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

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.

Includes bibliographical references. / 59 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Various articles published by John Robin Warren on the discovery and pathology of Helicobacter pylori. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 2000

Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7281. Adviser: Karen J. Goodman. Includes bibliographical references.

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.

Thesis (M.Sc.) (Honours) -- University of Western Sydney, Hawkesbury, 2001. / A thesis submitted for the degree of Master of Science (Honours), Centre for Farming Systems Research, University of Western Sydney, Hawkesbury, 2001. Bibliography : leaves 142-162.

Thesis (M. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (Ph.D.) -- University of Western Sydney, 2006. / A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliography.

Peu étudiée et méconnue, initialement décrite au début du XXe siècle, Helicobacter pylori fut redécouverte en 1982 par deux chercheurs australiens, JR Warren et BJ Marshall. Ils soutinrent que la plupart des ulcères gastro-duodénaux étaient causés par cette bactérie, et non par le stress ou la nourriture épicée, comme pensé auparavant. Cette découverte révolutionna le monde de la gastroentérologie et leur valut le prix Nobel de physiologie et de médecine 2005. Environ la moitié de la population mondiale est colonisée par H. pylori au niveau de l'estomac. Dans 10 à 20% des cas, l'infection peut évoluer vers un ulcère gastro-duodénal et dans certains cas vers une transformation maligne. Cette infection se soigne classiquement à l'aide d'une trithérapie associant 2 antibiotiques à un inhibiteur de la pompe à protons pour neutraliser l'acidité gastrique.<p>Notre travail de recherche a consisté à analyser la proportion de patients infectés par H. pylori dans une cohorte de plus de 22.000 patients, issus de divers groupes ethniques, vivant en Belgique. Ces souches de H. pylori, isolées dans notre laboratoire, à partir des biopsies gastriques, ont aussi servi à une étude pour suivre l'évolution de la résistance aux antibiotiques ces 20 dernières années afin de proposer des améliorations de la prise en charge thérapeutique de l'infection à H. pylori en Belgique. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished