Kupffers vesicle 발생 과정에서 AKAP12와 Claudin5의 역할 연구

학위논문 (박사)-- 서울대학교 융합과학기술대학원 분자의학 및 바이오제약학과, 2017. 8. 서영준.Left-right asymmetric organ development is important to establish a proper body plan of vertebrates. In zebrafish, the Kupffers vesicle (KV) is a fluid-filled sac which controls asymmetric organ development, and a properly inflated KV lumen by means of fluid influx is a prerequisite for the asymmetric signal transmission. KV is derived from the dorsal forerunner cells (DFCs), which collectively migrate from the margin of the embryonic shield to the tail bud region, and become polarized to form a rosette structure. Then, the lumen is formed at the apical point of the rosette structure and expanded by fluid influx through ion channels. Finally, nodal signal is exclusively restricted in left-lateral plate mesoderm and organ shows its laterality. However, the underlying mechanisms of the cell-cell interaction during KV formation are poorly studied. Here, I identified that akap12 was expressed in DFCs and downregulation of akap12β in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, KV lineage cell-specific knockdown of akap12β also exhibited the organ laterality defects. In akap12β morphants, cluster of DFC was fragmented which represents a failure of cell collectivity within DFCs, and Cdh1 expression was decreased. Thus, these findings suggest that akap12β regulates KV development by maintaining cell collectivity within DFCs via Cdh1-mediated adherens junction. Furthermore, I identified that the cldn5a is highly expressed at the apical surface of KV epithelial cells and tightly seals the KV lumen. Downregulation of cldn5a in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, accelerated fluid influx into KV by combined treatment of forskolin and 3-isobutyl-1-methylxanthine failed to expand the partially-formed KV lumen in cldn5a morphants. However, malformed KV lumen and defective heart laterality in cldn5a morphants were significantly rescued by exogenous cldn5a mRNA, suggesting that the tightness between the luminal epithelial cells is important for KV lumen formation. Thus, these findings suggest that cldn5a is required for KV lumen inflation and left-right asymmetric organ development. Taken together, these findings suggest that akap12β regulates KV development by maintaining cell collectivity within DFCs via Cdh1-mediated adherens junction and cldn5a regulates KV development by tight sealing the paracellular space between the KV epithelial cells.INTRODUCTION 1 1. Organ laterality and left-right organizer 1 2. Kupffers vesicle development 4 3. A-Kinase Anchoring Protein 12 (AKAP12) 7 4. Tight junction and Claudin5 10 PURPOSE OF THIS STUDY 12 MATERIALS AND METHODS 13 1. Ethics statement 13 2. Zebrafish 13 3. Reverse Transcription-Polymerase Chain Reaction 13 4. Quantitative-PCR 13 5. Morpholino injection 14 6. Pharmacological Reagents 14 7. In vitro transcription 14 8. Transgenesis of Tg(akap12β:mCheery-caax) 15 9. Genome editing using CRISPR/Cas9 system 15 10. genomic DNA isolation 15 11. T7E1 analysis 16 12. AlwN1 restriction analysis 16 13. Melting temperature analysis 16 14. Whole-mount in situ hybridization 16 15. Immunohistochemistry 18 16. Immunofluorescence 18 17. Statistical analysis 19 RESULTS 23 1. akap12β is the major isoform of akap12 during KV development. 23 2. Organ laterality was disrupted in akap12β morphants. 26 3. Organ laterality defects were also exhibited in DFC akap12β morphants. 31 4. KV was malformed in DFC akap12β morphants. 34 5. The cluster of DFC was fragmented in DFC akap12β morphants. 38 6. Generation of TALEN-mediated akap12 mutant. 45 7. Construction of Tg(akap12β:mCheery-caax) by BAC modification 52 8. cldn5a is expressed in KV lineage cells. 56 9. Organ laterality was disrupted in cldn5a morphants. 61 10. cldn5a is required for proper inflation of KV lumen. 69 11. Establishment of cldn5a crispant. 80 DISCUSSION 86 REFERENCES 92 요약 (국문초록 ) 100Docto

顧客滿足과 知覺된 서비스品質과의 關係에 關한 硏究 : 關與度와 購買經驗의 調整的 役割을 中心으로

학위논문(석사)--서울大學校 大學院 :經營學科 經營學專攻,1996.Maste

METHOD AND APPARATUS FOR COMPRESSING DATA BASED ON DEEP LEARNING

본 개시는 데이터를 압축하는 방법 및 장치에 관한 것으로, 본 개시의 일 실시예에 따른 데이터 압축 방법은, 데이터 스토리지 내의 적어도 일부 데이터 블록들에 대해 유사 데이터 블록들로 이루어지는 복수의 카테고리들을 형성하는 단계, 각각의 카테고리에 레이블을 할당하는 단계, 신규 데이터 블록이 속하는 카테고리를 판단하기 위한 학습모델을 레이블링된 카테고리들을 이용하여 훈련시키는 단계, 신규 데이터 블록을 입력 받는 단계, 학습모델을 이용하여 신규 데이터 블록에 적합한 카테고리를 선택하는 단계, 및 선택된 카테고리의 대표 데이터 블록에 기초하여 신규 데이터 블록의 델타 압축을 실행하는 단계를 포함할 수 있다

The effects of Ge on the discontinuous precipitation in an Al-18.6at%Zn

학위논문(석사) - 한국과학기술원 : 재료공학과, 1990.2, [ [iv], 60 p. ]한국과학기술원 : 재료공학과

Serratia sp. MK1이 생산한 포스포리파제 A1A_1을 이용한 라이소-포스포리피드의 생산

학위논문(석사) - 한국과학기술원 : 생물공학과, 1995.2, [ v, 52 p. ]For the phospholipase $A_1$ preparation as a reaction catalyst, factors affecting phospholipase $A_1$ production by Serratia sp. MK1 has been investigated. Serratia sp. MK1 showed maximum phospholipase $A_1$ production at $30^\circ C$ and at pH 7.0 in 14 hours. The M9 minimal medium supplemented with fructose and $(NH_4)_2HPO_3$ as a carbon and nitrogen sources, respectively, showed improved enzyme production. $FeSO_4$ stimulated enzyme production and its optimal concentration was 1$\mu M$ in a growth medium. As a result of above optimization procedure, improved minimal medium could be formulated, and this medium resulted in about seven fold increase enzyme production as compared to the initial complex medium. General properties of phospholipase $A_1$ were also examined. The phospholipase $A_1$ showed its hydrolysis optimum pH and temperature at 8.0 and $40^\circ C$, respectively. The bile salts showed either an activating or inhibitoring effects, depending on the concentration. However, below 3mM concentration, addition of cholate and deoxycholate showed stimulatory effect on the enzyme action. Divalent metal ion was essential for enzyme action. Finally, the production of lysophospholipid by hydrolysis of phospholipid using phospholipase $A_1$ was investigated in two-phase and emulsion systems. Considering the yield of reaction products and efficiency of reaction, aqueous-solvent two-phase system was more appropriate than emulsion system for the lysophospholipid production. Among the 13 organic solvents tested as a solvent-phase, butyl acetate was selected as the most suitable solvent in terms of catalytic activity and enzyme stability. The optimal substrate concentration in butyl acetate was 5-10\%. Under the selected conditions, the reaction was carried out and the result of TLC/FID showed that phospholipid was hydrolyzed completely to the lysophospholipid in this system.한국과학기술원 : 생물공학과

포스포리파제를 이용한 플라스마로젠의 농축과 포스포리피드의 합성

학위논문(박사) - 한국과학기술원 : 생물과학과, 1999.2, [ xii, 100 p. ]In order to purify plasmalogen of phospholipids from bovine heart and brain, hospholipase A1 from Serratia sp. MK1 was used. This enzyme hydrolyzes only the ester bond at sn-1 position of diacyl glycerophosphocholine, not the ether bond of plasmalogen. We, therefore, obtained higher purity of plasmalogen of phospholipids from bovine heart and brain with the phospholipase. For the selection of the suitable reaction system, the reaction was examined in a two-phase system and an emulsion system. The purity of plasmalogen in the two-phase system was found to be better than that obtained in the emulsion system. In the case of the emulsion system $CaCl_2$ was required and 60% of diacyl glycerophosphocholine was hydrolyzed. However, in the two-phase system no $CaCl_2$ was required and most of diacyl glycerophosphocholine was hydrolyzed within 45 min. It was found that Tris-Cl buffer was better than phosphate buffer for the reaction. In Among various solvents, butyl acetate showed best catalytic activity and enzyme stability. Therefore we selected butyl acetate as a solvent in a two-phase system. The different ratios of solvent-aqueous phase had no significant effects on the purity of plasmalogen. In case of ethanolamine containing substrate, diacyl glycerophosphoethanolamine was completely hydrolyzed. When crude plasmalogen containing phospholipid was used, 99% purified plasmalogen was obtained at 0.5% substrate concentration. Synthesis of useful phospholipid with phospholipase $A_2$ was investigated. We synthesized phospholipids with PUFA (poly unsaturated fatty acid) and selected EPA (eicosapentaenoic acid) as a PUFA. For the increasing yield of PUFA-phospholipid, optimal EPA and enzyme quantity was investigated. Optimal EPA (using EPA-ester as a acyl donor) quantity was 50 mg when LPC was 10 mg. Phospholipase quantity was determined to 5 mg in considering reactant viscosity and enzyme cost. There are various parameters that govern this PUFA-phospholipid synthesis ...한국과학기술원 : 생물과학과