Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

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

Cathelicidin, a pleiotropic host defense peptide, has been shown to promote cutaneous wound repair and reaches high levels in the gastric mucosa during acute Helicobacter pylori-associated inflammation. The expression of cathelicidin, nevertheless, has also been found to be down-regulated in gastric hyperplastic polyps, tubular adenomas, and adenocarcinomas. We therefore hypothesized that cathelicidin might contribute to gastric ulcer healing and suppress gastric cancer growth. In this study, the role of this peptide in gastric tissue repair and carcinogenesis was investigated. / Collectively, this study demonstrates for the first time that cathelicidin can promote tissue repair and suppress cancer growth in stomachs by eliciting differential cellular signaling and responses in normal and cancerous gastric epithelial cells. These unique biological activities may open up a novel therapeutic avenue for the treatment of these diseases. / Concerning gastric carcinogenesis, the human cathelicidin LL-37 lowered gastric cancer cell proliferation and delayed G1-S transition in vitro and inhibited the growth of gastric cancer xenograft in vivo. Knockdown or induction of endogenous LL-37 by RNA interference or 1alpha,25-dihydroxylvitamin D3, respectively, increased or suppressed cell proliferation. In this connection, LL-37 increased bone morphogenetic protein (BMP) signaling, manifested as increases in BMP4 expression and the subsequent Smad1/5 phosphorylation and the induction of Smad6 and Smad7. Moreover, LL-37 increased the expression of p21Waf1/Cip1, whose induction was abolished by the knockdown of BMP receptor II. Knockdown of BMP receptor II or p21Waf1/Cip1 also abrogated the anti-mitogenic action of LL-37. The activation of BMP signaling by LL-37 was accompanied with the inhibition of chymotrypsin-like and caspase-like activity of proteasome. In this regard, proteasome inhibitor MG-132 mimicked the effect of LL-37 by increasing BMP4 mRNA expression and Smad1/5 phosphorylation. In addition, cyclin E 2 was down-regulated by LL-37 via a BMP-independent mechanism. Further analysis of clinical samples revealed that LL-37 and p21Waf1/Cip1 mRNA expression were both down-regulated in gastric cancer tissues and their expression were positively correlated. These findings indicate that LL-37 inhibits gastric cancer cell proliferation through activation of BMP signaling via a proteasome-dependent mechanism. / In relation to gastric ulcer healing, results revealed that ulcer induction in rats increased the expression of rat cathelicidin rCRAMP in the gastric mucosa. Further increase in expression of rCRAMP by local injection of rCRAMP-encoding plasmid promoted ulcer healing by enhancing cell proliferation and angiogenesis. rCRAMP directly stimulated proliferation of cultured rat gastric epithelial cells (RGM-1), which was abolished by inhibitors of matrix metalloproteinase (MMP), epidermal growth factor receptors (EGFR) tyrosine kinase, or mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase. rCRAMP also increased EGFR and ERK1/2 phosphorylation via an MMP-dependent mechanism. Knockdown of transforming growth factor alpha (TGFalpha), which is a ligand of EGFR, by small interfering RNA completely nullified the mitogenic signals evoked by rCRAMP in RGM-1 cells. These findings suggest that rCRAMP exhibits pro-healing activity in stomachs through TGFalpha-dependent transactivation of EGFR and its related signaling pathway to induce proliferation of gastric epithelial cells. / Wu, Ka Kei. / Adviser: Joseph J. Y. Sung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0252. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 153-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Orientador: Cristiane Duque / Coorientador: Luciano Tavares Angelo Cintra / Banca: João Eduardo Gomes Filho / Banca: Thais Marchini de Oliveira Valarelli / Banca: Christine Men Martins / Resumo: Este trabalho foi dividido em dois capítulos que objetivou avaliar: 1) o efeito isolado ou combinado do flavonoide epigallocatechin-3-gallate (EGCG) em associação com o peptídeo LL-37 e seu análogo KR-12-a5 sobre a viabilidade celular de fibroblastos e sobre cultura planctônica, biofilme simples, dual-espécies e túbulos dentináios e 2) as interações sinérgicas do EGCG e proantocianidina do oxicoco (A-type cranberry proanthocyanidins, AC-PAC), quando usado em combinação com LL-37 ou KR-12-a5 sobre a viabilidade celular, a capacidade de migração e inibição das citocinas em cultura de fibroblastos (HGF-1), quando estimuladas ou não pelo lipopolissacarídeo de A. actinomycetencomitans (LPS). No capítulo 1, a concentração inibitória mínima (MIC), a concentração bactericida mínima (MBC) e concentração inibitória fracionária (FIC) de EGCG, LL-37 e KR-12-a5 foram determinadas a partir de valores decrescentes dos compostos por meio dos métodos de microdiluição e checkerboard contra Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Fusobacterium nucleatum após 24 horas de tratamento. Fibroblastos da linhagem L-929 foram expostos a combinações de EGCG com peptídeos em diferentes concentrações e o metabolismo celular avaliado por ensaios de MTT. Os compostos com melhor efeito antimicrobiano e citotóxico foram avaliados por 24-36h, isoladamente ou em combinação, em biofilmes individuais ou biofilmes de dual-espécies com E. faecalis formados em placas de poliestireno por 4... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study was divided in two chapters that aimed to evaluate: 1) the effect of flavonoid epigallocatechin-3-gallate (EGCG), cationic peptide LL-37 peptide and its analogue KR-12-a5, alone or in combination, on fibroblast cell viability and on bacteria in planktonic and single/dual-species biofilms/dentin tubules; 2) the synergistic interactions of EGCG and cranberry proanthocyanidins (A-type cranberry proanthocyanidins, AC-PAC), when used in combination with LL-37 or KR-12-a5 on cell viability, the ability to induce cell migration and inhibit cytokines in culture of fibroblasts (HGF-1) when stimulated or not by the lipopolysaccharide of A. actinomycetencomitans (LPS). For the chapter 1, Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and fractional inhibitory concentration (FIC) of EGCG, LL-37 and KR-12-a5 were determined from decreasing values of the compounds by Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii and Fusobacterium nucleatum against microdilution and checkerboard after 24 hours of treatment. L-929 fibroblasts were exposed to combinations of EGCG with peptides at different concentrations and cell metabolism assessed by MTT assays. The compounds if the best antimicrobial and cytotoxic effect were also evaluated for 24-36h, alone or in combination, in 48h single- or dual-species biofilms with E. faecalis formed on polystyrene plates by bacterial counting. E. faecalis biofilms were also cultured in dentin tubules f... (Complete abstract click electronic access below) / Doutor

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.