foi também observado na mucosa gástrica de ratinhos tratados com Pteridium aquilinum na presença de infeção por H. pylori. Várias glicosiltransferases envolvidas na biossíntese de antigénios simples das mucinas e antigénios terminais do tipo Lewis apresentaram uma expressão alterada, respetivamente, na ausência ou presença de H. pylori. Estes resultados foram também validados através de um aumento da expressão de Sialil-LewisX.Foram ainda observadas alterações em glicosiltransferases que estão envolvidas nas etapas iniciais de O-glicosilação. A ppGalNAcT6 apresentou uma expressão alterada em carcinomas gástricos, estando associada à presença de invasão venosa.No geral, estes dados suportam a evidência de que a exposição das células aos genotóxicos e carcinogénicos Pteridium aquilinum e ptaquilosídeo, tem um papel fundamental na promoção da carcinogénese gástrica. O ambiente sinergístico associado à infeção com H. pylori salienta a importância da interação entre os fatores de risco que dão origem a este processo.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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