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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳示國(Shih-Kuo Chen) | |
| dc.contributor.author | Chun-Hao Chien | en |
| dc.contributor.author | 簡君豪 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:18:09Z | - |
| dc.date.available | 2025-04-15 | |
| dc.date.copyright | 2020-04-15 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-03-30 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63750 | - |
| dc.description.abstract | 腸道菌 (gut microbiota) 是腸道中微生物的總稱，其組成與週期性受外在刺激與宿主的調控，也能影響宿主的多種生理功能，包含代謝、免疫系統與神經系統等。過去研究發現，腸道菌相的組成受到宿主與外在環境的影響，如基因與飲食。每日飲食熱量即是一種外界刺激，卡洛里限制飲食 (calorie restriction) 過去被發現能影響腸道菌相與週期性，也能提升記憶能力，但其中的關聯性並不清楚。本研究利用抗生素處理 (antibiotic treatment) 與糞便微生物移植 (fecal microbiota treatment) 調控腸道菌相，結合次世代定序 (Next Generation Sequencing) 及多種行為測試，發現限制飲食熱量的方式，會藉由改變腸道菌的組成與週期性，而提升小鼠的記憶能力。 除了飲食，光線亦是一種外界刺激，可調控腸道菌的組成與週期性。我們將野生型小鼠與基因轉殖鼠 (Lrrk2G2019S)飼養於不同光照週期下，發現迴腸的表皮細胞參與光線對於腸道菌週期性調控之可能性。此外，我們將帕金森氏症 (Parkinson’s disease) 模式小鼠 (SNCAA53T) 飼養於異常的光照週期，發現腸道菌的改變早於行為病症病狀出現之前，且帕金森氏鼠與控制組小鼠的腸道菌相差異會隨著病症進展而增加。 整體而言，此研究證實了腸道菌於卡洛里限制飲食提升記憶能力之角色，也揭示腸道菌與帕金森氏症病人中突觸核蛋白 (α-synuclein) 異常沈積具雙向調控的可能性，更為宿主調控腸道菌週期性的機制提供新研究方向。 | zh_TW |
| dc.description.abstract | The composition and rhythmicity of the gut microbiota, referring to all microorganism in the gut, affects physiological functions of the host, such as metabolism, immune system, and nervous system. It has been shown that internal and external cues can both regulate the composition of the gut microbiota, such as the host genetic background and food intake, respectively. Calorie restriction (CR) is reported as one of the environmental cues to influence the composition and rhythmicity of the gut microbiota, as well as elevate memory with unknown mechanism. In this study, we showed that gut microbiota modulation such as antibiotics treatment or fecal microbiota transplantation could influence the memory enhancement effect in mice. Combining Next Generation Sequencing and several behavior tests, we validate that CR improves memory of mice via the modification on the gut microbial composition and rhythmicity. Besides, light is also an environmental cue, which influences the composition and rhythmicity of the gut microbiota. By keeping the WT and Lrrk2G2019S mice under different light-dark cycle, we demonstrate the epithelium cells in the ileum may potentially participate in the gut microbiota rhythmicity regulation via NOD2 pathway. Furthermore, our preliminary results showed that the gut microbial variation initiates before the presence of motor disability in SNCAA53T mice, a Parkinson’s disease (PD) mice model. And the gut microbial difference between PD and control mice increases with PD progression. Overall, our study validates the role of the gut microbiota in the CR-induced memory enhancement. We also demonstrate the possible reciprocal regulation between the gut microbiota and the aberrant α-synuclein accumulation in PD subjects. In addition, this study reveals a novel perspective in the regulation of the gut microbiota rhythmicity by the host. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:18:09Z (GMT). No. of bitstreams: 1 ntu-109-R07b21005-1.pdf: 10521551 bytes, checksum: 0a0debf12493ae96a332b876897a1fbf (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員審定書 ii 致 謝 iii 摘 要 iv Abstract v Contents vii Chapter I Introduction 1 1.1 Circadian rhythm 1 1.1.1 Overview of the circadian rhythm 1 1.1.2 Molecular clock of the circadian rhythm 2 1.1.3 The central and peripheral clock of the circadian system 3 1.2 Gut microbiota 4 1.2.1 The composition of the gut microbiota 5 1.2.2 The rhythmicity of the gut microbiota 7 1.2.3 The gut microbiota and the immune system 9 1.2.4 The gut microbiota and Parkinson’s Disease (PD) 12 1.3 Calorie restriction (CR) 15 1.3.1 CR and the gut microbiota 15 1.3.2 CR and health 16 Statement of Purpose 19 Chapter II Materials and Methods 21 2.1 Animals 21 2.2 Experimental design 22 2.2.1 The relationship of the gut microbiota and CR-induced memory enhancement 22 2.2.2 The transformation of the gut microbiota in PD disease mice model 23 2.2.3 The regulation of the gut microbiota rhythmicity through NOD2 pathway 24 2.3 Illumina MiSeq sequencing 25 2.3.1 Total fecal DNA extraction 25 2.3.2 16S metagenomic library preparation 26 2.3.3 Pooling and quality control 28 2.3.4 Next Generation Sequencing (NGS) 29 2.4 Microbiota sequence analysis 29 2.4.1 Sequence assembling and identification 29 2.4.2 Composition analysis 31 2.4.3 Diversity analysis 31 2.4.4 Microbe abundance analysis 32 2.4.5 Circadian analysis of microbial oscillations 32 2.5 Quantification of the gut microbe 33 2.5.1 Standard preparation 33 2.5.2 Quantitative real-time polymerase chain reaction (qPCR) 34 2.6 Fecal microbiota transplantation (FMT) 35 2.7 Quantification of gene expression 35 2.7.1 RNA extraction 35 2.7.2 Reverse transcription 37 2.7.3 qPCR 37 2.8 Novel object recognition test (NOR) 38 2.9 Three chamber social test (3CH) 38 2.10 Open field test 39 2.11 Beam balance test 40 2.12 Rotarod test 41 2.13 Immunocytochemistry 42 2.13.1 Brain collection 42 2.13.2 Immunocytochemistry 42 2.13.3 Analysis of the PER2-positive cell number in the SCN 43 2.14 Statistical analysis 43 Chapter III Results - The Relationship of the Gut Microbiota and CR-induced Memory Enhancement 45 3.1 The gut microbiota is different between CR and ad libitum (AL) 45 3.2 The gut microbiota is involved in CR-induced memory enhancement 47 3.3 Rhythmicity is probably not involved in CR-improved memory 52 Chapter IV Results - The Transformation of the Gut Microbiota in PD Mice Model 55 4.1 PD progression in SNCAA53T mice (HET) is accelerated 55 4.2 Gut microbial variation starts at early pre-symptom stage and increases with PD progression 56 Chapter V Results - The Regulation of the Gut Microbiota Rhythmicity through NOD2 Pathway 59 5.1 Nod2 expresses in a circadian manner 59 5.2 The role of NOD2 pathway in the regulation of light on the gut microbiota rhythmicity needs further investigation 60 Chapter VI Discussion 63 6.1 The participation of the gut microbiota in CR-induced memory enhancement 63 6.2 The gut microbiota rhythmicity and CR-induced memory enhancement. 64 6.3 The biological meaning of ⍺ and β diversity 65 6.4 The gut microbial candidates in the CR-enhanced memory 67 6.5 The possible reciprocal regulation of the gut microbiota and α-synuclein accumulation in the gut 70 6.6 NOD2 pathway potentially regulates the rhythmicity of the gut microbiota 73 Significance of the Work 75 References 77 Figures 91 Figure 1. Experimental design of Chapter III (1). 91 Figure 2. The composition of the gut microbiota of mice treated with CR or ad libitum (AL). 92 Figure 3. The phylum level relative abundance of the gut microbiota of AL and CR mice. 93 Figure 4. The diversity of the gut microbiota of AL and CR mice. 94 Figure 5. Abundance analysis of the gut microbiota of AL and CR mice. 95 Figure 6. Circadian rhythmicity profile of the gut microbiota of AL and CR mice. 96 Figure 7. Quantification of total gut microbe abundance in the gut microbiota of mice treated with AL, CR, AL plus antibiotics cocktail (ALA) and CR plus antibiotics cocktail (CRA). 97 Figure 8. The results of novel object recognition test (NOR) of mice treated with AL or CR plus water or antibiotics cocktail (Abx). 98 Figure 9. The results of three chamber social test (3CH) of mice treated with AL or CR plus water or Abx. 99 Figure 10. Experimental design of Chapter III (2). 101 Figure 11. The composition of the gut microbiota of mice treated with AL, CR, AL plus AL fecal (FAL) and AL plus CR fecal (FCR) during the experiments. 102 Figure 12. Experimental design of Chapter III (3). 103 Figure 13. The composition of the gut microbiota of AL, CR, FAL and FCR mice. 104 Figure 14. The phylum level relative abundance of the gut microbiota of FAL and FCR mice. 105 Figure 15. The diversity of the gut microbiota of FAL and FCR mice. 106 Figure 16. Abundance analysis of the gut microbiota of FAL and FCR mice. 107 Figure 17. Circadian rhythmicity profile of the gut microbiota of FAL and FCR mice. 108 Figure 18. The results of NOR of FAL and FCR mice. 109 Figure 19. The results of 3CH of FAL and FCR mice. 110 Figure 20. The linear regression of the rhythmicity and discrimination index of NOR. 111 Figure 21. The linear regression of the rhythmicity and discrimination index of 3CH. 112 Figure 22. The linear regression of the genera and discrimination index of NOR. 113 Figure 23. The linear regression of the genera and discrimination index of 3CH. 114 Figure 24. Experimental design of Chapter III (4). 115 Figure 25. Circadian rhythmicity profile of the gut microbiota of mice treated with CR and kept under normal 12h/12h light-dark cycle (LDC) or constant dark environment (DDC). 116 Figure 26. The results of NOR of LDC and DDC mice. 117 Figure 27. The results of 3CH of LDC and DDC mice. 118 Figure 28. The composition of the gut microbiota of LDC and DDC mice. 119 Figure 29. The phylum level relative abundance of the gut microbiota of LDC and DDC mice. 120 Figure 30. The diversity of the gut microbiota of LDC and DDC mice. 121 Figure 31. Abundance analysis of the gut microbiota of LDC and DDC mice. 122 Figure 32. Experimental design of Chapter IV. 123 Figure 33. The results of Open Field Test of HET and NTG mice. 124 Figure 34. The results of rotarod test and beam balance test of HET and NTG mice. 125 Figure 35. The composition of the gut microbiota of HET and NTG mice at different age. 126 Figure 36. The phylum level relative abundance of the gut microbiota of HET and NTG mice at different age. 127 Figure 37. The diversity of the gut microbiota of HET and NTG mice. 128 Figure 38. Abundance analysis of the gut microbiota of HET and NTG mice. 129 Figure 39. Circadian rhythmicity profile of the gut microbiota of HET and NTG mice. 130 Figure 40. Experimental design of Chapter V (1). 131 Figure 41. Quantification of expression of Nod1, Nod2, Rab1a, Rab2a and Lrrk2 in the ileum of WT mice. 132 Figure 42. Quantification of expression of Nod2 in the ileum of WT mice. 133 Figure 43. Experimental design of Chapter V (2). 134 Figure 44. Representative figures of PER2-expressing cells in the suprachiasmatic nucleus (SCN) of Lrrk2G2019S (HETL) mice and Lrrk2NTG (NTGL) mice. 135 Figure 45. Quantification of PER2-positive cell number in the SCN of HETL mice and NTGL mice under different light treatments. 136 Figure 46. The composition of the gut microbiota of HETL and NTGL mice under different light treatments. 137 Figure 47. The phylum level relative abundance of the gut microbiota of HETL and NTGL mice at different light treatments. 138 Figure 48. The diversity of the gut microbiota of HETL and NTGL mice. 139 Figure 49. Abundance analysis of the gut microbiota of HETL and NTGL mice. 141 Figure 50. Circadian rhythmicity profile of the gut microbiota of HETL and NTGL mice. 142 Tables 143 Table 1. Primers used in metagenomics sample preparation 143 Table 2. Primers of Nextera® Index 144 Table 3. Primer List of the Preparation of Standard DNA 145 Table 4. Primer List of the Gut Microbe Quantification 146 Table 5. Primer List of the Gene Expression Quantification 147 Appendix I 16S Metagenomic Analysis Pipeline 149 Appendix II Posters 155 Poster 1. 2019 Poster of United Exhibition, College of Life Science, National Taiwan University, Taiwan 155 Poster 2. 2019 Taiwanese Society of Developmental Biology Retreat, Taiwanese Society of Developmental Biology, Taiwan 156 Poster 3. 2019 Taiwan Neuroscience Society Annual Meeting, Taiwan Neuroscience Society, Taiwan 157 Poster 4. 2019 Tsukuba Conference, University of Tsukuba, Japan 158 Poster 5. 2019 Annual Meeting Society For Neuroscience, Society For Neuroscience, United States of America 159 | |
| dc.language.iso | en | |
| dc.title | 腸道菌於卡洛里限制飲食提升記憶能力與帕金森氏症之研究 | zh_TW |
| dc.title | The Study of the Gut Microbiota in Calorie-restriction-induced Memory Enhancement and Parkinson’s Disease | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王培育(Pei-Yu Wang),林靜嫻(Chin-Hsien Lin),江皓森(Hao-Sen Chiang),周銘翊(Ming-Yi Chou) | |
| dc.subject.keyword | 腸道菌,生理時鐘,卡洛里限制飲食,學習型記憶,帕金森氏症, | zh_TW |
| dc.subject.keyword | gut microbiota,circadian rhythm,calorie restriction,learning memory,Parkinson’s disease, | en |
| dc.relation.page | 159 | |
| dc.identifier.doi | 10.6342/NTU202000715 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-03-30 | |
| 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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