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

A tuberculose ocular afeta 1 a 2% dos pacientes com diagnóstico de tuberculose sistêmica. O tratamento convencional consiste na administração oral dos agentes antituberculosos. Devido às barreiras oculares, o tratamento tópico requer dose elevada e repetidas administrações para atingir efeito terapêutico no olho. Assim, a toxicidade, nos tratamentos convencionais, pode ser relevante. Considerando tais limitações, o desenvolvimento de preparações que permitam a obtenção de produtos com maior eficácia e segurança é de fundamental importância. Nesse sentido, o presente trabalho teve como objetivos o desenvolvimento, a caracterização físico-química e microbiológica de nanoemulsão contendo rifampicina (NR) revestidas empregando cloreto de quitosana (NR-Quit) e sulfato de polimixina B (NR-Poli) com potencial aplicação para o tratamento da tuberculose ocular por via tópica. A NR foi preparada empregando método por homogeneização a alta pressão e apresentou diâmetro hidrodinâmico médio (DHM), índice de polidispersão (IP), potencial zeta (PZ) e pH entre 131,0 e 137,3 nm, entre 0,19 e 0,24, entre -31,0 e -35,4 mV e entre 5,10 e 5,26, respectivamente. A eficiência de encapsulação da rifampicina, determinada por espectrofotometria UV-vis, foi de 82,5 % m/v. Para obtenção de NR-Quit e NR-Poli foi empregado planejamento fatorial completo. Foi observada alteração do PZ de NR após adição de cloreto de quitosana de -35,4 para +51,3 mV. No caso da adição de sulfato de polimixina B o PZ foi alterado de -35,4 para +5,5 mV. Nesse sentido, a abordagem metodológica elucidou que a concentração do cloreto de quitosana e sulfato de polimixina B influenciou significativamente no potencial zeta da NR-Quit e da NR-Poli. Além disso, foi observada a relação linear entre a concentração dos agentes catiônicos empregados e o potencial zeta da NR-Quit e NR-Poli. Essas preparações, no estudo de estabilidade, mostraram aspecto visual, DHM, IP e PZ inalterados por período maior que 90 dias. Também, os valores de pH, viscosidade e osmolalidade foram ajustados entre 4,07 e 4,55, 1,03 e 1,08 cP, 209,7 e 213,4 mOsm/Kg, respectivamente. A atividade antimicrobiana realizada frente ao Mycobacterium tuberculosis H37Rv da NR, NR-Quit, NR-Poli e solução padrão de rifampicina, determinada pela concentração mínima inibitória (CMI), revelou CMI igual 0,125 µg/mL para todas as preparações. Tal resultado demonstrou que os processos de obtenção da nanoemulsão e do revestimento não alteraram a eficácia antimicrobiana da rifampicina. O presente trabalho permitiu o desenvolvimento de preparações inovadoras para o tratamento da tuberculose ocular, por via tópica, com potencial maior eficácia e segurança. / Ocular tuberculosis affects 1 to 2% of the patients diagnosed with systemic tuberculosis. The conventional treatment is the oral administration of the anti-tuberculosis agents. Due to eye barriers, topical treatment requires high dose and repeated administrations to achieve a therapeutic effect on the eye. Thus, toxicity is a major concern in these conventional treatments. Considering these limitations, development of preparations that enable products with higher efficacy and safety is of fundamental importance. In this sense, the present work aimed preparation, physicochemical properties evaluation and microbiological characterization of the rifampicin nanoemulsion (RN) coated using chitosan chloride (RN-Chit) and polymyxin B sulfate (RN-Poly) with potential application for topical treatment of ocular tuberculosis. The RN was prepared by high-pressure homogenization and present droplet mean size (DMS), polydispersity index (PdI), zeta potential and pH between 131.0 and 137.3 nm, between 0.19 and 0.24, between -31.0 and -35.4 mV and between 5.10 and 5.26, respectively. The encapsulation efficiency of rifampicin determined of using spectrophotometric UV-vis method was 82.5% w/v. For preparations RN-Chit and RN-Poly factorial experimental design was employed. The change in the zeta potential of RN-Chit was observed after the addition of chitosan chloride, from -35.4 to +51.3 mV. In the other case, an addition of polymyxin B sulfate changed the PZ from -35.4 to +5.5 mV. Therefore, the methodological approach elucidated that the concentration of chitosan chloride and polymyxin B sulfate significantly influenced the zeta potential of RN-Chit and RN-Poly. Furthermore, the linear relationship between the concentration of cationic agents employed and the zeta potential of RN-Chit and RN-Poly was observed. These preparations, in the stability study, showed visual appearance, DMS, PdI and PZ unchanged for a period greater than 90 days. Additionally, pH, viscosity and osmolality values were adjusted between 4.07 and 4.55, 1.03 and 1.08 cP, 209.7 and 213.4 mOsm/kg, respectively. The antimicrobial activity against Mycobacterium tuberculosis H37Rv of RN, RN-Chit, RN-Poly and the standard solution of rifampicin, determined by the minimum inhibitory concentration (MIC), showed MIC of 0.125 µg/mL for all preparations. This result demonstrated that the processes of nanoemulsion preparation and coating did not affect the antimicrobial efficacy of rifampicin. The present work allowed the development of innovative preparations for the treatment of topical ocular tuberculosis with potential high efficacy and safety.

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.

Uma série de porfirinas meso-substituídas tetracatiônicas foi sintetizada e caracterizada, com o objetivo de estudar o papel da anfifilicidade e a presença de zinco em propriedades que podem influenciar na eficácia dos mesmos como FSs na terapia fotodinâmica. Observamos que as propriedades fotofísicas dos compostos são semelhantes (máximos de absorção na mesma região, red. quantico fl0,02; rend. quantico ox. singlete ~0.8). O aumento na lipofilicidade e a presença de zinco no centro do anel porfirínico aumentam a incorporação dos FSs em vesículas e células, uma vez que a presença de zinco possibilita a coordenação pelos grupos fosfato dos fosfolipídeos (os resultados os estudos das monocamadas de Langmuir e dos filmes de Langmuir-Blodgett corroboram com esta afirmação). A incorporação em mitocôndrias é também dependente da lipofilicidade do FS e é influenciada pelo potencial eletroquímico de membrana. A presença do zinco mostrou diminuir a incorporação em mitocôndrias isoladas e em mitocôndrias nas células HeLa, devido às características particulares da membrana mitocondrial interna. A fototoxicidade aumenta proporcionalmente ao aumento da eficiência da incorporação em membranas, que é atribuída às interações favoráveis entre os FSs e as membranas, permitindo a fotooxidação mais eficiente das mesmas. Para esta série de compostos, a eficiência fotodinâmica é diretamente proporcional à ligação em membranas e incorporação em células, mas não está totalmente relacionada ao acúmulo seletivo em mitocôndrias. Os resultados preliminares de permeação e retenção cutâneos mostram que apesar destes FSs apresentarem baixa penetração e retenção na pele, quando adequadamente formuladas passam a apresentar penetração e retenção cutâneas adequadas de maneira que poderiam ser utilizados na TFD tópica no tratamento de câncer de pele. / A series of photosensitizers (PS), which are meso-substituted tetra-cationic porphyrins, was synthesized and characterized in order to study the role of amphiphilicity and zinc insertion in PDT efficacy. The photophysical properties of all compounds are quite similar (absorption maxima in the same region of the spectra, f 0.02; ~0.8). An increase in lipophilicity and the presence of zinc in the porphyrin ring result in higher vesicle and cell uptake, because zinc can be complexed by the phosphate groups of the phospholipids. The results from the study of Langmuir monolayers and Langmuir-Blodgett mixed films corroborate this affirmation. Binding in mitochondria is dependent on the PS lipophilicity and on the electrochemical membrane potential. Zinc insertion was also shown to decrease the interaction with isolated mitochondria and with the mitochondria of HeLa cells, an effect that has been explained by the particular characteristics of the mitochondrial internal membrane. Phototoxicity was shown to increase proportionally with membrane binding efficiency, which is attributed to favorable membrane interactions between FSs and membranes, which allow for more efficient membrane photooxidation. For this series of compounds, photodynamic efficiency is directly proportional to membrane binding and cell uptake, but it is not totally related to mitochondrial targeting. Preliminary results of skin permeation and retention show that besides presenting low permeation and retention when suitably formulated, FSs can cross the EC barrier and accumulate in deeper regions, thus being applicable to topical PDT in the treatment of skin cancer.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-12-05T19:27:25Z No. of bitstreams: 1 AquilesAbreu.pdf: 1827634 bytes, checksum: d2d7ee078cf8878dfe676a53e1e8bdf0 (MD5) / Made available in DSpace on 2017-12-05T19:27:25Z (GMT). No. of bitstreams: 1 AquilesAbreu.pdf: 1827634 bytes, checksum: d2d7ee078cf8878dfe676a53e1e8bdf0 (MD5) Previous issue date: 2017-10-30 / CAPES / Biofilms based on sodium alginate (AS) crosslinked with cationic polyacrylamide (PAMc) of high molar mass were made by the casting technique. In this study, the central rotational compound design (DCCR) was used and 11 trials were performed. The biofilms produced were characterized with respect to moisture (ω), water solubility (S), water vapor permeability (PVA), thickness (σ), absorption spectroscopy in the infrared region with Fourier transform (FTIR), microscopy scanning (SEM) and mechanical properties. The experimental results showed that biofilms with lower glycerol contents obtained lower moisture content. It was observed that for the solubility and permeability to water vapor, the lower values observed were influenced by the concentrations of PAMc. Assay 5 (AS 6.0g, GLI 1.0g, PAMc 2.5%) presented lower values of ω and PVA, with possible application as food coatings and assay 10 (AS 6.0g, GLI 3.0g; PAMc 2.5%) higher S, and can be applied in biodegradable packages. The results obtained through FTIR confirmed the chemical interaction between AS and PAMc. Morphological analyzes showed that biofilms showed heterogeneity when the concentrations of MAP were increased. Regarding the mechanical properties, tensile strength (TR) and Young's modulus (E) were found to increase when the PAMc concentrations were higher and the deformation decreased when glycerol concentrations were high. Trial 11 (AS 6.0g, GLI 3.0g, PAMc 2.5%) showed higher TR (14.06 MPa) and E (21.17 MPa), with potential for applications as biodegradable bags. / Biofilmes à base de alginato de sódio (AS) reticulados com poliacrilamida catiônica (PAMc) de alta massa molar foram confeccionados pela técnica casting. Nesse estudo foi utilizado o delineamento composto central rotacional (DCCR) realizando-se 11 ensaios. Os biofilmes produzidos foram caracterizados com relação à umidade (ω), solubilidade em água (S), permeabilidade ao vapor de água (PVA), espessura (σ), espectroscopia de absorção na região do infravermelho com transformada de Fourier (FTIR), microscopia eletrônica de varredura (MEV) e propriedades mecânicas. Os resultados experimentais mostraram que os biofilmes com menores teores de glicerol obtiveram menores teores de umidade. Observou-se que para a solubilidade e permeabilidade ao vapor de água, os menores valores observados foram influenciados pelas concentrações de PAMc. O ensaio 5 (AS 6,0g; GLI 1,0g; PAMc 2,5%) apresentou menores valores de ω e PVA, com possível aplicação como coberturas de alimentos e o ensaio 10 (AS 6,0g; GLI 3,0g; PAMc 2,5%) maior S, podendo ser aplicado em embalagens biodegradáveis. Os resultados obtidos através FTIR confirmaram a interação química entre o AS e a PAMc. As análises morfológicas mostraram que os biofilmes apresentaram heterogeneidade quando as concentrações de PAMc foram aumentadas. Quanto às propriedades mecânicas, verificou-se que a tensão à ruptura (TR) e o módulo de Young (E) aumentaram quando as concentrações de PAMc foram maiores e a deformação diminuiu quando as concentrações de glicerol foram elevadas. O ensaio 11 (AS 6,0g; GLI 3,0g; PAMc 2,5%) apresenta maiores TR (14,06 MPa) e E (21,17 MPa), com potenciais para aplicações como sacolas biodegradáveis.

Nanoemulsões catiônicas têm sido consideradas como potenciais sistemas carreadores para oligonucleotídeos (ON) antisenso. O objetivo do presente trabalho foi desenvolver nanoemulsões catiônicas como um sistema de liberação para ON anti-topoisomerase II de Plasmodium falciparum. Primeiramente, nanoemulsões constituídas de triglicerídeos de cadeia média, lecitina de gema de ovo, glicerol e água contendo os lipídeos catiônicos oleilamina ou DOTAP (2 mM) foram obtidas através do procedimento de emulsificação espontânea. Este procedimento resultou em formulações monodispersas com diâmetro de gotícula de 200-260 nm e potencial zeta de +50 e +55 mV. Após, um método espectrofotométrico no UV para quantificação dos ON em série fosfodiéster (PO) ou fosforotioato (PS) foi validado. O método mostrou-se linear, específico, preciso e exato para a determinação de PO e PS, sem diferenças significativas entre os ON. Nas condições validadas, as isotermas de adsorção dos ON às nanoemulsões foram obtidas através da determinação dos ON na fase aquosa externa das nanoemulsões, após ultrafiltração/centrifugação dos complexos. A taxa de recuperação através das membranas de ultrafiltração de celulose regenerada (30 kDa) foi superior a 92%. Os resultados indicam a adsorção progressiva dos ON com as nanoemulsões, até cerca de 60 mg/g de fase interna para o complexo DOTAP-PS. Finalmente, evidências adicionais da adsorção de PO e PS às nanoemulsões foram detectadas pelo aumento do diâmetro de gotícula, inversão do potencial zeta e morfologia das gotículas avaliada por microscopia eletrônica de transmissão. O conjunto dos resultados obtidos demonstra que ON de série PO e PS anti-topoisomerase II de P. falciparum podem ser adsorvidos eficientemente às nanoemulsões catiônicas. / Cationic nanoemulsions have been recently considered as a potential delivery system for antisense oligonucleotides (ON). The aim of the present work was to evaluate cationic nanoemulsions as a delivery system for ON against the Plasmodium falciparum topoisomerase II gene. Firstly, nanoemulsions composed of medium chain triglycerides, egg yolk lecithin, glycerol and water, containing the cationic lipids oleylamine or DOTAP (2 mM) were obtained through spontaneous emulsification process. This procedure resulted in monodisperse formulations with droplet size of 200-260 nm and zeta potential of +50 and +55mV. After that, an UV spectrophotometric method for the quantification of either phosphodiester (PO) or phosphorothioate (PS) ON was validated. The method was linear, specific, precise, and accurate for the determination of PO and PS, without significant differences between both ON. In the validated conditions, ON adsorption isotherms with nanoemulsions were obtained through the ON determination in the external phase of nanoemulsions, after ultrafiltration/centrifugation of complexes. The recovery through regenerated cellulose membranes (30kDa) was higher than 92%. The results showed a progressive ON adsorption to the nanoemulsions up to approximately 60mg/g of internal phase for DOTAP-PS complexes. Finally, additional evidences of PO and PS adsorption to nanoemulsions could also be detected by the increase of the mean droplet size, the inversion of the zeta potential and the morphology of the oil droplets obtained by transmission electron microscopy. The overall results showed that PO and PS ON against P. falciparum anti-topoisomerase II gene can be efficiently adsorbed to the cationic nanoemulsions.

Nanoemulsões catiônicas têm sido recentemente propostas como sistemas carreadores de DNA. O presente trabalho teve por objetivo avaliar a influência de diferentes fosfolipídeos sobre propriedades dos complexos formados entre nanoemulsões e pDNA (pTracerTMCMV2). Em uma primeira etapa, nanoemulsões catiônicas constituídas de triglicerídeos de cadeia média, estearilamina, lecitina de gema de ovo ou fosfolipídeos isolados (DSPC, DOPC, DSPE ou DOPE), glicerol e água foram preparadas através do procedimento de emulsificação espontânea. Independente do tipo de fosfolipídeo empregado, esse procedimento conduziu à obtenção de nanoemulsões catiônicas monodispersas com diâmetro de gotícula e potencial zeta de cerca de 250 nm e +50 mV, respectivamente. A complexação do pDNA com as nanoemulsões catiônicas, avaliada através do retardamento de migração do pDNA em gel de agarose por eletroforese, foi total quando o complexo apresenta uma relação de cargas [+/-] ≥ 1,0. Nestas condições, os complexos formados foram protegidos da degradação pela enzima DNase I. Em uma segunda etapa, a citotoxicidade das nanoemulsões e dos complexos com o pDNA sobre células Hep G2 foi avaliada, através do ensaio de MTT. Os resultados obtidos demonstraram que a adição de quantidades crescentes das nanoemulsões, conduz a uma toxicidade progressiva sobre as células, independente do pH do meio. Dentre as formulações estudadas, aquelas estabilizadas pelos fosfolipídeos DSPC e DSPE, de elevada temperatura de transição de fases, foram marcadamente menos tóxicas, em comparação com as formulações obtidas com lecitina, DOPC e DOPE. Essa mesma tendência foi detectada para os complexos formados com o pDNA. Em uma última etapa, foi realizado um estudo preliminar de transferência gênica em células Hep G2, utilizando a técnica de PCR em tempo real. Dentre as diferentes formulações testadas, a maior quantidade de DNA de GFP detectada parece ser para a formulação obtida com o fosfolipídeo DSPC, ilustrando as potencialidades de uso das nanoemulsões desenvolvidas como reagentes de transfecção de pDNA em células Hep G2. Em conclusão, o conjunto dos resultados obtidos demonstra o efeito dos fosfolipídeos empregados sobre propriedades físico-químicas, complexação, estabilidade, citotoxicidade e transfecção de nanoemulsões catiônicas como sistemas carreadores de pDNA. / Cationic nanoemulsions have been recently considered as potential delivery systems for DNA. The aim of the present work was to evaluate the influence of different phospholipids on the properties of complexes formed between nanoemulsions and pDNA (pTracerTM-CMV2). First, cationic nanoemulsions composed of medium chain triglycerides, stearlyamine, egg lecithin or isolated phospholipids (DSPC, DOPC, DSPE or DOPE), glycerol and water were prepared through spontaneous emulsification process. Independently of the type of phospholipid used this procedure results in monodisperses cationic nanoemulsions with droplet size and zeta potential of about 250 nm and +50 mV, respectively. The complexation of pDNA with cationic nanoemulsions, analyzed by agarose gel retardation assay was total when the complex possesses a charge relation [+/-] ≥ 1.0. In these conditions the complexes were protected from enzymatic degradation by DNase I. After that, the cytotoxicity of the nanoemulsion and the complexes with pDNA in Hep G2 cells was evaluated through MTT assay. The results showed that the addition of increasing amount of nanoemulsion leads to a progressive toxicity on the cells independently of the media’s pH. Among the studied formulations the ones stabilized with the phospholipids DSPC and DSPE, that have elevated phase transition temperatures, were much less cytotoxic in comparison with the formulations obtained with lecithin, DOPC and DOPE. This same trend was detected for the complexes formed with pDNA. Finally a preliminary study of gene transfer to Hep G2 cells was performed using real-time PCR technique. Among the different formulations tested, the major quantity of reporter DNA detected seems to be for the formulation obtained with the DSPC phospholipid. This shows the potentialities of the use of nanoemulsions as transfection reagents of pDNA in Hep G2 cells. In conclusion, the overall results show the effect of the phospholipids on physicochemical properties, complexation, stability, cytotoxicity and transfection of cationic nanoemulsions as delivery systems for pDNA.

A malária é um problema de saúde mundial. Esta doença é causada pela infecção dos eritrócitos com o protozoário do gênero Plasmodium. A atovaquona (ATQ) é um análogo estrutural da coenzima Q, usado como monoterapia ou em combinação com proguanil no tratamento da malária. A incorporação de fármacos de reduzida hidrossolubilidade, como a ATQ, em veículos tradicionais, é frequentemente limitada devido a razões de solubilidade. Neste contexto, nanoemulsões oferecem uma alternativa promissora devido a sua efetividade na solubilização de fármacos, aumentando a eficácia, e reduzindo os efeitos adversos. Assim, o objetivo deste trabalho foi desenvolver nanoemulsões catiônicas contendo ATQ para administração intravenosa. Nanoemulsões compostas de ATQ, triglicerídeos de cadeia média, lecitina de gema de ovo, 1,2-dioleoil-3-trimetil amônio propano (DOTAP), glicerol, polissorbato 80 e água foram obtidas pelo método de emulsificação espontânea. Este método levou à obtenção de nanoemulsões monodispersas com um diâmetro médio de aproximadamente 200 nm, como confirmado pela microscopia eletrônica de transmissão. O potencial  das nanoemulsões foi positivo (> 30 mV em pH 5). Um método isocrático de cromatografia líquida de fase reversa foi validado para quantificar ATQ. O método foi específico, linear, preciso e exato para a quantificação de ATQ. O teor de fármaco para todas as formulações foi próximo a 95%. A ATQ liberada a partir das nanoemulsões foi avaliada em dois meios diferentes (RPMI-1640 + 0,5% albumina ou PBS + 0,5% polissorbato 80) após a separação de ATQ livre através das membranas de ultrafiltração (poro de 0,1m). Independente do meio utilizado, ATQ foi progressivamente liberada a partir das formulações em função do fator de diluição e permaneceu constante em função do tempo. A maior liberação de ATQ foi observada para nanoemulsões contendo polissorbato 80 e no meio contendo este tensoativo não iônico em condições sink. O diâmetro das nanoemulsões permaneceu inalterado nas condições de liberação para nanoemsulões contendo polissorbato 80 enquanto uma inversão do - potencial de valores positivos para negativos foi detectado. Tais formulações mostraram uma inibição do crescimento do parasita in vitro frente a cepas de P. falciparum resistentes à ATQ. / Malaria is a global public health problem. This disease is caused by infection of red blood cells with protozoan parasites of the genus Plasmodium. Atovaquone (ATQ) is a structural analogue of coenzyme Q used as monotherapy or in combination with proguanil in malaria treatment. The incorporation of poorly-water soluble drugs such as ATQ in well-accepted vehicles is frequently limited due to solubility reasons. In this context, nanoemulsions offer an appealing alternative due to their effectiveness in drug solubilization, improved efficacy, and reduced side effects. The main purpose of this study was to develop cationic nanoemulsions containing ATQ for intravenous administration. Nanoemulsions composed of ATQ, medium chain triglycerides, egg lecithin, dioleoyl trimethylammonium propane (DOTAP), glycerol, polysorbate 80 and water were obtained by means of spontaneous emulsification. This procedure led to obtaining monodisperse nanoemulsions with mean droplet size of approximately 200 nm, as attested by transmission electron microscopy. - potential of nanoemulsions was positive (> 30mV at pH 5). An isocratic reversed – phase liquid chromatography method was validated for ATQ quantification. The method was specific, linear, precise, and accurate for ATQ quantification. The drug content in all formulations was close 95%. The ATQ release from nanoemulsions was evaluated in two different media (RPMI-1640 + 0.5% albumin or PBS + 0.5% polysorbate 80) after separation of free ATQ on ultrafiltration membranes (cut off 0.1μm). Whatever the media used, ATQ was progressively released from formulations upon the dilution ratio and remained constant over time. A higher release of ATQ was observed for nanoemulsions containing polysorbate 80 and in the media containing this non-ionic surfactant in sink conditions. The droplet size of nanoemulsions remained unchanged in the release conditions for nanoemulsions containing polysorbate 80 whereas an inversion of the -potential from positive to negative values was detected. Such formulations showed in vitro an inhibition of parasite growth against an ATQ-resistant P. falciparum strain.

In 1982, routine screening of the Pseudopterogorgia elizabethae stirred the scientific community by showing the presence of cytotoxic metabolites with antimicrobial activity. Since this discovery a vast amount of research has been conducted in synthesising metabolites of the soft coral. Herein we report the developments towards the synthesis of two metabolites (+)-Erogorgiaene and (+)-Elisabethadione utilising three key reactions in setting up the molecules three chiral centres. The use of asymmetric allylation, oxy-Cope rearrangement and cationic cyclisation was utilised to set up the desired stereocentres from a starting cinnamyl aldehyde. Natural elisabethadione was synthesised in a racemic form as a 2:1 mixture of diastereoisomers at the C-13 stereocentre.