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研究生:林均儒
研究生(外文):Jun-Ru Lin
論文名稱:探討突變基因DCX在大腦神經發育和平腦症中的影響
論文名稱(外文):Subcellular Roles of DCX Mutations in Brain Development and Lissencephaly
指導教授:蔡金吾
指導教授(外文):Jin-Wu Tsai
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:腦科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:45
中文關鍵詞:平腦症DCX微管連結蛋白大腦神經遷移
外文關鍵詞:LissencephaplyDCXMicrotubule binding proteinNeuronal migration
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Contents
Acknowledgements 誌謝…………………………………………………………………i
Chinese Abstract 中文摘要…………………………………………………………v
English Abstract………………………………………………………………………vi
Contents………………………………………………………………………………vii
List of Figures……………………………………………………………………………ix
List of Supplemental Figures………………………………………………………………ix

Chapter 1: Introduction…………………………………………………1

1-1 Development of human cerebral cortex………………………………………1
1-2 Somal translocation in neuronal migration of brain development…………………2
1-3 Genetic variants in neuronal migration disorders…………………………2
1-4 Doublecortin (DCX) and its microtubule binding domain………………………4
1-5 The relationship between microtubule and DCX……………………5
1-6 The variants and mutations on DCX…………………………………………5
1-7 Significance……………………………………………………………6

Chapter 2: Material and Methods…………………………………………7

2-1 The approval and registration from clinical patients was in standard procedure…………………………………………………………………7
2-2 DNA Sanger sequencing…………………………………………………………7
2-3 Plasmids and Mutagenesis construct……………………………………7
2-4 Cell culture……………………………………………………………………9
2-5 Cell transfection………………………………………………………………9
2-6 Protein purification……………………………………………………………9
2-7 Microtubule spin down assay………………………………………………10
2-8 In vitro GST-pull down assay………………………………………………12
2-9 Western Blotting………………………………………………………………13
2-10 Microtubule regrowth assay………………………………………………13
2-11 Immunocytochemistry…………………………………………………14
2-12 Image analysis……………………………………………………………14
2-13 Statistical analysis……………………………………………………………15

Chapter 3: Result……………………………………………………………16

3-1 Identified DCX mutations in lissencephaly patients by whole exome sequencing or Sanger sequencing…………………………………………16
3-2 DCX mutations affect the binding ability to microtubules………………………17
3-3 Localization of WT and mutant DCX in cells……………………………18
3-4 Expression of DCX mutants delayed microtubule growth……………………18

Chapter 4: Conclusion………………………………………………………21

Chapter 5: Discussion………………………………………………………22

5-1 A similar three-dimensional fold in N- and C-terminal domain of doublecortin……………………………………………………22
5-2 GST-DCX pull down assay may not suitable to see microtubule binding ability…………………………………………………………………………23
5-3 Aggregation or denaturation of DCX in microtubule binding assay………24
5-4 DCX mutants may affect the speed of growing microtubules…………………25
5-5 Defecting microtubules might cause neuronal migration delay or post-mitotic cells damaged…………………………………………………………………26

Chapter 6: Reference…………………………………………………………27



List of Figures

Figure 1. Pedigrees and MRI images from the lissencephaly and subcortical band heterotopia patients……………………………………………………………30
Figure 2. The variants on DCX of lissencephaly (LIS) and subcortical band heterotopia X-linked (SBHX) patients…………………………………………………32
Figure 3. Alignment of DCX in different species …………………………………………34
Figure 4. DCX mutations affect the binding ability to microtubule………………………………35
Figure 5. Transfected EGFP-DCX wild type or mutants in U2OS cell…………37
Figure 6. DCX mutations caused microtubule growing slowly in the growth stage…………38
Figure 7. DCX mutations defected the growing speed of microtubule till the stabilization stage………………………………………………………….41
Figure 8. DCX mutations defected the formation of microtubules may delay the neuronal migration during brain development……………………………45

List of Supplemental Figures

Figure S1. In vitro GST pull down assay on GST-DCX wild type or mutants…………46
Figure S2. Similarity in N- and C-terminal binding domain of doublecortin………………48
Figure S3. DCX mutations might delay microtubule growing in the stabilization stage……………………………………………………………………………49
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