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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 邱浩傑(Hao-Chieh Chiu) | |
| dc.contributor.author | Cheng-Yun Hsu | en |
| dc.contributor.author | 許誠允 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:47:09Z | - |
| dc.date.available | 2025-08-17 | |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79139 | - |
| dc.description.abstract | 可入侵宿主細胞內進行感染的病原菌是全球一個嚴重的威脅。在侵入宿主細胞後，胞內細菌會藉由表現致病因子躲避細胞的先天性免疫防衛而得以存活。因此，針對胞內菌致病因子為標的之透過新穎殺菌機制的抗菌藥物開發是不可或缺的。我們之前的研究發現抗精神病用藥―樂賜平，具有抗小鼠巨噬細胞內沙門氏菌存活的效果。為了保留樂賜平的抗菌活性並去除其作為抗精神病用藥的活性，我們設計合成一系列的樂賜平衍生物，再利用高內涵影像分析平台來評估衍生物的抗菌活性及細胞毒性。經過一系列的篩選，我們找到一個新的化合物―SW14具有比樂賜平更為良好的抗胞內沙門氏菌的活性。除此之外，SW14也對胞內的福氏志賀氏菌、單核球增多性李斯特菌、抗甲氧苯青黴素金黃色葡萄球菌、小腸結腸耶氏菌，還有多重抗藥性的沙門氏菌具有良好的抗菌活性，並且在小鼠的沙門氏菌感染模型中SW14也展現其活性。機制方面，我們發現抗氧化劑可以反轉SW14對胞內沙門氏菌的抑制，並證實SW14具有抑制細菌外排幫浦的活性，且會增加細菌對於氧化壓力的感受性，此些發現表明SW14可能是藉由抑制細菌透過外排幫浦的抗氧化能力來達到降低胞內感染的細菌量，後續可發展為新一類的抗菌藥物。 | zh_TW |
| dc.description.abstract | Pathogenic bacteria capable of invading and proliferating inside host cells have been a serious threat worldwide. After entering host cells, these intracellular pathogens can express a variety of virulence factors to evade the innate immunity of host cells. In light of this, the development of a novel virulence-targeted antibacterial drug has been a potential strategy to control the infections of intracellular bacteria. Previously, we showed that an antipsychotic drug, loxapine, exhibited potent antibacterial activity against intracellular Salmonella enterica in murine RAW264.7 macrophagic cells. To improve loxapine’s antibacterial activity, a series of loxapine derivatives were designed and synthesized. The antibacterial activity and cytotoxicity of these compounds were simultaneously evaluated using an image-based high-content assay. The screening identified a new compound SW14 which showed a multifold increase in antibacterial activity against intracellular multiple-antibiotic-resistant S. enterica, Shigella flexneri, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and Yersinia enterocolitica. Moreover, SW14 is also efficacious in the murine salmonellosis model. Subsequent mechanism studies indicated that the ROS scavengers could reverse the antibacterial effect of SW14 against intracellular Salmonella. Also, SW14 exhibited suppressive activity on the bacterial efflux pump and increase the susceptibility of bacteria to oxidative stresses. Together, our data suggested that SW14 might suppress intracellular bacteria via impeding the efflux pump-mediated resistance to oxidative stresses. The structure of SW14 represents a promising scaffold for the development of a new virulence-targeted antibacterial agent against intracellular bacteria. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:47:09Z (GMT). No. of bitstreams: 1 U0001-1708202015292200.pdf: 3291301 bytes, checksum: 14cb999af16f0c1cd7a9ff110b763282 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 致謝 i 中文摘要 ii Abstract iii Contents v 1. Introduction 1 1.1 Difficulties in intracellular pathogenic bacterial therapy 2 1.2 Salmonella enterica serovar Typhimurium 3 1.3 Dibenzoxazepines 4 1.4 Specific aims 5 2. Materials and Methods 6 2.1 Cell culture 7 2.2 Bacteria strains 7 2.3 Drugs and reagents 8 2.4 High-content screening assay 9 2.5 ROS scavenger assay 11 2.6 Colony-forming assay 11 2.7 Cell viability assay 13 2.8 Hoechst 33342 accumulation assay 13 2.9 Determination of intracellular reactive oxygen species (ROS) 14 2.10 Sensitivity of S. Typhimurium to hydrogen peroxide (H2O2) 16 2.11 Murine salmonellosis model 16 2.12 Intracellular persister assay 17 2.13 Gene expression assay 19 2.14 Statistical analysis 20 3. Results 21 3.1 SW14 was the most potent derivative identified through high-content assay 22 3.2 The results of high-content assay were consistent with colony-forming assay and cell viability assay 23 3.3 SW14 could inhibit intra-macrophagic Salmonella together with either fluoroquinolone or cephalosporin 24 3.4 SW14 was also active against intracellular MDR or fluoroquinolone-resistant Salmonella 24 3.5 SW14 had a broad-spectrum effect on other intracellular bacteria 25 3.6 SW14 decreased intracellular persister population 26 3.7 SW14 could extend the survival time of Salmonella-infected mice 27 3.8 SW14 didn’t inhibit bacterial growth in the broth 28 3.9 SW14 didn’t induce autophagy flux and its antibacterial activity wasn’t affected by atg7 knockdown 28 3.10 SW14 was active against both vacuolic and cytosolic bacteria 29 3.11 SW14 inhibited efflux pump activity of Salmonella 30 3.12 ROS scavengers reduced the inhibitory effect of SW14 on intracellular Salmonella 32 3.13 SW14 didn’t increase the ROS level in RAW264.7 cells 33 3.14 SW14 enhanced the susceptibility of bacteria to H2O2 33 4. Discussion 34 4.1 The colony-forming units of Shigella flexneri were much lower than others in colony-forming assay 35 4.2 The similarity of LPM to the intracellular environment 35 4.3 The mechanism how SW14 inhibited the efflux pump activity 36 4.4 Cytotoxicity and dibenzoxazepines 37 4.5 Conclusion and future works 37 5. Reference 39 6. Tables 49 Table 1. Antibacterial activity and cytotoxicity of loxapine derivatives 50 Table 2. Susceptibility of antibiotic-resistant Salmonella Typhimurium strains to antibiotics 51 Table 3. The structure-relationship of compounds with different core structures 52 7. Figures 53 Figure 1. The results of high-content assay were consistent with that of colony-forming assay and cell viability assay. 54 Figure 2. SW14 exhibited suppressive activity against intracellular Salmonella in combined with cefixime or ciprofloxacin. 57 Figure 3. SW14 inhibited intracellular proliferation of not only MDR strains but also fluoroquinolone-resistant strains of S. Typhimurium. 58 Figure 4. SW14 had a broad-spectrum effect of inhibiting intracellular bacterial proliferation. 60 Figure 5. SW14 decreased the population of intracellular Salmonella persisters. 62 Figure 6. SW14 could extend the survival time of Salmonella-infected mice. 64 Figure 7. SW14 didn’t inhibit bacterial growth in the culture broth. 66 Figure 8. SW14 didn’t induce autophagy flux and its antibacterial activity wasn’t affected by atg7 knockdown. (Western blot data from Shih-Hsiu Chou) 68 Figure 9. SW14 inhibited the efflux pump activity. 70 Figure 10. ROS scavengers reversed the suppressive effect of SW14 on intracellular S. Typhimurium. 73 Figure 11. SW14 didn’t increase ROS production directly. 74 Figure 12. SW14 elevated the susceptibility of S. Typhimurium to H2O2. 76 Figure 13. The order of magnitude of Shigella flexneri intracellular growth curve was constant. 77 Figure 14. The expression profile of efflux pumps wasn’t similar at all between S. Typhimurium cultured in RAW264.7 cells and LPM. 78 | |
| dc.language.iso | en | |
| dc.subject | 外排幫浦 | zh_TW |
| dc.subject | 氧化壓力 | zh_TW |
| dc.subject | 高內涵 | zh_TW |
| dc.subject | 胞內細菌 | zh_TW |
| dc.subject | 樂賜平 | zh_TW |
| dc.subject | loxapine | en |
| dc.subject | high-content assay | en |
| dc.subject | oxidative stress | en |
| dc.subject | efflux pump | en |
| dc.subject | intracellular bacteria | en |
| dc.title | 以新穎二苯氧氮平類化合物去削弱巨噬細胞內沙門氏菌透過外排幫浦的抗氧化能力 | zh_TW |
| dc.title | Novel Dibenzoxazepines Attenuate the Efflux Pump-Mediated Resistance to Oxidative Stresses of Intra-macrophagic Salmonella | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張永祺(Yung-Chiy Chang),蕭崇瑋(Chung-Wai Shiau),蘇伯琦(Po-Chi Soo),陳振暐(Jenn-Wei Chen) | |
| dc.subject.keyword | 樂賜平,胞內細菌,高內涵,外排幫浦,氧化壓力, | zh_TW |
| dc.subject.keyword | loxapine,intracellular bacteria,high-content assay,efflux pump,oxidative stress, | en |
| dc.relation.page | 79 | |
| dc.identifier.doi | 10.6342/NTU202003770 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-17 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| dc.date.embargo-lift | 2025-08-17 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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