자가 면역 뇌척수염 마우스 모델에서 deferoxamine을 전처리한 개의 지방유래 중간엽줄기세포로부터 유래한 세포외소포체의 면역 조절 효과

학위논문(박사) -- 서울대학교대학원 : 수의과대학 수의학과, 2023. 2. 윤화영.Mesenchymal stem/stromal cells (MSCs) are effective therapeutic agents that ameliorate inflammation through paracrine effect. In particular, several studies have been tried to apply MSC to autoimmune neurological diseases such as multiple sclerosis. Multiple sclerosis is a disease in which nerve damage occurs when inflammatory cells infiltrate nerve tissue due to loosing self-regulated immune system. Also in dogs, there is a similar disease, which is a meningoencephalitis of unknown etiology (MUE). The cause of MUE is not yet clear, but it is assumed to be caused by immune problem, and in this regard, main treatment is administrating non-specific immunosuppressants. Although about 25% of dogs which has neurological disease struggle with MUE, treatment has not been developed significantly. Non-specific immunosuppressants, including steroids, has several problems such as gastrointestinal disorders and hormonal secretion disorders, on the other hand, immunosuppressants treatment effect is not guaranteed, one of the difficulties in treating MUE. In this respect, extracellular vesicles (EVs) derived from MSCs have been investigated as a treatment option for autoimmune diseases. However, further study is needed on clinical efficacy of EV. To improve the secretion of anti-inflammatory factors from MSCs, preconditioning with hypoxia or hypoxia-mimetic agents has been attempted. Moreover, the molecular changes in preconditioned MSC-derived EVs have been explored and its clinical efficacy has not been proven. This study aimed to evaluate the therapeutic effect of EVs derived from deferoxamine (DFO)-preconditioned canine adipose tissue-derived (cAT)-MSCs (EVDFO) in an experimental autoimmune encephalomyelitis (EAE) mouse model and explore the mechanism underlying immunomodulation function of EV. This dissertation is composed of three parts. The first part of dissertation revealed that cAT-MSCs preconditioned with DFO (MSCDFO) can more effectively direct and reprogram macrophage polarization into the M2 anti-inflammation state by paracrine effect. MSCDFO exhibited enhanced secretion of anti-inflammatory factors such as prostaglandin E2 and tumor necrosis factor-α-stimulated gene-6. To evaluate the interaction between MSCDFO and macrophages, RAW 264.7 cells were co-cultured with cAT-MSCs using the transwell system, and changes in the expression of factors related to macrophage polarization were analyzed using the quantitative real-time PCR and western blot assays. When RAW 264.7 cells were co-cultured with MSCDFO, the expression of M1 and M2 markers decreased (iNOS, 1.32 fold, p<0.01; IL-6, 3.46 fold, p<0.05) and increased (CD206, 2.61 fold, p<0.001; Ym1, 4.92 fold, p<0.01), respectively, compared to co-culturing with non-preconditioned cAT-MSCs. Thus, cAT-MSCs preconditioned with DFO can more effectively direct and reprogram macrophage polarization into the M2 phase, an anti-inflammatory state. The second part of dissertation is designed to evaluate that EVDFO regulated macrophage through activating signal transducer and transcription3 (STAT3) phosphorylation. In MSCDFO, Hypoxia-inducible factor 1-alpha was found to accumulate and expression of Cyclooxygenase-2 (COX-2) was increased (16.77 fold, p<0.001). Changes in expression of COX-2 were reflected in the derived EVs as well. The canine macrophage cell line, DH82, was treated with EVnon and EVDFO after lipopolysaccharide stimulation and polarization changes were evaluated with quantitative real-time PCR and immunofluorescence analyses. When DH82 was treated with EVDFO, the expression of M1 marker was reduced (IL-1β, 2.45 fold, p<0.001; IL-6, 17.26 fold, p<0.001) while that of M2 surface marker was enhanced (CD206, 7.24 fold, p<0.001) compared to that when DH82 was treated with EVnon. Further, phosphorylation of STAT3 expression was increased more when DH82 cells were treated with EVDFO (1.79 fold, p<0.001). EV derived from cAT-MSC treated with si-COX2 showed similar effect with EV and the effect of immunomodulation was decreased than EVDFO (IL-1β, 2.21 fold, p<0.001; IL-6, 1.43 fold, p<0.001; CD206, 2.27 fold, p<0.001). Thus, COX-2 in EV may be one of key factor to regulate STAT3 and modulate macrophage. The last part of dissertation demonstrates that EVDFO treatment has a relatively higher efficacy in reducing inflammation than non-preconditioned EV treatment and could modulate immune system through regulating STAT3 in EAE model. EAE mice were divided into different groups based on intranasal administration of EVs or EVDFO (C57BL/6, male, control=6, EAE=8, EAE+EV=8, EAE+EVDFO=8, 10 μg/day;14 injections). On day 25 post-EAE induction, the mice were euthanized, and the spleen, brain, and spinal cord were analyzed into histopathologic and expression of RNA and protein level. Histologically, in the EV and EVDFO groups, the infiltration of inflammatory cells decreased significantly (EV, 1.38 fold, p<0.01; EVDFO, 1.72 fold, p<0.01), and demyelination was alleviated (EV, 2.96 fold, p<0.05; EVDFO, 5.28 fold, p<0.05). Immunofluorescence staining showed that the expression of CD206 and Foxp3, markers of M2 macrophages and regulatory T (Treg) cells, respectively, increased significantly in the EVDFO group compared to the EAE and EAE+EV group. In the EAE group, the number of CD4+CD25+Foxp3+ Treg cells in the spleen decreased significantly compared with the naïve group (2.74 fold, p<0.001). In contrast, the number of Treg cells showed a greater increase in the EAE+EVDFO group than in the EAE+EV group (1.55 fold, p<0.05). The protein expression of STAT3 and pSTAT3 increased in the spleen in the EAE groups compared to the naïve group (STAT3, 2.02 fold, p<0.001; pSTAT3, 2.14 fold, p<0.001). However, following EV treatment, STAT3 expression decreased compared to the EAE group (1.32 fold, p<0.001), especially reduction of STAT3 was evident in EVDFO compared to EV group (1.90 fold, p<0.001). Therefore, EV could regulate STAT3 expression and EVDFO has more effect than EV. In conclusion, that preconditioned with DFO in cAT-MSC is an effective method to improve immunomodulation effect of EVs. Also, EVDFO is potential therapeutic option for multiple sclerosis through regulating STAT3 pathway and modulating immune system. These findings suggest a new approach to cell free therapy with preconditioned EV in other autoimmune diseases as well as multiple sclerosis. Furthermore, this study is a major basis that EVDFO can be applied as a new treatment for MUE in dogs and the cornerstone to the development of autoimmune disease treatment in veterinary medicine.중간엽줄기세포(Mesenchymal stem cell; MSC)의 분비 능력은 염증을 개선하는데 있어서 효과적이라 보고되어, 이를 염증 치료제로 개발하기 위해 활발히 연구되고 있다. 특히나 다발성 경화증과 같이 자가면역 신경질환에서도 줄기세포 치료를 적용하려는 노력들이 이어지고 있다. 다발성 경화증은 사람의 자가면역 신경질환 중 하나로, 자기 조절 면역세포의 이상으로 인해 신경조직을 외부요인으로 오인하고 염증 세포들이 침윤되면서 신경손상이 생기는 질환이다. 개에서는 유사한 질환으로 원인 불명의 비감염성 뇌수막염이 있다. 해당 질환의 원인은 아직 명확하게 밝혀지지 않았으나 면역 이상으로 인해 생기는 것으로 추정되며, 이와 관련해 비특이적 면역억제제로 치료한다. 개의 신경질환 중 약 25%를 차지할 정도로 많은 수의 개가 해당 질환으로 투병을 하나 아직까지 치료법이 크게 발달되어 있지 않다. 스테로이드를 포함한 비특이적 면역억제제는 위장관 장애, 호르몬 분비 장애 등의 문제를 유발하는 것이 가장 큰 문제이며, 그에 반해 치료 효과가 확실하게 보장되지 않는다는 것이 뇌수막염 치료의 어려운 점이다. 자가면역 신경질환에서 비특이적 면역억제제 치료의 단점을 보완할 다른 치료 요법들에 대한 연구 개발들이 이어지고 있으며, 그중 하나가 MSC에서 유래된 세포외소포체(extracellular vesicle; EV)이다. 그러나 아직까지는 세포외소포체의 임상적 효능이 충분히 입증되지 않아 실제 임상에서 적용되지 못하고 있다. 이를 해결하기 위해 MSC의 항염증 인자 분비를 촉진시키는 방법들이 고안되었고, 그중 한 가지 방법이 저산소 배양 혹은 저산소증 모방제를 사용한 전처리 방법이다. 전처리한 MSC에서 유래한 세포외소포체 내의 분자 변화를 분석하는 연구들이 진행되고 있으나, 아직까지 이것의 임상적 효능이 충분히 밝혀지지 않아 추가적인 전임상 및 임상 적용 연구가 필요하다. 본 연구는 실험적 자가면역성 뇌척수염(experimental autoimmune encephalomyelitis; EAE) 마우스 모델에서 deferoxamine (DFO)로 전처리한 개 지방조직유래(canine adipose tissue derived; cAT)-MSCs에서 유래된 세포외소포체(EVDFO)의 치료 효과를 확인하고 세포외소포체의 면역조절기능의 작용원리를 탐색하였다. 첫 번째, DFO가 전처리된 cAT-MSC (MSCDFO)가 분비 효과를 통해 대식세포를 더 효과적으로 M2 항염증 상태로 유도할 수 있음을 밝혔다. MSCDFO와 대식세포의 상호작용을 평가하기 위해 공배양 시스템을 이용하여 RAW 264.7 세포와 MSCDFO를 같이 배양하였으며, 중합효소 연쇄반응 기법과 western blot 분석 기법을 이용하여 대식세포 분극과 관련된 인자의 발현 변화를 분석하였다. RAW 264.7 세포를 MSCDFO와 공배양한 경우, 전처리 하지 않은 cAT-MSC와 공배양한 경우에 비해 M1 및 M2 마커의 발현이 각각 감소(iNOS, 1.32배, p<0.01; IL-6, 3.46배, p<0.05) 및 증가(CD206, 2.61배, p<0.001; Ym1, 4.92배, p<0.01)하였다. 따라서 DFO로 전처리한 cAT-MSC는 대식세포 분극을 더 효과적으로 항염증 상태인 M2 단계로 유도할 수 있다. 두 번째, EVDFO가 STAT3의 인산화 활성화를 통해 대식세포를 조절한다는 것을 밝혔다. MSCDFO에서는 HIF-1α가 축적되고 COX-2의 발현이 증가(16.77배, p<0.001)하였다. COX-2의 발현 변화는 MSCDFO에서 유래한 세포외소포체에도 반영되었다. 개 대식세포주 DH82를 LPS로 자극한 뒤 EVnon 및 EVDFO를 처리하여 그 변화를 중합효소 연쇄반응 기법 및 면역 형광 염색 분석을 통해 평가하였다. DH82를 EVDFO로 처리한 경우, M1 관련된 지표의 발현은 감소(IL-1β, 2.45배, p<0.001; IL-6, 17.26배, p<0.001)하였으나, M2 관련된 지표의 발현은 EVnon으로 처리한 경우보다 향상(CD206, 7.24배, p<0.001) 되었다. 또한 DH82 세포를 EVDFO로 처리했을 때 STAT3 발현의 인산화는 더 증가하였다(1.79배, p<0.001). si-COX2로 처리된 cAT-MSC에서 유래한 EV는 아무 처리하지 않은 EV와 유사한 효과를 보였으며, 대식세포 조절 효능은 EVDFO보다 감소하였다(IL-1β, 2.21배, p<0.001; IL-6, 1.43배, p<0.001; CD206, 2.27배, p<0.001). 따라서 EV 내에 있는 COX-2는 STAT3를 조절하여 대식세포를 변화시킬 수 있는 핵심 인자 중 하나로 추정된다. 마지막으로, EVDFO 치료가 전처리 하지 않은 EV보다 상대적으로 더 높은 항염증 효능을 가지며 EAE 모델에서 STAT3 조절을 통해 면역 체계를 조절할 수 있음을 밝혔다. 실험 비교를 위해 EAE 그룹과 EV 또는 EVDFO를 비강 투여한 그룹으로 나누었다(C57BL/6, male, control=6, EAE=8, EAE+EV=8, EAE+EVDFO=8, 10 μg/일/14회). 질환을 유도한지 25일 차에 쥐를 안락사 시키고 비장, 뇌, 척수를 조직병리학적, RNA 및 단백질의 발현 정도를 분석하였다. 조직학적으로 EV 및 EVDFO군에서는 척수에서의 염증 세포의 침윤이 현저히 감소하였고(EV, 1.38배, p<0.01; EVDFO, 1.72배, p<0.01) 탈수초화 현상이 완화되었다 (EV, 2.96배, p<0.05; EVDFO, 5.28배, p<0.05). 면역 형광 염색을 통해 M2 대식세포와 조절 T 세포(Treg)의 지표인 CD206과 Foxp3의 발현이 EAE와 EAE+EV 그룹에 비해 EVDFO 그룹에서 증가하였다. EAE 그룹에서 비장의 CD4+CD25+Foxp3+ Treg 세포의 수는 naïve 그룹에 비해 유의하게 감소했다(2.74배, p<0.001). 반면, Treg 세포의 수는 EAE+EV 그룹보다 EAE+EVDFO 그룹에서 더 큰 증가를 보였다(1.55배, p<0.05). STAT3와 pSTAT3의 단백질 발현은 naïve 그룹과 비교하였을 시 EAE 그룹의 비장에서 크게 증가하였다(STAT3, 2.02배, p<0.001; pSTAT3, 2.14배, p<0.001). 그러나 EV 처리한 그룹에서는 EAE 그룹에 비해 STAT3 발현이 감소하였으며(1.32배, p<0.001), 특히 EV 그룹에 비해 EVDFO가 주입된 그룹에서 STAT3의 감소가 뚜렷하게 나타났다(1.90배, p<0.001). 따라서 EV는 STAT3 발현을 조절할 수 있는 것으로 추정되며, EVDFO는 EV보다 그 효과가 크다고 종합할 수 있다. 결론적으로, cAT-MSC를 DFO로 전처리 하는 것은 MSC와 세포외소포체의 면역조절 효과를 향상시켜 치료능을 효율적으로 올릴 수 있는 방법이다. 또한, EVDFO가 면역세포 내의 STAT3를 유동적으로 변화시키고 이를 통해서 면역 체계를 조절할 수 있다는 점에서, STAT3를 조절하는 것이 다발성 경화증의 치료법의 중요 원리로 제시될 수 있다. 이러한 발견은 다발성 경화증뿐만 아니라 다른 자가면역 질환에서의 새로운 무세포 치료법을 제시한다. 더 나아가 개의 질환 중 이와 유사한 질환인 원인 불명의 비감염성 뇌척수막염에도 EVDFO가 새로운 치료제로 적용될 수 있음을 보여주는 주요한 첫 근거이며, 수의학에서의 자가 면역 질환 치료 발전의 토대가 되는 연구이다.LITERATURE REVIEW 1 1. Generalities of preconditioned mesenchymal stem cell (MSCs) 1 2. Properties of extracellular vesicles (EVs) derived from MSCs 2 3. Immunomodulation function of EVs derived from MSCs 3 4. Preclinical and clinical application of MSC derived EVs in immune disorders 5 Chapter Ⅰ. Preconditioning of canine adipose tissue-derived mesenchymal stem cells with deferoxamine potentiates anti-inflammatory effects by directing/reprogramming M2 macrophage polarization 12 1. Introduction 12 2. Material and methods 14 2.1. Isolation and characterization of canine adipose tissue-derived (cAT)-MSCs 14 2.2. Cell culture and expansion 15 2.3. Cell viability analysis 15 2.4. RNA extraction, cDNA synthesis, and the quantitative real-time polymerase chain reaction (qRT-PCR) 16 2.5. Protein extraction, cell fractionation, and western blot analysis 16 2.6. ELISA 17 2.7. Co-culture of macrophages with preconditioned cAT-MSCs 18 2.8. Statistical analyses 18 3. Results 18 3.1. Viability of DFO preconditioning in cAT-MSC 18 3.2. DFO induces hypoxic response in cAT-MSCs 19 3.3. DFO preconditioning increases the expression and secretion of anti-inflammatory factors 19 3.4. MSCDFO direct macrophage polarization in vitro 20 4. Discussion 21 5. Table and Figures 27 Chapter Ⅱ. Extracellular vesicles derived from DFO-preconditioned canine AT-MSCs reprogram macrophages into M2 phase 40 1. Introduction 40 2. Materials and methods 42 2.1. Cell preparation and culture 42 2.2. Transfection of cAT-MSCs with siRNA 43 2.3. Isolation and characterization of EVs derived from cAT-MSCs 44 2.4. RNA extraction, cDNA synthesis, and quantitative real-time polymerase chain reaction (qRT-PCR) 45 2.5. Protein extraction, cell fractionation, and western blotting 45 2.6. Immunofluorescence analyses 46 2.7. Statistical analyses 47 3. Results 47 3.1. Characterization of cAT-MSC derived EVs 47 3.2. Elevation of HIF-1α/COX-2 expression in MSCDFO 48 3.3. cAT-MSC-derived EVs transport COX-2 to DH82 and activate the phosphorylation of STAT3 48 3.4. Change of polarization of DH82 when treated with preconditioned EVs 49 4. Discussion 50 5. Table and Figures 54 Chapter Ⅲ. Deferoxamine preconditioned cAT-MSC derived EV alleviate inflammation in EAE mouse model through regulating STAT3 66 1. Introduction 66 2. Material and Methods 68 2.1 Cell isolation and culture 68 2.2. Isolation and characterization of EVs from cAT-MSCs 69 2.3. EAE induction and therapy 70 2.4. Histological analysis 71 2.5. Immunohistochemistry analysis 72 2.6. RNA extraction, cDNA synthesis, and real-time PCR 73 2.7. Protein extraction and western blotting 73 2.8. Isolated splenocytes and activation 74 2.9. Cytokine assay 74 2.10. Flow cytometry analysis of the Treg cell population 75 2.11. Obtaining PBMCs and treatment with EVs 75 2.12. Statistical analyses 76 3. Results 76 3.1. Characterization of cAT-MSC-derived EVs and elevation of protein levels in MSCDFO 76 3.2. cAT-MSC-derived EVs and EVDFO alleviated clinical signs and histological changes in the EAE mouse model 77 3.3. Cytokine and protein level changes in the spinal cord and brain of EV-treated mice 79 3.4. EVs altered the Treg cell population in the EAE mouse model 80 3.5. Cytokine and protein level changes in the spleen in EV-treated mice 80 3.6. Evaluation of the effect of EVs in canine PBMCs through RNA and protein expression analyses 81 4. Discussion 82 5. Table and Figures 89 GENERAL CONCLUSION 107 REFERENCES 111박

선택적 아로마타제 조절제의 대체재로서 약콩껍질추출물에 대한 연구

학위논문(석사)--서울대학교 대학원 :농업생명과학대학 농생명공학부(바이오모듈레이션전공),2019. 8. 이기원.에스트로겐은 여성의 생식과 성적 발달에 중요한 역할을 하는 스테로이드 호르몬이다. 여성은 노화가 진행됨에 따라 난소의 기능이 저하되고 에스트로겐의 분비가 감소하므로, 정상적인 에스트로겐 수준을 유지하는 것이 여성 건강에 중요하다. 아로마타제는 에스트로겐 합성에 필수적 효소이며, 조직 특이적으로 발현한다. 선택적 아로마타제 조절제 (selective aromatase modulator; SAM)는 에스트로겐 합성을 필요로 하는 부위에서 아로마타제를 발현할 수 있고, 에스트로겐 의존성 암 조직에서 아로마타제 과발현을 억제할 수 있다. 따라서 본 연구의 목적은 갱년기 및 폐경기 여성의 에스트로겐 저하 증상을 개선하기 위해 체내에서 에스트로겐을 근본적으로 생성하고, 에스트로겐 생성이 필수적인 조직에서 아로마타제의 발현을 증가시키는 천연물을 발견하는 것이다. 더불어 아로마타제 발현을 조직 특이적으로 조절하는 선택적 아로마타제 조절제로서의 작용을 확인하고자 한다. 본 연구를 통해, 약콩껍질추출물이 사람의 부신 피질 암세포주인 NCI H295R 세포와 쥐의 난소에서 유래한 난포에서 17 베타-에스트라디올 생합성을 증가시키는 것을 관찰하였다. 약콩껍질추출물은 스테로이드 호르몬 생합성 경로에서 스테로이드 호르몬 생성 효소인 3β-HSD 와 CYP19A1 의 발현을 증가시킴으로써 17 베타-에스트라디올 생합성을 촉진하였다. 약콩껍질추출물은 부신 및 난소에서는 에스트로겐을 생합성 하였지만, 에스트로겐 의존성 암세포로 알려진 유방암 및 난소암세포에서는 세포 증식을 억제하였다. 더불어 H295R 세포에서 CYP19A1 의 발현을 증가시켰지만 단일 배양된 지방세포 및 유방암세포와 공동 배양된 지방세포에서 CYP19A1 의 발현에 영향을 주지 않았다. 결론적으로, 본 연구는 약콩껍질추출물이 조직 특이적으로 에스트로겐 생합성 및 아로마타제 발현을 촉진하는 에스트로겐 저하 증상 개선용 천연물 의약품 기능성 소재로 사용될 수 있음을 시사한다.Estrogen is a steroid hormone that plays an important role in female reproductive function and sexual development. As women get older, the secretion of estrogen decreases and estrogen supplementation is crucial to alleviate symptoms related to estrogen deficiency. CYP19 (also called aromatase) is the key enzyme responsible for estrogen production and it is expressed in a tissue-specific manner. Aromatase should be expressed in sites that require estrogen synthesis and inhibited overexpression in estrogen-dependent cancer tissues. Therefore, it is necessary to investigate natural products that can supplement estrogen and regulate aromatase expression tissue specifically for menopausal womens health. In this study, we found that Yak-Kong seed coat extract increases 17β-estradiol production in human adrenocortical carcinoma NCI-H295R cells and mouse antral follicles. It promotes 17β-estradiol biosynthesis by increasing the protein and gene expression of CYP19A1 and 3β-HSD in steroidogenesis pathway. Furthermore, it suppresses proliferation of breast and ovarian cancer cells known as estrogen dependent cancer cells and regulates aromatase expression of adipocytes followed a different pattern from that of H295R cells. These results suggest that the extract of Yak Kong seed coat could be a potential alternative to selective aromatase modulator for menopausal women.CONTENTS ABSTRACT .................................................................................................................... ⅰ CONTENTS .................................................................................................................... ⅲ Ⅰ. INTRODUCTION........................................................................................................ 1 Ⅱ. MATERIALS AND METHODS .......................................................................... 7 1. Soybean materials .................................................................................................... 7 2. Animals ....................................................................................................................... 8 3. Cell culture ................................................................................................................. 8 4. Antral follicle culture.............................................................................................. 11 5. Hormone measurement ........................................................................................ 12 6. Real-time quantitative PCR ................................................................................. 12 7. Western blotting ...................................................................................................... 14 8. Cell viability assay .................................................................................................. 15 9. 3T3-L1 and MCF-7 coculture .............................................................................. 16 10. Statistical analysis ................................................................................................. 18 Ⅲ. RESULTS .................................................................................................................. 19 1. Yak-Kong seed coat extract has the most estradiol-stimulating effect in H295R cells among soybean extracts ..................................................................... 19 2. Yak-Kong seed coat extract promotes production of 17β-estradiol in a dosedependent manner in H295R cells and mouse antral follicles ........................... 22 3. Yak-Kong seed coat extract increases protein and mRNA levels of CYP19A1 in H295R cells ........................................................................................ 26 4. Yak-Kong seed coat extract increases protein and mRNA levels of 3β-HSD in H295R cells .............................................................................................................. 29 5. Yak-Kong seed coat extract does not increase protein levels of CYP11A1, CYP17A1, or 17β-HSD in H295R cells .................................................................. 32 6. Yak-Kong seed coat extract decreases viability of human breast and ovarian cancer cells .................................................................................................... 34 7. Yak-Kong seed coat extract does not stimulate CYP19A1 expression in 3T3-L1 adipocytes, both mono- and co-cultured with MCF-7 cells ................ 37 Ⅳ. DISCUSSION ......................................................................................................... 40 Ⅴ. REFERENCES ....................................................................................................... 46 국문 초록 .......................................................................................................................... 51Maste

가변 토폴로지 트러스 로봇의 안정적인 주행 알고리즘 개발

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 기계공학과, 2020. 8. 김종원.Variable Topology Truss (VTT) is truss structured modular robot that can self-reconfigure its topology and geometric configuration, which can be usefully applied to rescuing work in disaster site. In this thesis, design of VTT is introduced and stable rolling locomotion algorithm for VTT is proposed. To achieve self-reconfiguration feature, VTT are composed specially designed members and nodes. VTTs members consist of Spiral Zippers which are novel linear actuators that has high extension ratio, light weight and high strength. VTTs nodes consist of Passive Member-Ends and Master Member-Ends. Passive Member-Ends are linkage type spherical joint with large angle range that can accommodate many members. Master Member-Ends are spherical manipulators that built in Sphere and it move member to change topology of VTT. Rolling locomotion of VTT is achieved by controlling the center of mass by geometric reconfiguration. However, the locomotion planning is complex problem, because VTT is parallel mechanism with high degree of freedom and many constraints, which makes it difficult to predict and avoid constraints for feasible planning. Thus, it needs stable algorithm that can find locomotion trajectory even in complicated and large environment. In addition, since VTT has many sophisticated components, the algorithm must prevent VTT being damaged from ground by tumbling. To meet the requirements, proposed locomotion algorithm is composed of 3 steps; support polygon planning, center of mass planning and node position planning. In support polygon planning, support polygon path is planned by newly proposed random search algorithm, Polygon-Based Random Tree (PRT). In center of mass planning, trajectory of desired projected center of mass is planned by maximizing stability feature. Planned support polygon path and center of mass trajectory guide VTT to have good-conditioned shape which configuration is far from constraints and makes locomotion planning success even in complex and large environment. In node position planning, Non-Impact Rolling locomotion algorithm was developed to plan position of VTTs nodes that prevent damage from the ground while following planned support polygon path and center of mass trajectory. The algorithm was verified by two case study. In case study 1, locomotion planning and simulation was performed considering actual constraints of VTT. To avoid collision between VTT and obstacle, safety space was defined and considered in support polygon planning. The result shows that VTT successfully reaches the goal while avoiding obstacles and satisfying constraints. In case study 2, locomotion planning and simulation was performed in the environment having wide space and narrow passage. Nominal length of VTT was set to be large in wide space to move efficiently, and set to be small in narrow passage to pass through it. The result shows that VTT successfully reaches the goal while changing its nominal length in different terrain.가변 토폴로지 트러스 (Variable Topology Truss, VTT)는 토폴로지와 기하학적 형상의 재구성이 가능한 트러스 구조의 모듈 로봇이다. 본 논문에서는 VTT의 설계 구조를 소개하고 VTT의 안정적인 주행을 알고리즘을 제안한다. VTT는 토폴로지와 기하학적 형상의 재구성을 위해 특수한 구조의 멤버와 노드를 가진다. VTT의 멤버는 높은 압축비, 가벼운 중량, 높은 강도를 가진 신개념 선형 구동기인 스파이럴 지퍼로 구성되어 있다. VTT의 노드는 패시브 멤버 엔드와 마스터 엔드로 구성되어 있다. 패시브 멤버는 링키지 구조의 3 자유도 관절로, 넓은 각도 구동 범위를 가지고 있고 많은 수의 멤버를 연결할 수 있다. 마스터 멤버 엔드는 노드 부의 내장된 구형 매니퓰레이터로, 토폴로지 재구성 시 멤버를 이동시키는데 사용된다. VTT는 기하학적 형상을 변화하여 구르는 움직임을 통해 주행한다. VTT의 주행 알고리즘은 서포트 폴리곤 계획 단계, 무게 중심 계획 단계, 노드 위치 계획 단계로 이루어진다. 서포트 폴리곤 계획 단계에서는 새롭게 제안된 무작위 탐색 (random search) 알고리즘인 Polygon-Based Random Tree (PRT)을 적용해 서포트 폴리곤의 경로를 계획한다. 무게 중심 계획 단계에서는 안정성을 최대화하는 VTT의 무게 중심 궤적을 계획한다. 계획된 서포트 폴리곤 경로와 무게 중심 궤적을 VTT가 제한 조건으로부터 먼 좋은 상태의 형상을 유지하게 하여 복잡한 환경에 대해서도 경로 계획이 실패하지 않고 안정적으로 이루어질 수 있도록 한다. 노드 위치 계획 단계에서는 서포트 폴리곤 경로와 노드 위치의 궤적을 추종하는 노드 위치 궤적을 계획한다. 이 과정에서 비충격 롤링 이동 알고리즘 (Non-Impact Rolling locomotion algorithm)을 적용하여 지면과의 충돌로 인한 충격이 일어나지 않는 궤적을 계획한다. 실제 VTT의 제한 조건을 반영한 모델에 본 알고리즘을 적용하여 시뮬레이션을 수행한 결과, VTT가 모든 제한 조건을 만족하고 장애물을 회피하면서 목표 지점에 도달할 수 있음을 확인하였다.Chapter 1. Introduction 1 1.1 Motivation 1 1.2 Previous Truss Type Modular Robot 4 1.3 Previous Research on VTTs Locomotion 8 1.3.1 Heuristic Based Methods 9 1.3.2 Optimization Based Method 10 1.4 Objectives of Locomotion Algorithm 12 1.5 Contribution of Thesis 13 1.5.1 Introduction to Hardware Design of VTT 13 1.5.2 Stable Rolling Locomotion of VTT 15 Chapter 2. Design of Variable Topology Truss 17 2.1 Member Design 18 2.1.1 Spiral Zipper 20 2.1.2 Tensioner 26 2.2 Node Design 28 2.2.1 Passive Member-End and Sphere 29 2.2.2 Master Member-End 36 2.3 Control System 40 2.4 Node Position Control Experiment 44 Chapter 3. Mathematical Model of Variable Topology Truss 47 3.1 Configuration and Terminology 47 3.2 Inverse Kinematics 50 3.3 Constraints 51 3.4 Stability Criteria 64 Chapter 4. Locomotion Algorithm 66 4.1 Concept of Locomotion Algorithm 67 4.1.1 Method for Successful Planning and Obstacle Avoidance 67 4.1.2 Method to Prevent Damage from the Ground 71 4.1.3 Step of Locomotion Algorithm 72 4.2 Support Polygon Planning 73 4.2.1 Polygon-Based Random Tree (PRT) Algorithm 73 4.2.2 Probabilistic Completeness of PRT Algorithm 79 4.3 Center of Mass Planning 85 4.4 Node Position Planning 86 4.4.1 Concept of Non-Impact Rolling Locomotion 86 4.4.2 Planning Algorithm for Non-Impact Rolling Locomotion 89 4.4.3 Optimization Problem of Moving Phase 94 4.4.4 Optimization Problem of Landing Phase 98 4.4.5 Optimization Problem of Transient Phase 99 Chapter 5. Experimental Verification 100 5.1 Case Study 1: Actual VTT Prototype 101 5.1.1 Simulation Condition 101 5.1.2 Obstacle Avoidance Method 103 5.1.3 Simulation Result 104 5.2 Case Study 2: Environment with Narrow Passage 111 5.2.1 Simulation Condition 111 5.2.2 Support Polygon Planning with Varying Nominal Length 114 5.2.3 Simulation Result 117 Chapter 6. Conclusion 126 Bibliography 129 Abstract in Korean 134Docto

그래핀기반 물질을 이용한 인간 지방유래 기질세포의 연골 분화 효과 연구

학위논문(석사)--서울대학교 대학원 :농업생명과학대학 농생명공학부(바이오모듈레이션전공),2019. 8. 임정묵.Cartilage is a tissue consisting of chondrocytes and extracellular matrix (ECM), which is highly elastic, buffers against a given force, and the friction coefficient of joint cartilage is very low, helping the joints to move in a state of little friction. But cartilage is a kind of expendable body part that wears out as much as it is used, and it is also damaged by inflammation, trauma, and aging. Cartilage has very limited self-renewal because there are no blood vessels, nerves, or lymphatic vessels. Thus, cartilage cannot easily stop when it starts to damage, and damaged cartilage has many limitations about regenerating into cartilage with normal function and structure. Also, the damaged cartilage area becomes more vulnerable to mechanical pressure, so it is easily broken and worn, resulting in larger defects. As such, a number of cartilage-related diseases develop and progresses faster than other tissues. Some typical diseases include degenerative arthritis, which causes pain due to cartilage wear, and others include rheumatoid arthritis, achondroplasia, pyogenic arthritis, and chondrosarcoma. Furthermore, these cartilage-related diseases can lead to bone-related complications if they persist for a long time. These diseases can lead to a reduction in the quality of life and life expectancy of patients, and the loss of substantial medical costs. Therefore, it is very important to regenerate damaged cartilage early, but there are many deficiencies in current cartilage treatment. Although many researchers continue to suggest ways to treat cartilage-related diseases, they are still far from normal and perfect cartilage regeneration. Up until now, studies have been actively conducted to regenerate damaged cartilage tissue using an ideal biomaterial that can mimic and replace cartilage tissue with various cells. Among them, cells that are widely used in the field of regenerative medicine are human adipose-derived stromal cells (hASCs). This cell has a great advantage that it can be easily obtained from any tissue of the human body, and it is suitable as a material for a cell therapy agent because it does not take much time for cell proliferation and can be repeatedly collected. And recently, graphene (G), a carbon-based material, is emerging as a biomaterial in tissue engineering and regenerative medicine. In addition, a variety of graphene-based materials (GBMs) have also been recognized for their research value as biomaterials. GBMs are derivatives of G. Most GBMs are made through the process of oxidation. GBMs that have the advantage of oxidation are more applied in various fields than G. In particular, GBMs play a role as biomaterials due to their various functional groups, biocompatibility, mechanical stability, and other characteristics. And GBMs have been extensively studied in the field of tissue regeneration and repair. However, since G was discovered in 2004 and is a new material only about 20 years old, there is a limit to the lack of prior research related to cartilage. Therefore, cartilage regeneration studies related to GBMs are essential. And because accurate standard indicators for GBMs have not yet been created, characterization and comparison of various GBMs is critical. Based on an understanding of the characteristics of GBMs, this study was conducted to apply to cartilage regeneration studies. The GBMs used in this study were graphene oxide (GO), nano-graphene oxide (nGO) and graphene quantum dot (GQD). All are oxidized GBMs. These are the forms in which parts of the surface have been replaced with oxygen as graphene is oxidized, and the great advantage is that the binding force of each layer of graphite is reduced and distributed well in the solution. Since it can be synthesized in the solution phase, it can be mass-produced and overcome the disadvantages of graphene, which is a very expensive material and has a very high production cost. And because it is easier to attach new functional groups to oxygen than to carbon, it is possible to make more functionalized graphene derivatives. As hydrophobic graphene turns into hydrophilic, oxidized GBMs, affinity with cells increases, and it has the advantage of being easily mixed into culture medium. Therefore, since these advantages allow me to study GBMs as biomaterials, I analyzed the characteristics of each GBMs using Fourier Transform Infra-Red (FT-IR), X-ray Photoelectron Spectroscopy (XPS), and Electrophoretic Light Scattering (ELS). Functional group analysis confirmed that all GBMs were oxidized by showing binding to carboxyl groups, hydroxyl groups and epoxy groups common to surface of GBMs. Through C1s spectra analysis, the degree of oxidation can be determined by the binding and binding ratio of carbon and oxygen. It was found that oxidation is high in the order of nGO, GO, and GQD. Hydrodynamic radius analysis was used to confirm the hydrodynamic radius of each material. This means the radius of the particles in the solution. Therefore, the size of each particle in the solution can be known, and the order in which it is large was confirmed to be GO, nGO and GQD. Finally, Zeta potential analysis was used to confirm the dispersion stability of the particles in solution. The larger the negative value, the higher the dispersion stability. Generally, over –30 means having good stability, so I found that GO and nGO had better dispersion stability than GQD. Therefore, the dispersion stability is high in the order of nGO, GO, and GQD. These data show that all three GBMs are oxidized and help to understand and compare the physical and chemical properties of each particle. Based on the unique properties of these GBMs, I applied them to chondrogenic differentiation of hASCs. Although studies on cartilage regeneration using various cells and biomaterials have been actively studied, there are still no standardized therapies related to cartilage as various problems such as potential side effects on regenerative therapy and verification of effects. So the study was conducted to investigate the effect on the cartilage differentiation of hASCs by applying GBMs, which have recently emerged as biomaterials in the field of regenerative medicine, to cartilage research. First, I conducted a study on the characterization of hASCs. Microscopic photographs showed that hASCs had fibroblastic morphology, and the ability to differentiate into osteocyte, adipocyte and chondrocyte was confirmed through three different dyeing reagents. And based on the GO with the largest particle size, I confirmed the concentration suitable for chondrogenic differentiation of hASCs. As a result, it was confirmed that a concentration of 10 μg / ml or less was suitable. Thus, GO, nGO, and GQD at concentrations of 1 and 10 μg / ml were applied to hASCs. As a result, the size of the chondrocyte pellet was not different from the induction group. However, it was confirmed that Glycosaminoglycans (GAGs) were significantly increased in the 1 μg / ml group of GO and 1 μg / ml of nGO group compared with the induction group through alcian blue staining and toluidine blue staining. These results suggest that GO and nGO, the oxidized graphene, support Chondrogenic differentiation of hASCs. The main purpose of this study was to determine the effect of GBMs on chondrogenic differentiation of hASCs. Particularly, it was confirmed that the effect of chondrogenic differentiation is different according to the particle size and degree of oxidation of GBMs. These studies will help to understand GBMs, which are bio-new materials, and will greatly contribute to the development of new cartilage therapies using these materials.본 논문에서는 3가지의 다른 종류의 그래핀 기반 물질들의 물리적, 화학적 특성을 분석하고 비교를 통해 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점에 대한 이해에 대한 연구와 그래핀 기반 물질들을 생체 소재로 활용한 인간 지방유래 기질세포의 연골 분화에 미치는 영향에 대한 조직 공학 연구를 진행하였다. Chapter 3에서는 그래핀 기반 물질들의 특성을 이해하기 위해 물리적, 화학적 분석에 대한 연구를 진행한 후, 분석된 그래핀 기반 물질들을 이용하여 인간 지방유래 기질세포의 연골 분화에 대한 효과를 확인하였다. 그래핀은 발견된 지 20년이 채 되지 않은 바이오신소재로, 참고할 만한 이전 연구가 많이 없다. 그래서 그래핀 유도체들의 물리, 화학적 표준 지표가 마련되어 있지 않기 때문에 각 각의 특성들을 정확하게 분석하고 이해하는 것이 매우 중요하다. 먼저, Fourier Transform Infra-Red (FT-IR)를 이용하여 각 물질들의 표면에 어떤 작용기들로 구성되어 있는지 확인하였다. 3 종류의 그래핀의 표면에서 공통적으로 카르복실기, 하이드록실기, 에폭시기를 가진다는 것을 확인하였다. 이는 3종류의 그래핀 기반 물질들이 모두 산화되었음을 보여준다. X-ray Photoelectron Spectroscopy (XPS)를 이용해서는 C1s spectra 분석을 하였다. 이를 통해 탄소와 산소의 결합과 비율로 산화 정도를 확인할 수 있었고, 그 결과, 산화 정도는 나노 산화 그래핀, 산화 그래핀, 그래핀 양자점 순서로 큰 것을 확인하였다. Electrophoretic light scattering (ELS)를 이용해서는 각 물질들의 유체역학적 반경을 측정하였다. 이는 용액 속에서 입자가 영향을 주는 반경을 의미하기 때문에, 입자의 크기를 알 수 있다. 그 결과, 입자의 크기는 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점 순서로 큰 것을 확인하였다. 마지막으로, ELS를 이용하여 zeta potential 값을 측정하였다. 제타 포텐셜 값이 높은 음의 값을 가질수록 용액에서의 분산 안정성이 높으며, 일반적으로 –40이 넘어가면 안정성이 좋다는 것을 의미한다. 그 결과, 분산 안정성이 높은 순서는 나노 산화 그래핀, 산화 그래핀, 그래핀 양자점 순서임을 확인하였다. 이러한 결과들을 통해 그래핀 기반 물질들의 표면 작용기, 산화 정도, 입자의 크기, 분산안정성에 대해 이해할 수 있었고, 이는 그래핀 기반 물질들을 다양한 분야에서 적용시킬 때 많은 도움을 줄 것으로 생각한다. 그리고 분석한 그래핀 기반 물질들을 바이오소재로서 이용하여 인간 지방유래 기질세포의 연골 분화에 미치는 영향에 관한 연골 재생 연구를 하였다. 소수성인 그래핀을 산화시켜 만든 그래핀 기반 물질들은 친수성으로 성질이 바뀌면서 세포와의 친화성이나 배양 배지에 쉽게 분산될 수 있는 장점을 가져 조직공학 분야에 접목시키기 유리하다. 그래서 나는 먼저, 인간 지방유래 기질세포의 형태학적인 분석과 골, 지방, 연골로의 분화능에 대한 분석을 하였다. 인간 지방유래 기질세포는 섬유아세포와 같은 모양을 가졌으며, 골, 지방, 연골로의 분화능을 가지는 것으로 확인하였다. 그리고 나서 인간 지방유래 기질세포의 연골 분화에 적합한 산화 그래핀의 농도를 확인하였다. 산화 그래핀을 기준으로 한 이유는 입자의 크기가 가장 크고, 조직 공학과 관련된 참고문헌이 가장 많기 때문이다. 그 결과, 10 ㎍/㎖의 이하의 농도에서 적합한 것을 확인하였다. 그래서 1과 10 ㎍/㎖의 농도의 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점을 이용해 인간 지방유래 기질세포의 연골 분화 영향을 확인하였을 때, 콘드로사이트의 펠렛 크기에는 영향을 주지 않았다. 하지만 alcian blue staining과 toluidine blue staining으로 조직학적 분석을 하였을 때, 1 ㎍/㎖의 농도의 산화 그래핀과 나노 산화 그래핀 군에서 양성대조군에 비해 GAGs의 함량이 유의적으로 증가함을 확인하였다. 그 결과, 100 nm 이상의 크기를 가진 산화 그래핀 기반 물질들이 인간 지방유래 기질세포의 연골분화에 효과가 있음을 확인하였다. 그러므로 이 연구를 통해 재생 의학이나 조직공학 분야에서 떠오르는 바이오 신소재로 자리매김을 할 수 있는 가능성을 확인하였다. 앞으로 그래핀 기반 물질들이 세포와 세포 외 기질 등 주변 환경에 미치는 영향이나 세포 독성, 적합한 배양 조건에 관한 깊이 있는 연구가 더 진행된다면 충분히 조직 공학적 응용이 가능해 질 것이다PART I : GENERAL INTRODUCTION & LITERATURE REVIEW 1 CHAPTER 1 : General Introduction 2 CHAPTER 2 : Literature Review 9 1. Cartilage 10 1.1. Definition 10 1.2. Composition 10 1.3. Cartilage-related diseases 15 2. Tissue engineering 22 2.1. Definition 22 2.2. Materials . 22 2.2.1 Cells . 23 2.2.2 Biomaterials . 26 3. Cartilage regeneration 29 PART II : EFFECT OF GRAPHENE-BASED MATERIALS ON CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STROMAL CELLS 31 CHAPTER 1 : Effect of graphene-based materials on chondrogenic differentiation of human adipose-derived stromal cells 32 1. Introduction 33 2. Materials and Methods 39 3. Results 44 4. Discussion 58 PART III : GENERAL DISCUSSION & CONCLUSION 61 1. General discussion and conclusion 62 REFERENCES 66 SUMMARY IN KOREAN 95Maste

사람 보행 분석 연구와 그 결과를 활용한 휴머노이드 로봇 보행 패턴 생성

학위논문 (박사) -- 서울대학교 대학원 : 융합과학기술대학원 융합과학부(지능형융합시스템전공), 2020. 8. 박재흥.발의 미끄러짐은 보행의 안정성을 떨어트리는 요인 중 하나이다. 보행 중 발에 발생하는 수평 전단력이 발과 지면 사이의 마찰력보다 커지면, 발은 접촉을 상실하고 미끄러지게 된다. 여기서, 발과 지면 사이의 마찰력은 발에 작용하는 수직력에 의해 결정되게 된다. 즉, 휴머노이드 로봇 보행 패턴 생성의 측면에서 보자면, 로봇 발에 발생하는 수평력과 수직력을 어떻게 설계하는지에 따라 보행 중 미끄러짐의 가능성이 바뀐다는 것이다. 선형 역진자 모델은 휴머노이드 로봇의 무게 중심 궤적 생성을 위해 자주 사용되어왔다. 선형 역진자 모델은 로봇의 무게 중심 높이를 일정하게 유지하도록 제한한다. 무게 중심의 높이 제한 때문에 로봇의 수직 방향의 가속도는 보행 속도와 관련 없이 항상 중력 가속도가 된다. 그러나 수평 방향의 가속도는 보행 속도가 증가하면 비례하여 증가한다. 따라서 빠른 보행 속도에서는 수직력에 비례하는 마찰력에 비해 수평 전단력이 커지면서 발의 미끄러짐이 발생할 수 있다. 선형 역진자 모델에 의한 일정한 수직 높이 구속 조건이 로봇 발의 미끄러짐을 유발할 수 있다는 것을 시사한다. 무게 중심의 적절한 수직 움직임을 생성함으로써 휴머노이드 로봇 보행 중 발의 미끄러짐을 줄일 수 있다. 인간공학 분야에서는 Available Coefficient of Friction(aCOF)과 Utilized Coefficient of Friction(uCOF)을 이용하여 사람 보행 중 발의 미끄러짐 가능성을 예측하는 연구들이 수행됐다. 여기서, aCOF는 두 물체의 재질이나 상태에 의해 결정되는 마찰 계수이다. 반면, uCOF는 보행 중 지지하는 발에 가해지는 수평 전단력과 수직력의 비이다. 인간공학 연구들에 따르면, uCOF가 aCOF를 초과할 때 발은 접촉을 상실하고 미끄러지게 된다. 로봇 발의 미끄러짐 감소를 위해서는 로봇 보행 중 발에 발생하는 uCOF가 로봇 발과 지면 사이의 aCOF 보다 작아지도록 적절한 수직 방향의 무게 중심 궤적을 생성하는 것이 필요하다. 다양한 형태의 수직 방향의 무게 중심 궤적 생성이 가능한데, 간단하면서도 효율적인 방법은 무게 중심의 에너지가 보존되도록 수직 방향의 무게 중심 궤적을 생성하는 것이다. 기존 선형 역진자 모델을 이용해 수평 방향의 무게 중심 궤적을 생성하고, 운동 에너지와 위치 에너지가 교환되면서 전체 에너지가 보존되는 수직 방향의 무게 중심 궤적을 추가하는 것이다. 무게 중심의 에너지 보존 원리를 이용하여 무게 중심의 양의 일(Mechanical Work) 생성을 최소화함으로써 관절의 양의 일 생성을 감소시키고, 이를 통해 보행 중 에너지 효율을 높이는 것이 가능하다. 이 논문은 발과 지면 사이의 aCOF 보다 작도록 보행 중 uCOF를 유지하면서 무게 중심의 양의 일을 최소화하는 적절한 수직 방향의 무게 중심 궤적을 생성하는 것을 목표로 한다. 발의 미끄러짐이 감소하면서 에너지 효율이 높은 휴머노이드 로봇 보행 패턴 생성을 위해, 먼저 사람 보행 중 uCOF에 관한 연구와 사람 보행 중 관절의 일에 관한 연구를 선행한다. 사람 보행에 관한 분석 연구와 사람 보행의 원리 이해를 통해 최적화 알고리즘 기반 수직 방향의 무게 중심 궤적 생성 방법이 제시된다. 제시된 알고리즘을 이용하여 구해진 수직 방향의 무게 중심 궤적을 휴머노이드 로봇 보행 실험에 적용한다. 궁극적으로 이 논문은, 수직 방향의 무게 중심 궤적을 추가함으로써 기존 선형 역진자 모델의 한계를 극복하여, 미끄러짐의 가능성이 감소하고 에너지 효율이 높은 휴머노이드 로봇 보행 패턴을 생성한다.Foot slippage is one of the factors responsible for the increasing instability during human walking. A slip occurs when the horizontal shear force acting on the foot becomes greater than the frictional force between the foot and the ground, which is proportional to the vertical force. For humanoid robot walking, the possibility of a slip depends upon how the horizontal shear force and vertical force both acting on the foot are designed. In the linear inverted pendulum model (LIPM), which is commonly used to generate the center of mass (COM) trajectory of humanoid robots, the vertical height of the COM is kept constant. The constant height of the COM restricts that the vertical force is always equal to the gravitational force at any walking speed. However, upon increasing the walking speed, the horizontal ground reaction force increases in proportion with the forward and lateral accelerations of the COM. This increase in the horizontal ground reaction force, while the vertical ground force is being constant, suggests that the robot-foot slippage can occur because of the restriction of the vertical motion by the LIPM constraint. By generating the appropriate vertical motion, the robot-foot slippage can be reduced during humanoid robot walking. Researchers in the field of ergonomics have been conducted studies on the relationship between the available coefficient of friction (aCOF) and the utilized coefficient of friction (uCOF) to predict the potential for a slip during human walking. The aCOF is both the static and dynamic coefficient of friction between two objects in contact, and it depends on the properties of the objects. The uCOF is the ratio of the horizontal shear force to the vertical force applied by the supporting foot. Foot slippage occurs when the uCOF exceeds the aCOF. Various types of vertical motion can set the maximum value of the uCOF to be less than the aCOF between the foot and floor for humanoid robot walking. One of the simple and energy-efficient methods is to minimize the mechanical work of the COM by introducing added vertical motion. Therefore, the COM pattern would become more energy efficient by exchanging kinetic energy and potential energy. This thesis aims to generate the appropriate vertical motion of the COM to maintain the utilized coefficient of friction (uCOF) less than the available coefficient of friction between the foot and the ground, and to minimize the mechanical work during humanoid robot walking. Before generating a slip-safe and energy-efficient COM trajectory for humanoid robot walking, studies on analyzing the COM patterns, mechanical work, and uCOF during human walking are conducted to understand the principle of walking. Vertical motions at various speeds are generated using an optimization method. Subsequently, the generated COM motion patterns are used as reference trajectories of the COM for humanoid robot walking. This thesis suggests a way to generate slip-safe and energy-efficient COM patterns, which, in turn, overcome the limitations of the LIPM by adding vertical COM motion.Chapter 1 Introduction 1 1.1 Research Background 1 1.2 Contributions of Thesis 3 1.3 Overviews of Thesis 4 Chapter 2 Dynamics of Walking 5 2.1 Walking Model 5 2.1.1 Linear Inverted Pendulum Model 5 2.1.2 Spring-Loaded Inverted Pendulum Model 6 2.1.3 Extrapolated Center of Mass Dynamics 9 2.2 Walking Theory 11 2.2.1 Step-to-Step Transition 11 Chapter 3 HumanWalking Analysis 13 3.1 Motion Capture for Walking 13 3.1.1 Motion Capture Technology 13 3.1.2 Joint Kinematics and Kinetics 15 3.2 Joint and COM During Human Walking 17 3.2.1 Introduction 17 3.2.2 Methods 19 3.2.3 Change of Joint Angle and the COM 20 3.2.4 Discussion 26 3.3 Slipping During Human Walking 27 3.3.1 Introduction 27 3.3.2 Methods 31 3.3.3 Change of uCOF and GRF 34 3.3.4 Interaction Effect Between Heel Area and Speed 36 3.3.5 Discussion 39 3.4 Mechanical Work During Human Walking 44 3.4.1 Introduction 44 3.4.2 Methods 46 3.4.3 Calculation for Joint Mechanical Work 48 3.4.4 Change of Joint Mechanical Work 51 3.4.5 Change of Stride Parameters 53 3.4.6 Discussion 54 Chapter 4 Robot Walking Pattern Generation 59 4.1 Introduction 59 4.2 Forward and Lateral COM 61 4.2.1 XcoM Method 61 4.2.2 Preview Control Method 63 4.3 Vertical COM 64 4.3.1 Calculation for uCOF 64 4.3.2 Calculation for ZMP 65 4.3.3 Calculation for COM Mechanical Work 66 4.3.4 Optimization for Vertical COM Generation 68 4.3.5 Results of Optimization for Vertical COM 73 4.4 Slipping During Robot Walking 75 4.4.1 Robot Simulation 75 4.4.2 Robot Experiments 77 4.5 Mechanical Work During Robot Walking 81 4.5.1 Robot Simulation 81 4.5.2 Robot Experiments 82 4.6 Discussion 87 4.6.1 Tracking Errors in Robot Experiments 87 4.6.2 Effect of Vertical Motions on Real Net Power 91 4.6.3 Trade-Off Between Efficiency and Stability 92 4.6.4 Difference Between Human and Robot 93 Chapter 5 Conclusions 95 Bibliography 97 Abstract (Korean) 111Docto

Calcium silicate 근관 충전 재료의 실시간 나노 누출

학위논문 (박사) -- 서울대학교 대학원 : 치의학대학원 치의과학과, 2021. 2. 이우철.1. Objectives This study aimed to investigate the real-time nanoleakage of calcium silicate (Ca-Si) based root canal filling materials. 2. Materials and Methods Extracted human teeth (n = 30) were decoronated and enlarged apically to size #40 (taper 0.06) using Protaper Next X4 instruments (Dentsply, USA). The teeth were randomly divided into 3 experimental groups and obturated with respective materials; Gutta Percha (GP) with AH 26 sealer using the continuous wave of condensation technique, GP with EndoSeal MTA sealer using the continuous wave of condensation technique, and solely obturated with orthograde Biodentine. The roots were embedded into acrylic resin except the apical 2 mm to prevent leakage through lateral surface and a metal tube was fixed on the coronal orifice by using flowable composite resin. The prepared specimen was connected to a nanoFlow device (IB Systems, Seoul, Korea) under hydrostatic pressure (40 cm∙H2O) and fluid flow was traced through the filled roots. Data were detected at the nanoscale twice per second and automatically recorded in units of nL/s. Leakage was quantified as the mean slope after the curve stabilized over time. Data were statistically analyzed using the Kruskal-Wallis test. The level of significance was set at 5%. 3. Results The calculated leakage values were 0.0670 ± 0.0516 nL/s for GP/AH26, 0.1397 ± 0.1579 nL/s for GP/EndoSeal MTA, and 0.0358 ± 0.0538 nL/s for Biodentine, with no statistically significant differences among the root filling materials (P > 0.05). 4. Conclusion Real-time measurements under hydrostatic pressure with the nanoFlow device enabled precise fluid flow tracing through the root canal filling material. In terms of nanoleakage, the tested root canal filling materials showed no significant differences.1. 목적 본 연구의 목적은 Calcium silicate (Ca-Si) 기반 근관 충전재의 실시간 나노 누출을 조사하는 것이다. 2. 재료 및 방법 30개의 발치된 사람의 단근치의 치관을 제거하고 Protaper Next X4를 사용하여 근관을 확대한 후 세 그룹으로 나누어 각각 Gutta percha (GP) 와 AH26 실러, GP 와 EndoSeal MTA 실러, Biodentine으로 충전하였다. 불필요한 누출을 막기 위해 치근단 2mm 를 제외한 부분을 아크릴릭 레진에 매몰하고 근관 입구에 유동성 복합레진을 사용하여 금속 관을 고정한 뒤, 40cm∙H2O의 정수압 하에서 nanoFlow 장치(IB syetems, Seoul, Korea) 에 연결하여 근관을 통한 미세 누출을 측정하였다. 데이터는 초당 2회, 나노 단위로 감지되었으며 nL/s 단위로 자동 기록되었다. 누출 양은 누출 곡선이 평탄화 된 이후의 평균 기울기로 계산되었다. 데이터는 Kruskal-Wallis 테스트를 사용하여 통계적으로 분석되었다. 유의 수준은 5%로 설정하였다. 3. 결과 계산된 나노 누출 값은 GP/AH26의 경우 0.0670 ± 0.0516 nL/s, GP/EndoSeal MTA의 경우 0.1397 ± 0.1579 nL/s, Biodentine의 경우 0.0358 ± 0.0538 nL/s 로, 재료 사이에 유의한 차이는 나타나지 않았다(P > 0.05). 4. 결론 nanoFlow 장치를 사용함으로써 보다 정확한 근관 충전 재료의 실시간 미세 누출의 측정이 가능하였다. 나노 누출 측면에서 각 근관 충전재 사이에는 큰 차이가 나타나지 않았다.I. Introduction 1 II. Materials and Methods 7 1. Ethics 7 2. Sample size calculation 7 3. Sample preparation 7 4. Nanoleakage measurement . 9 5. Statistical analysis 10 III. Result 11 IV. Discussion 12 V. Conclusion 18 Reference 19 Tables & Figures 27 Korean Abstract 31Docto

효율적이고 안전한 에너지 저장을 위한 리튬이온전지 및 슈퍼커패시터 용 전극물질의 개발과 아키텍쳐링

학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2016. 2. 이종협.Occasional reports on accidental explosions that appear in the news, increases the safety concerns of using Li ion batteries (LIBs). The scope of LIB is expanding toward large-scale energy storage systems such as electrified transportation and smart grids. Considering these facts, manufacturers and consumers now have concerns regarding the safe use of such devices. The high voltage anode materials have greatly relieved the unstable state of LIB during operation because they can exclude the uneven plating of lithium and reductive electrolyte decomposition. Although various titanium based oxides have been developed as high voltage anode, low capacity or poor potential flatness still severely hinder their industrial usage. In this dissertation, two types of titanium-based crystals are proposed, and they exhibited enhanced Li storage performance as high voltage anode materials as follows. A c-channel that is formed inside stacked (001) planes in rutile TiO2 exhibits the lowest energy barrier for Li migration. In this regard, the rational design of a TiO2 architecture for stacked (001) planes is needed in order to maximize Li storage. Here, a three-dimensional and dendritic TiO2 sphere comprised of c-channel specialized nanorods is proposed, which can be prepared via the specific adsorption of chlorine ion on the (110) plane. Along with a confined Li pathway, such radially assembled TiO2 nanorods show a low intercrystalline resistance. When fabricated into an electrode, the three-dimensional and dendritic TiO2 sphere is capable of delivering an almost 100% Coulombic efficiency in conjunction with long cycle charge/discharge stability (300 cycles). This approach, which is based on theoretical studies and experimental validation, provides guidance for tailoring electrode materials for use in Li storage systems. H2Ti12O25 (HTO), a recently discovered anode material for LIB, has outstanding electrochemical performance compared to other high voltage materials. However, its thermodynamic/kinetic properties as a Li host have not been thoroughly investigated yet. In this study, the Li storing behavior of HTO was intensively characterized by a combined theoretical experimental study. In addition, the strong dependence of electrochemical performance on Li diffusion kinetics stimulated us to develop a nanostructured HTO which provides incorporated Li with a short diffusion length inside a nano-crystal. As a result, the nanostructured HTO showed upgraded Li storage performance. This work suggests that the HTO is one of the most competitive material found to date for the construction of advanced LIB with excellent safety and stability. Electrochemical capacitors (so-called supercapacitors), with high power density and superior cycling ability, are considered to be one of the most stable and safe energy storage systems. High power as well as reasonably high energy density are provided, supercapacitors are likely to be regarded as a representative energy storage system along with batteries. The common challenge in supercapacitor design is the resistive behavior originated from sluggish ion transport, poor electrical conductivity, and high charge transfer resistance, which increases equivalent series resistance of overall cell. In addition, a variety of activation process for the increase of surface area not only are involved in harmful chemicals or toxic metals but also lower its cost-effectiveness. In this dissertation, two strategies for resolving above-mentioned challenging points are proposed3D bicontinuous metal-carbon hybrid and organic gel-wrapped MnO2 thin fim electrode. The three dimensionally aligned bicontinuous carbon and metal hybrids are synthesized. The resulting material shows a significantly high rate capability up to 1,000 V s-1. The proposed strategy exploits an agarose gel as a template for the simultaneous construction of three dimensional (3D) carbon structures filled with a metal-lined architecture in supercapacitors. The carbon framework with three dimensional and interconnected metal lines inside minimizes both empty inner pores and electron transfer barriers, which results in a substantial reduction of the overall electrode resistance. In addition, it can be used for the filteration of voltage ripples due to the ultrafas7t response with high efficiency. A robust hybrid film containing MnO2 was prepared for achieving large areal capacitances. An agarose gel, as an ion-permeable and elastic layer coated on a current collector, plays a key role in stabilizing the deposited pseudocapacitive MnO2. Cyclic voltammetry and electrochemical impedance spectroscopy data indicate that the hybrid electrode is capable of exhibiting a high areal capacitance up to 52.55 mF cm-2, with its superior structural integrity and adhesiveness to the current collector being maintained, even at a high MnO2 loading. The emergence of body-centric power generators such as piezoelectric/solar cell and the functional/morphological evolution of smart phones have created a need for advanced devices for energy storage. In order to meet the power demand of such devices, the construction of a series of unconventional energy storage systems have been developed in the past few years, which enable one to achieve flexible, foldable, and stretchable characteristics. In order to secure their stability and safety of energy storage platform under mechanically stressed conditions, the primary requirements of electrode materials include robust electrical connectivity and mechanical endurance. In the last part of this dissertation, a lint taping method is described for the fabrication of thin layer of conducting network using graphite felt. An all-solid-state, completely foldable and washable energy storage platform was fabricated. By adopting it as a supercapacitor electrode, the physical characteristics and electrochemical properties of such a GFCN are identified. A constructed graphitic fiber network derived from conventional graphite felt was readily assembled into a full-cell by its self-adhering architecture. The as-prepared system exhibits high mechanical properties under various folding motifs and washable characteristics without capacitance fading by virtue of the robust electrical connectivity of the fibrous graphite network and intimate contact between the polymeric gel electrolyte and the electrodes. The collected results suggest that this supercapacitor system is a promising candidate for practically available and wearable energy storage systems with high cost-effectiveness and scalability.Chapter 1. Introduction 1 1.1 Toward safe and efficient Li-ion batteries: Development of high voltage anode materials 1 1.1.1 Development of Li-related batteries from a safety standpoint 1 1.1.2 Challenges in development of high voltage anode materials for use in LIBs 2 1.1.3 Strategy 1: c-channel specialized TiO2 sphere as high voltage anode materials 4 1.1.4 Strategy 2: H2Ti12O25 nanostructures as high voltage anode materials 6 1.2 Toward safe and efficient supercapacitors: Development of hybrid electrode materials 14 1.2.1 Development of supercapacitors for safe and reliable energy storage systems 14 1.2.2 Challenges facing supercapacitors 15 1.2.3 Strategy 1: Bicontinuous metal/carbon hybrid as high power supercapacitor electrodes 15 1.2.4 Strategy 2: Hybrid MnO2 film for enhancing the structural integrity of thin film supercapacitor electrodes 17 1.3 A system level approach to construct robust energy storage platform enduring mechanical stress 23 1.4 Objectives 26 Chapter 2. Radial Alignment of c-Channel Nanorods in 3D Porous TiO2 for Eliciting Enhanced Li Storage Performance 28 2.1 Experimental 28 2.1.1 Preparation of TiO2 materials 28 2.1.2 Characterizations 29 2.1.3 Electrochemical measurements 29 2.1.4 Computational details 30 2.2 Results and discussion 32 2.2.1 Li diffusion dynamics inside rutile TiO2 32 2.2.2 Synthesis of 3D-TS 33 2.2.3 Characterization of 3D-TS 35 2.2.4 Anisotropic growth of TiO2 nanorods 37 2.2.5 Electrochemical analyses 38 Chapter 3. Nanostructured H2Ti12O25 as a Superior High Voltage Anode Materials for Li-Ion Batteries 67 3.1 Experimental 67 3.1.1 Synthesis of H2Ti12O25 67 3.1.2 Characterizations 68 3.1.3 Electrochemical measurements 68 3.1.4 Computational details 69 3.2 Results and discussion 70 3.2.1 Structural evolution during synthesis of HTO 70 3.2.2 Characterizations of HTO made from phase-pure TiO2 70 3.2.3 Diffusion properties of Li inside HTO 72 3.2.4 Effect of operating temperature on Li storage performance 74 3.2.5 Kinetic gap of structural transformation from TiO2 to Na2Ti3O7 75 3.2.6 Synthesis and Li storage performance of nanostructured HTO 78 Chapter 4. 3D Bicontinuous Metal/Carbon Hybrid using an Agarose Gel for Ultra-Fast Charge/Dischargeable Supercapacitor Electrodes 105 4.1 Experimental 105 4.1.1 Fabrication of bicontinuous carbon and 3D Au composites 105 4.1.2 Physicochemical characterizations 105 4.1.3 Electrochemical characterizations 106 4.1.3 Calculations 106 4.2 Results and discussion 107 4.2.1 Preparation of the 3D metal/carbon composite 107 4.2.2 Electrochemical properties of the 3D metal/carbon composite 109 Chapter 5. Hybrid MnO2 Film with Agarose Gel for Enhancing the Structural Integrity of Thin Film Supercapacitor Electrodes 125 5.1 Experimental 125 5.1.1 Fabrication of hybrid MnO2 electrodes 125 5.1.2 Characterizations 125 5.1.3 Calculations 126 5.1.4 3D finite elemental method modelling 127 5.1.5 Density functional theory calculations 128 5.2 Results and discussion 129 5.2.1 Synthesis and characterization of agarose gel-wrapped MnO2 electrode 129 5.2.2 Electrochemical measurements 131 Chapter 6. Robust Energy Storage Platform with Foldability and Washability 147 6.1 Experimental 147 6.1.1 Preparation of graphit felt-based conducting network (GFCN) electrode 147 6.1.2 Assembly of a full-cell system 147 6.1.3 Oxidation of graphite felt 148 6.1.4 Characterizations 149 6.1.5 Calculations 149 6.2 Results and discussion 151 6.2.1 Selection of optimal adhesive substrate 151 6.2.2 Assembly of full-cell 154 6.2.3 Electrochemical measurements 155 6.2.4 Folding tests 156 6.2.5 Washing tests 157 6.2.6 Improvement of capacitance via surface treatments 159 Chapter 7. Summary and Conclusions 179 Chapter 8. Recommendation for Further Research 182 Bibliography 183 요약 (국문초록) 198 List of publications 204Docto

Perception and Inner Struggle Experienced by Nursing Students in Relation with Infection Management through Observation and Performance of Infection Control Activities

Purpose: To study the internal psychological conflicts among nursing students during an infection control protocol carried out in the hospital by measuring their observation skills and performance during clinical training. Methods: Investigation of both pre- and post- infection control was conducted using questionnaires for clinical infection practices. We identified and evaluated the students observation skills, clinical performance, clinical perception,and internal conflict regarding clinical infection control. We also interviewed the students as part of our study. Results: Among parameters such as clinical performance, observation skills, clinical perception, and internal conflict, the average observation skills (t=5.49, p<.001) were significantly lower, while internal conflict among students (t=-7.23, p<.001) was significantly higher than expected prior to clinical training. Generally, there was a negative correlation between observation skills and internal conflict in every aspect of infection control practice (r=-.281, p=.031). Internal conflict was significantly higher than expected in the context of hand hygiene (t=-2.135, p=.037), personal hygiene (t=-3.48, p=.002), and ventilator management (t=-3.69, p<.001). Clinical performance of students in the context of hand hygiene (t=4.69, p<.001), personal hygiene (t=2.06, p=.044), and ventilator management (t=2.68, p<.001) was significantly lower than expected prior to clinical training. Conclusion: Our findings showed that internal psychological conflict is higher when infection control practices are observed or performed to a lesser degree. Therefore, reinforcing education regarding infection control among students, such as developing a systematic program, or consecutive training and monitoring, is suggested

마우스에서 결핵균 감염 상태에 따른 유전자 발현의 차이

Dept. of Medical Science/석사Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. One-third of the world's population is estimated to be latently infected with M. tuberculosis; however, only approximately 10% of latently infected people will develop this overt disease, and their aerosol droplets can be transmitted through the air. Current tests available for the diagnosis of TB are limited by their inability to differentiate between latent tuberculosis infection (LTBI) and active TB disease. Therefore, an accurate and effective marker for differential diagnosis should be developed for predicting disease progression. Given the increasing interest in identifying new biomarkers against TB, we examined promising biomarkers that could distinguish between chronic/reactivation TB disease and LTBI using an M. tuberculosis-infected mouse model. The lung tissue and serum of C57BL/6 mice were used to identify the differences of gene expression among healthy, LTBI, chronic TB, spontaneous reactivation, and immunosuppressive drug-treated groups. First, microarray analysis using isolated mRNAs from mice tissues showed several important immune-related genes with different expression levels between the chronic/latent and healthy control groups (p < 0.05). Next, quantitative reverse transcription-polymerase chain reaction to validate the results obtained by the microarray was performed. Based on the cDNA microarray results, 17 candidate genes were selected and clustered into four groups: 1) chemokines excluding monocyte chemoattractant proteins (MCPs) (CXCL9, CXCL10, CXCL11, CCL5, CCL19); 2) MCPs (CCL2, CCL7, CCL8, CCL12); 3) Receptors (IL2Rβ, IL7R, IL12Rβ1, IL12Rβ2, IL21R, IL27Rα); and 4) TNF and IFN- genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the latent stage. CXCL9, CCL7, CCL12 were noticeably increased in the chronic stage compared with those in the latent stage. Therefore, these three significantly increased cytokines in lung tissue from the mouse TB model might be candidates for biomarkers that distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient TB markers. We used Luminex assays to confirm the same tendency of gene expression in mice serum. Second, there is a substantial need for biomarkers to distinguish latent, chronic TB from spontaneous reactivation, for predicting disease progression. To identify the immunological status of the latent, chronic, and reactivation stages, immunological genes were analysed in lung tissues from mice infected with M. tuberculosis. Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR using isolated microRNAs from mice tissues. In the result, 10 microRNAs (mmu-miR-1a-3p, mmu-miR-133a-5p, mmu-miR-133a-3p, mmu-miR-206-3p, mmu-miR-133b-3p, mmu-miR-3064-5p, mmu-miR-450b-3p, mmu-miR-26b-3p, mmu-miR-181a-2-3p, mmu-miR-8114) were likely to be the biomarkers that can distinguish between latent and reactivation TB. Specifically, mmu-miR-206-3p was noticeably increased in the reactivation stage compared with the latent and chronic stage. And mmu-miR-1a-3p is expected to be a biomarker candidate that can simultaneously distinguish latent, chronic, and reactivation stages. In conclusion, these findings suggest that the protective mechanisms against TB infection may be related to chemokines, MCPs, chemokine receptors and microRNAs that modulate the activity of immune responses, and some of which are potential biomarkers distinguishing different stages of M. tuberculosis infection. 결핵의 원인은 대부분 Mycobacterium tuberculosis 이며, 감염성 질병 중에서 사망률이 세계 2위에 랭크되어 있는 심각한 질병이다. WHO는 2013년까지 M. tuberculosis 에 감염되어 발병된 사람은 900만 명, 사망자는 150만 명이라고 밝혔다. 현재 전체 인구의 약 1/3, 즉 20억 명 이상이 TB 잠복 감염자인 것이다. 그 중 5-10% 만이 활동성 결핵 환자가 되는데, 대부분 ...ope

이찬해 작곡 「해금 독주를 위한 '속죄제'」 분석 연구

학위논문 (석사)-- 서울대학교 대학원 : 음악과, 2017. 2. 양경숙.현재에 이르러 창작곡은 필수불가결한 국악의 한 장르로 자리 잡았다. 따라서 연주가들은 올바른 음악 분석을 통해 작품의 음악적 가치를 객관적으로 파악하고, 근거 있는 음악적 사고가 바탕이 된 연주를 해야 한다. 본 논문은 이찬해 작곡 「해금 독주를 위한 속죄제」를 연구 분석하여 작곡가의 작품세계를 바탕으로 속죄제의 의미에 따른 악기 편성과 흐름을 고찰하였으며, 작품의 전체 구조를 파악한 뒤 프랙탈 분석 이론에 근거하여 작품을 이루는 음들의 핵심구조를 알아내었다. 이러한 연구를 바탕으로 효과적인 운지법, 운궁법 및 특수기법이 포함된 연주법을 살펴보았으며 다음과 같은 결론을 얻었다. 첫째, 단악장 형식인 「해금 독주를 위한 속죄제」는 템포 변화와 선율을 구성하는 핵심구조, 악구의 특징에 따라 Introduction, Ⅰ, Ⅱ, Cadenza, Ⅲ, CodaⅠ, CodaⅡ 크게 7단락으로 나누었다. 둘째, 「속죄제」는 해금으로 인간의 죄와 회개를 나타내고자 하였으며, 징은 하나님의 목소리, 임재(臨在)를 표현하였다. 작곡가의 기독교적 세계관을 투영하여 작품을 해석하였을 때, 징(혹은 공)의 반주가 있는 악구와 징 반주가 없는 악구 두 부분으로 나눌 수 있으며, 첫 번째 악구는 하나님과 인간의 화합과 뜻 일치의 내용으로 볼 수 있고, 두 번째 악구는 인간이 하나님의 통제에서 벗어나 죄를 짓는 부분으로 해석할 수 있다. 셋째, 음악작품의 형성과정에서 핵심이 되는 구조나 선율이 전체를 이룰 것이라 보는 프랙탈 구조이론에 의해 「속죄제」를 구조 분석하였을 때, 작품의 핵심 구조는 A♭을 중심음으로 위․아래 완전 5도의 3개 음으로 구성된 관계이다. 이는 Introduction단락에서 핵심 구조의 선율이 명확하게 드러나며, 전개 되는 작품 속에 확장이나 음옮김과 같은 변형 속에서 끊임없이 존재하고 있었다. 따라서 는 자기유사성질을 지닌 핵심구조의 반복으로 부분과 전체가 같다는 프랙탈 구조의 특징과 닮아있으며, 통일성과 응집성이 내재되어 있는 작품임을 증명하였다. 넷째, 이찬해는 「속죄제」에서 주로 글리산도(Glissando), 트레몰로(Tremolo), 중음주법(Double-stop)을 사용하여 풍부한 음향과 현대적인 색채를 이끌어내고자 하였다. 특히 중음주법(Double-stop)에서는 아직 해금에 보편화 되지 않은 기법들로 이루어져 있어 연주가들에게 높은 역량을 요구하며, 작곡가가 의도한 음향과 음색에 맞게끔 다양한 기법을 개발할 수 있게 하였다. 필자는 작품의 구조와 의미 분석을 통해 알맞은 연주법을 제시하였다. 이와 같이 「해금 독주를 위한 속죄제」 분석을 통하여 작곡가 이찬해의 첫 번째 해금 작품 연구가 이루어졌다. 작품의 구조 분석이 실질적인 연주법에 적용하는데 도움이 되길 바라며, 형식이 뚜렷하게 정해지지 않은 현대음악 작품 분석에 있어 방법론의 다양화를 모색해볼 수 있길 바란다.Ⅰ. 서 론 1 1. 연구목적 1 2. 연구방법 및 연구범위 5 Ⅱ. 이찬해의 작품세계 7 Ⅲ. 「해금 독주를 위한 속죄제」 분석 12 1. 프랙탈 분석 이론 12 2. 구조 분석 18 1. Introduction 단락 구조 분석 20 2. Ⅰ단락, Ⅱ단락 구조 분석 23 3. Cadenza 단락 구조 분석 30 4. Ⅲ단락 구조 분석 33 5. CodaⅠ, CodaⅡ단락 구조 분석 36 Ⅳ. 연주법 40 1. 악기편성 40 2. 운지법 54 3. 운궁법 64 4. 특수주법 71 Ⅴ. 결 론 77 참고문헌 79 첨부악보 81 Abstract 89Maste