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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 江皓森(Hao-Sen Chiang) | |
| dc.contributor.author | Chieh Lin | en |
| dc.contributor.author | 林婕 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:56:59Z | - |
| dc.date.available | 2023-08-01 | |
| dc.date.copyright | 2018-08-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-27 | |
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Hall, An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science, 1995. 269(5228): p. 1270. 36. Liberto, M., D. Cobrinik, and A. Minden, Rho regulates p21CIP1, cyclin D1, and checkpoint control in mammary epithelial cells. Oncogene, 2002. 21: p. 1590. 37. Chiang, H.-S., et al., GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses. Nature Immunology, 2013. 15: p. 63. 38. Fine, N., et al., GEF-H1 is necessary for neutrophil shear stress–induced migration during inflammation. The Journal of Cell Biology, 2016. 215(1): p. 107-119. 39. Papayannopoulos, V., Neutrophil extracellular traps in immunity and disease. Nature Reviews Immunology, 2017. 18: p. 134. 40. Kakiashvili, E., et al., GEF-H1 Mediates Tumor Necrosis Factor-α-induced Rho Activation and Myosin Phosphorylation: ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY. The Journal of Biological Chemistry, 2009. 284(17): p. 11454-11466. 41. Hakkim, A., et al., Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat Chem Biol, 2011. 7(2): p. 75-7. 42. Branzk, N., et al., Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol, 2014. 15(11): p. 1017-25. 43. Urban, C.F., et al., Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans. PLoS Pathogens, 2009. 5(10): p. e1000639. 44. Kanaho, Y., et al., Molecular mechanisms of fMLP-induced superoxide generation and degranulation in mouse neutrophils. Advances in Biological Regulation, 2013. 53(1): p. 128-134. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71176 | - |
| dc.description.abstract | 嗜中性球在免疫系統扮演非常重要的角色，近年來有研究發現發現嗜中性白血球有個特殊的機制，會將細胞核的染色體結構解旋，將上面帶有抗微生物蛋白的DNA釋放到胞外，稱為嗜中性胞外網狀結構(neutrophil extracellular traps, NETs)，這種網狀結構會纏繞病原，阻止其擴散並促進清除和分解。由於過去已知細胞骨架會調控NETs的形成，而guanine nucleotide exchange factor H1 (GEF-H1)可以藉由調節RhoA GTPases的活性來影響下游微小管(microtubule)和肌動蛋白(actin)的動態平衡，因此本論文欲探討GEF-H1在NETs形成時所扮演的角色。我們從缺乏GEF-H1的基因改造小鼠分離出嗜中性球，經實驗確認GEF-H1缺陷並不會影響嗜中性球的分化以及穩定狀態下的細胞骨架型態。我們以phorbol 12-myristate 13-acetate (PMA)刺激嗜中性球，發現GEF-H1缺陷的嗜中性球有較低的胞外DNA訊號，雖然顆粒球(granules)中的蛋白能夠遷移到細胞核內，缺少GEF-H1的嗜中性球卻無法將其釋放到細胞外。且經由定量分析，得知GEF-H1缺陷嗜中性球所生成之NETs大多為擴散型(diffused)而非散佈型(spread)。實驗結果顯示GEF-H1並非藉由調控活性氧化物(reactive oxygen species, ROS)影響NETs的產生。進一步的研究發現GEF-H1會與散佈型NETs結合，並一起被釋放到胞外。綜合以上結果，可推論GEF-H1會調控NETs的釋放。 | zh_TW |
| dc.description.abstract | Neutrophils play an important role in the first line of immune defense. Apart from conventional pathways, neutrophil extracellular trap (NET), a newly discovered mechanism that neutrophil releases chromosomal DNA and specific anti-microbial proteins to the extracellular space and captures pathogens was demonstrated. The formation of NET, a process called NETosis, is accepted as a special form of cell death. However, the detailed mechanism of NETosis has not been fully understood.
Previous studies suggest that actin and microtubule networks are important for NETosis. Given that the guanine nucleotide exchange factor GEF-H1 is crucial in coupling microtubule dynamics to RhoA GTPase activation and actin polymerization, we hypothesized GEF-H1 plays a role in NETosis by regulating cytoskeleton dynamics. Here we found that the area of extracellular DNA was significantly reduced in GEF-H1-deficient mouse neutrophils after phorbol 12-myristate 13-acetate (PMA) incubation. While neutrophil elastase (NE), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) were detected in the nucleus of PMA-incubated GEF-H1-deficient neutrophils, those neutrophils failed to release granule proteins and chromatins extracellularly. Quantification analysis further indicated they had a significantly decreased percentage of spread form NET when compared to wild-type (WT) neutrophils in the presence of PMA. Moreover, we found that GEF-H1 was released and bound to NET components in response to PMA stimulation. Interestingly, the amount of ROS production was comparable between PMA-incubated WT and GEF-H1-deficient neutrophils further indicated that there were profound defects in the late stage of spread NET formation in GEF-H1-deficient neutrophils. Overall, our data suggest that GEF-H1 is the critical regulator of NET release. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:56:59Z (GMT). No. of bitstreams: 1 ntu-107-R05b21002-1.pdf: 7372552 bytes, checksum: 7297ad927b11bd4de5e98742289309ac (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | Contents vi
List of figures ix Chapter 1 Introduction 1 1.1. Neutrophils 1 1.1.1. Neutrophils in innate immune response 1 1.1.2. Neutrophil extracellular traps 2 1.2. GEF-H1 6 1.2.1. The functions of GEF-H1 6 1.2.2. The role of GEF-H1 in immune cells 7 1.3. Specific aim 8 Chapter 2 Materials and Methods 9 2.1. Mice 9 2.2. Isolation of mouse genomic DNA 9 2.3. Genotyping of the Arhgef2+/+, Arhgef2+/−, and Arhgef2−/− mice 10 2.4. Isolation of neutrophils from mouse bone marrow 10 2.5. Fungal culture 11 2.6. Quantification of extracellular DNA release 12 2.7. Flow cytometry 12 2.8. Immunofluorescence staining 13 2.9. Quantification of different NET forms 15 2.10. Phagocytosis Assay 16 Chapter 3 Results 17 3.1. GEF-H1 is dispensable in the generation of neutrophils in mouse bone marrow 17 3.2. GEF-H1 deficiency has no impact on the cytoskeleton pattern in neutrophils in naïve state 17 3.3. GEF-H1-deficient neutrophils show impaired extracellular DNA release upon PMA stimulation 18 3.4. GEF-H1-deficient neutrophils exhibit MPO and citH3 colocolization but are impaired to form spread NETs in response to PMA stimulation 19 3.5. Arhgef2-/- neutrophils show a decreased amount of spread form NETs after PMA stimulation 20 3.6. ROS production in response to PMA stimulation remains intact in GEF-H1-deficient neutrophils 22 3.7. GEF-H1 is released with NET in response to PMA stimulation 23 3.8. GEF-H1 deficiency has no impact in the phagocytosis ability in neutrophils when infected with yeast-locked efg1∆ C. albicans 24 3.9. GEF-H1-deficient mice are more susceptible to Candida albicans infection compared to WT mice 25 Chapter 4 Discussion 27 Chapter 5 Conclusion 31 References 32 Figures 39 Tables 58 Appendix 60 | |
| dc.language.iso | en | |
| dc.subject | 活性氧化物 | zh_TW |
| dc.subject | 嗜中性白血球 | zh_TW |
| dc.subject | 嗜中性球胞外網狀結構 | zh_TW |
| dc.subject | GEF-H1 | zh_TW |
| dc.subject | Reactive oxygen species | en |
| dc.subject | Neutrophils | en |
| dc.subject | Neutrophil extracellular traps | en |
| dc.subject | GEF-H1 | en |
| dc.title | 探討GEF-H1調控嗜中性球胞外網狀結構之釋放 | zh_TW |
| dc.title | Determining the impact of GEF-H1 on the release of neutrophil extracellular traps | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 伍安怡(Betty An-Ye Wu-Hsieh),陳斯婷(Szu-Ting Chen) | |
| dc.subject.keyword | 嗜中性白血球,嗜中性球胞外網狀結構,GEF-H1,活性氧化物, | zh_TW |
| dc.subject.keyword | Neutrophils,Neutrophil extracellular traps,GEF-H1,Reactive oxygen species, | en |
| dc.relation.page | 66 | |
| dc.identifier.doi | 10.6342/NTU201802048 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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