Dictyostelium discoideum 분화과정에서 glutathione 매개 cAMP 신호 전달체계

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부 미생물학과, 2017. 2. 강사욱.Glutathione (GSH, γ-glutamyl-L-cysteinylglycine) is the most prevalent reducing thiol-containing compound in eukaryotic cells and plays indispensable roles in multicellular development. However, the function of glutathione synthetase (gshB/GSS), the final-step enzyme of GSH biosynthesis, on development of Dictyostelium discoideum has not been clearly investigated. Herein, it was found that the overexpression of gshB (gshBoe) facilitates a significant increase of the intracellular GSH levels during both early and late development, which leads to developmental defects, such as the mound-arrest phenotype and the reduced spore formation. Moreover, in wild-type (KAx3) cells, addition of exogenous GSH led to similar defects to those characteristic of gshBoe cells. To investigate the effects of increased intracellular GSH level by gshB overexpression on Dictyostelium development in more detail, the cAMP-mediated aggregation and cell-type differentiation were examined. First, when cells were allowed to develop at low cell densities, gshBoe cells were not able to form aggregates and streams. gshBoe cells exhibited multiple pseudopodia, indicating a lack of axial polarity during aggregation. In addition, gshBoe cells exhibited abnormal fluctuating pattern in F-actin polymerization in response to cAMP stimulation. Next, during differentiation, gshBoe cells showed remarkable differences in the cell-type proportioning with KAx3 cells. In the mounds of gshBoe cells, pre-stalk cells were increased relative to those in KAx3 mounds, whereas pre-spore cells were remarkably decreased, which was consistent with the reduced sporulation of gshBoe cells. Based on these results, the expression pattern of the essential components of the cAMP signaling pathway (carA/cAR1/cAMP receptor 1, gpaB/Gα2/G protein alpha subunit 2, gpbA/Gβ/G protein beta subunit, and acaA/ACA/adenylyl cyclase A), which are developmental regulators, was examined in gshBoe cells. As the result, the expression level of carA and gpaB was upregulated, especially after 8 h of development. Similarly, in gshBoe cells, the intracellular cAMP concentration were significantly 4.5-fold higher than those of KAx3 cells at 8 h after the onset of development. To further investigate the effect of gshB overexpression on the cAMP signaling pathway, the mutant strains which constitutively expressed carA and gpaA (carAoe and gpaBoe, respectively) were generated. During developmental processes, gpaBoe cells displayed the defects in multicellular aggregation and F-actin polymerization characteristic of gshBoe cells, which may be as a secondary response to abnormally elevated cAMP concentrations, but carAoe cells did not. Furthermore, gpaBoe cells exhibited phenocopies of gshBoe cells in cell-type proportioning, such as the increased pre-stalk cells and the decreased pre-spore cells. Interestingly, gpaB knockdown in gshBoe cells (gpaBas/gshBoe) partially rescued the defects of gshBoe cells. gpaBas/gshBoe cells produced fruiting bodies with small sori and were able to form aggregates and streams at low cell densities. These results suggest that the developmental defects associated with gshBoe cells may be caused by increased gpaB expression and high intracellular cAMP concentrations. Additionally, there was the distinction in Gα2-GFP localization between KAx3 and gshBoe cells. In KAx3 cells, Gα2-GFP was uniformly expressed prior to cAMP stimulation and rapidly translocated to the plasma membrane after cAMP stimulation. However, in gshBoe cells, Gα2-GFP was strongly localized at the plasma membrane both before and after the addition of cAMP, implying that GSH could also regulate Gα2 localization. Taken together, these findings suggest that GSH regulates developmental stage and cell-type proportioning by upregulating cAMP signaling pathway via modulation of Gα2 expression and localization during the development of Dictyostelium. These results will provide insights into the general mechanisms underlying cell development involved in GSH-mediated signaling.I. INTRODUCTION 1 1. Glutathione 2 1.1. An overview of gluathione 2 1.2. Glutathione biosynthesis 3 1.3. Glutathione metabolism 6 1.4. The roles of glutathione in cellular functions 7 1.5. The roles of glutathione in development 9 2. Dictyostelium discoideum 11 2.1. An overview of Dictyostelium discoideum 11 2.2. The early events during development in D. discoideum 13 2.2.1. The cAR1-mediated cAMP signaling 14 2.2.2. Chemotaxis in D. discoideum 16 2.3. Cell differentiation in D. discoideum 20 2.3.1. Control of cell-type induction 20 2.3.2. Cell fate and patterning 21 2.3.3. Subdivision of the multicellular structures 22 2.3.4. Terminal differentiation (Culmination) 26 3. Aims of this study 27 II. MATERIALS AND METHODS 29 1. Chemicals used in this study 30 2. D. discoideum culture and development 30 3. Generation of overexpression and/or knockdown strains 31 4. RNA extraction and northern blot analysis 34 5. Measurement of intracellular GSH level 34 6. F-actin staining assay 35 7. Neutral red and histochemical staining 36 8. Quantitative RT-PCR 36 9. Measurement of intracellular cAMP level 37 10. GFP fluorescence microscopy 37 11. Cell fate choice 38 III. RESULTS 39 1. Effects of intracellular GSH level on development of D. discoideum 40 1.1. Generation of mutant cells having high or low GSH contents in D. discoideum 40 1.2. An increase of intracellular GSH level inhibits Dictyostelium development 40 2. Developmental properties of gshBoe cells 46 2.1. Effects of high GSH level on aggregation processes 46 2.2. Effects of high GSH level on cell differentiation 48 2.3. Effects of high GSH level on the regulation of cAR1-mediated cAMP signaling 54 3. The role of gshB overexpression in the regulation of gpaB expression 57 3.1. Developmental properties of gpaBoe cells 57 3.2. Effects of gpaB overexpression on aggregation processes 57 3.3. Effects of gpaB overexpression on cell differentiation 62 3.4 The relationship between the expression of gshB and gpaB 67 3.4.1. gpaB knockdown can rescue the phenotypic defects in gshBoe cells 67 4. The effect of gshB overexpression on the regulation of Gα2 localization 73 IV. DISCUSSION 76 V. REFERENCES 87 국문초록 105Docto

사이버윤리의 도덕과교육에서의 적용에 관한 연구

학위논문(석사)--서울대학교 대학원 :국민윤리교육과,2002.Maste

An Analysis of the Economic Effects of the Pilot Project for Multiple-Purpose Utilization of Paddy Fields Focusing on Income and Welfare Changes

The food self-sufficiency rate of agricultural products in Korea, excluding rice, is around 20%, and the government is promoting various policies including a Multiple-Purpose Utilization of Paddy Fields project, to increase the self-sufficiency rate of major grains. The project for Multiple-Purpose Utilization of Paddy Fields is being promoted as a part of a program to create farmland infrastructure to facilitate the cultivation of crops other than rice in rice paddies, and pilot projects were started in four regions in 2020. The purpose of this study is to analyze the economic effects of the pilot project for Multiple-Purpose Utilization of Paddy Fields, and to propose policies to increase the effectiveness of the project. In order to analyze the economic effect, we estimated the change in farm income generated by switching from rice to other crops, and measured the effect of welfare change using the Equilibrium Displacement Model (EDM). As a result of the analysis, social welfare is expected to increase when the pilot project for Multiple-Purpose Utilization of Paddy Fields is implemented, and the income of the beneficiary farmers is also expected to improve compared to that of single-cropping when double-cropping is implemented. However, it was found that the economic feasibility of the project differs depending on the crops converted. Juksan-myeon, Gimje-si, which is an area where soybean production was successful, was analyzed from the viewpoint of increasing the economic feasibility of the pilot project. Their success factors were analyzed into four major factors: infrastructure, farming methods, education, and collaboration with local agricultural organizations. If such a success story can be utilized in the future project implementation process, it can contribute to the improvement of farm household income and national economic welfare.N

2012년도 하계학술대회 발표 논문집 '차기 정부의 농정과제' (안희정,이정환,이명현,박시현,김동환,이정희,황성혁,유학열,이영옥,정현희,장재봉,최지현,조현진,김윤식,Jun Hee Lee,Yoon,Seok-Gon,Hirogi Fujishima,K

2012년도 하계학술대회 발표 논문집 '차기 정부의 농정과제' [특별강연] 농어업·농어촌의 새희망 「3농혁신」 (안희정 충청남도 도지사) [기조강연] 지금 우리는 어디에, 그리고 어디로? (이정환 GS&J 인스티튜트) ￭차기정부의 농정과제 : 농가소득의 안정 및 복지 (이명현 인천대학교 교수) ￭신 정부의 농정과제 -농촌정책- (박시현 한국농촌경제연구원 선임연구위원) ￭차기 정부의 농정 방향 -농산물 유통 및 협동조합 분야-  (김동환 안양대학교 교수/농식품신유통연구원 원장) ￭식품산업정책의 나아갈 방향 (이정희 중앙대 경제학부 교수,황성혁 농협경제연구소 부연구위원) ￭농어업 6차산업화의 개념 및 성공요인 도출 -충남 선진사례를 중심으로- (유학열,이영옥 충남발전연구원) ￭광역 공동상표의 방향성 정립 및 시(군)도간 상호 발전 방안 -으뜸Q마크를 중심으로-  (정현희 충남발전연구원) ￭값싼 수다(Cheap talk)가 가상적 편의(Hypothetical bias)에 미치는 영향 : 실험선호추출법  (장재봉 영남대학교 자연자원대학 식품자원경제학과 조교수,최지현 한국농촌경제연구원 선임연구위원) ￭유가와 시설작물 재배농가 소득률 상관관계 분석  (조현진 경남발전연구원 전문연구원,김윤식 교신저자,경상대 농업경제학과 부교수) ￭Problems and Solution Measures for Wholesale Market System in Japan (Jun Hee Lee Graduate School in Tokyo Univ,Yoon,Seok-Gon Namseoul Univ, Hirogi FujishimaTokyo Univ, Kazuyuki Miyabe Nihon Univ) ￭Reducing the Social Cost of Federal Crop Insurance : A Role for Government Hedging with Weather Derivatives (Wongo Chung Research Fellow Korea Rural Economic Institute) ￭고랭지 배추 재배 농가의 생산 효율성 분석  (이향미 강원대 농업자원경제학과 박사과정,고종태 강원대 농업자원경제학과 교수) ￭체재형 주말농장 입주에 대한 지불의사금액 추정 (박지연 경북대학교 교수) ￭일별 외식여부에 따른 소비자의 칼로리 섭취 효과 분석  (박미성 고려대 식품자원경제학과 대학원,안병일·한두봉 고려대 식품자원경제학과 교수) ￭기후 변수가 주요 품목의 생산성에 미치는 영향 : 준모수적 분석 (조현경,조은빛 서울대 대학원 농경제사회학부 농업·자원경제학 전공  권오상,노재선 서울대 농경제사회학부 농업·자원경제학 교수)1. [주제발표] ￭차기정부의 농정과제 : 농가소득의 안정 및 복지 (이명현 인천대학교 교수) ￭신 정부의 농정과제 -농촌정책- (박시현 한국농촌경제연구원 선임연구위원) ￭차기 정부의 농정 방향 -농산물 유통 및 협동조합 분야-  (김동환 안양대학교 교수/농식품신유통연구원 원장) ￭식품산업정책의 나아갈 방향 (이정희 중앙대 경제학부 교수,황성혁 농협경제연구소 부연구위원) 2. [특별세션] ￭농어업 6차산업화의 개념 및 성공요인 도출 -충남 선진사례를 중심으로- (유학열,이영옥 충남발전연구원) ￭광역 공동상표의 방향성 정립 및 시(군)도간 상호 발전 방안 -으뜸Q마크를 중심으로-  (정현희 충남발전연구원) 3. [자유주제] ￭값싼 수다(Cheap talk)가 가상적 편의(Hypothetical bias)에 미치는 영향 : 실험선호추출법  (장재봉 영남대학교 자연자원대학 식품자원경제학과 조교수,최지현 한국농촌경제연구원 선임연구위원) ￭유가와 시설작물 재배농가 소득률 상관관계 분석  (조현진 경남발전연구원 전문연구원,김윤식 교신저자,경상대 농업경제학과 부교수) ￭Problems and Solution Measures for Wholesale Market System in Japan (Jun Hee Lee Graduate School in Tokyo Univ,Yoon,Seok-Gon Namseoul Univ, Hirogi FujishimaTokyo Univ, Kazuyuki Miyabe Nihon Univ) ￭Reducing the Social Cost of Federal Crop Insurance : A Role for Government Hedging with Weather Derivatives (Wongo Chung Research Fellow Korea Rural Economic Institute) ￭고랭지 배추 재배 농가의 생산 효율성 분석  (이향미 강원대 농업자원경제학과 박사과정,고종태 강원대 농업자원경제학과 교수) ￭체재형 주말농장 입주에 대한 지불의사금액 추정 (박지연 경북대학교 교수) ￭일별 외식여부에 따른 소비자의 칼로리 섭취 효과 분석  (박미성 고려대 식품자원경제학과 대학원,안병일·한두봉 고려대 식품자원경제학과 교수) ￭기후 변수가 주요 품목의 생산성에 미치는 영향 : 준모수적 분석 (조현경,조은빛 서울대 대학원 농경제사회학부 농업·자원경제학 전공  권오상,노재선 서울대 농경제사회학부 농업·자원경제학 교수