박테리아의 뉴클레오타이드 이차신호전달 물질인 (p)ppGpp와 cyclic di-GMP의 대사역학

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 생물물리 및 화학생물학과, 2019. 2. 석영재.박테리아 뉴클레오타이드 이차신호전달 물질은 세포내 또는 세포외 환경 신호를 전달한다. 이러한 신호 전달 시스템은 박테리아 생리에 광범위한 영향을 미치기 때문에 박테리아는 이차신호전달 물질의 대사와 신호 전달을 조절하는 다양한 효소를 지니고 있다. 이 효소를 이용하여, 박테리아는 다양한 주위환경 신호에 반응하여 이차신호전달 물질의 세포 내 농도를 정교하게 조절 하며, 만약 이 물질의 양이 제대로 조절되지 않으면 세포의 성장 억제 및 세포사망, 불균형 표현형을 야기하기 때문에 이 신호물질의 정교한 생체 제어는 세포 생리학에 매우 중요하다. 현재까지 cAMP의 대사와 신호 메커니즘에 관한 연구는 비교적 잘 알려져 있지만, 박테리아 내 다른 뉴클레오타이드 이차신호전달 물질들의 조절 기작은 거의 알려져 있지 않다. 대장균에서 (p)ppGpp 신호 전달은 두 가지 효소, (p)ppGpp를 합성하는 RelA 단백질과 (p)ppGpp를 합성 또는 분해하는 SpoT 단백질에 의해 매개된다. 다양한 영양 스트레스에 대응하여, 이 두 단백질들은 세포 내 (p)ppGpp 농도를 정교하게 조절하여 박테리아 세포가 스트레스 조건에 적응할 수 있게 한다. 특히, SpoT 단백질은 대장균내 (p)ppGpp를 가수분해 할 수 있는 유일한 효소이며, 세포 내 (p)ppGpp가 과량 축적되면 심각한 세포 성장 억제 및 세포사를 야기하기 때문에 SpoT 단백질의 활성은 매우 정밀하게 조절되어야 한다. 하지만, 현재까지 SpoT 단백질의 활성이 어떻게 조절 되는지는 거의 알려져 있지 않다. 조절 기작을 찾기 위해 ligand fishing 실험을 수행 하였고, 이를 통해 대장균의 Rsd 단백질이 SpoT 단백질과 직접 상호작용하여 SpoT 단백질의 (p)ppGpp 를 부수는 활성을 증가 시킨다는 것을 확인 하였다. 또한, 이러한 조절은 PEP 의존 당 수송 시스템의 일반적인 구성요소인 HPr 단백질의 인산화 상태에 의해 제어된다는 사실을 밝혔다. 본 연구에서는 이러한 실험 결과를 통해, Rsd 단백질은 주변 탄소원에 따라 대장균의 긴축 반응을 조절하는 새로운 조절자임을 밝혔다. c-di-GMP 신호 전달에서, phosphodiesterases A (PDE-As) 효소는 c-di-GMP를 pGpG로 분해한다. 하지만, 세포 내 pGpG가 축적되면 PDE-A 효소의 활성을 저하 시키기 때문에 세포는 이 신호 전달 시스템 내의 세포 내 pGpG 농도를 낮추기 위한 추가적인 phosphodiesterases B (PDE-Bs) 효소를 필요로 한다. 녹농균에서 Orn 단백질이 pGpG를 분해 함으로써 PDE-B 활성을 가진다고 보고 되었다. 하지만 Orn 단백질은 pGpG 뿐만 아니라 다양한 2~5개의 뉴클레오타이드 (nanoRNAs)를 부수는 광범위한 기질특이성을 가지고 있다. 본 연구에서는 생화학적 실험 방법 및 구조 분석을 통해 pGpG만을 특이적으로 부수는 PggH 단백질의 존재를 비브리오 콜레라균에서 새로이 확인 하였다. 이러한 실험 결과를 통해 이 단백질이 c-di-GMP 신호 전달을 완료하기 위해 필요한 새로운 PDE-B 효소로서 기능을 가진다는 사실을 밝혔다Bacterial nucleotide second messengers transduce intracellular or extracellular environmental signals to appropriate cellular responses. These signal transduction system has a wide range of effects on bacterial physiology. Thus, bacteria are equipped with a variety of enzymes that regulate its metabolism and signaling. Using these enzymes, bacteria elaborately modulate cellular concentration of second messengers in response to various cues. Since uncontrolled level of theses signal molecules causes growth inhibition, cell death and imbalanced phenotypes, its homeostatic control is very crucial for cellular physiology. While the metabolism and signaling mechanisms of cAMP have been relatively well documented to date, those of other nucleotide second messengers are not well understood. (p)ppGpp signaling is mediated by two enzymes, (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT proteins in Escherichia. coli. In response to various nutritional stresses, these two stringent factors fine tune the cellular (p)ppGpp concentrations, thus allowing bacterial cells to adapt to stress conditions. SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in E. coli. Therefore, its activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which causes severe growth inhibition and cell death. To date, however, little is known about how SpoT (p)ppGpp hydrolase activity is regulated in E. coli. The ligand fishing experiment revealed that Rsd directly interacts with SpoT and stimulates its (p)ppGpp-degrading activity. This regulation is controlled by the phosphorylation state of HPr, a general component of the PEP-dependent sugar transport system. Together, these data propose that Rsd is a carbon source-dependent regulator of the stringent response in E. coli. In c-di-GMP signaling pathway, phosphodiesterases A (PDE-As) specifically degrades c-di-GMP into pGpG. However, excess amounts of pGpG inhibit phosphodiesterase A (PDE-A) activity to limit the completion of c-di-GMP signal transduction. Therefore, cells require additional phosphodiesterases B (PDE-Bs) to adjust the cellular pGpG concentrations in this signaling cascade. Orn protein has been demonstrated to mediate PDE-B activity in Pseudomonas aeruginosa. However, it exhibits broad substrate specificity to degrade various two to five nucleotides (nanoRNAs) including pGpG. Biochemical and structural analysis identified PggH as a new pGpG-specific phosphodiesterase and characterized its functional role as a PDE-B enzyme. These data indicate that PggH as a new PDEB enzyme required to complete c-di-GMP signaling pathway in Vibrio choleraeAbstract ··················································································· i Contents ··················································································· iv List of Figures ··································································· ix List of Tables ······································································ xii Abbreviations ········································································· xiii Chapter I. Literature Review ························································· 1 1. Overview of bacterial nucleotide second messenger ·························· 2 1.1. Bacterial nucleotide second messenger ···································· 2 1.2. cAMP ··········································································· 2 1.3. (p)ppGpp ········································································ 3 1.4. c-di-GMP ··································································· 4 1.5. c-di-AMP ······································································ 5 1.6. cGAMP ······································································ 5 1.7. cGMP ······································································ 6 2. (p)ppGpp metabolism and signaling ········································· 7 2.1. RSH enzyme ······························································ 7 2.1.1. RelA ······································································· 7 2.1.2. SpoT ······································································ 8 2.1.3. Rel ········································································· 9 2.2. Physiological roles of (p)ppGpp ········································ 9 2.2.1. Transcription regulation by (p)ppGpp ······························· 9 2.2.2. Toxin-antitoxin systems and persistence ··························· 10 2.2.3. Persistence and virulence ·········································· 12 3. c-di-GMP metabolism and signaling ···································· 13 3.1. Diguanylate cyclases (DGCs) ··············································· 13 3.2. c-di-GMP-specific phosphodiesterases (PDEs) ··························· 14 3.3. Physiological roles of c-di-GMP ········································ 14 3.3.1. Development and morphogenesis ································ 14 3.3.2. Motile-sessile transition ·············································· 15 3.3.3. Bacterial virulence ·················································· 16 3.4. c-di-GMP effectors ···························································· 17 4. The aims of this study ······························································ 18 5. References ································································· 20 Chapter II. Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift in Escherichia coli ····························· 31 1. Abstract ················································································ 32 2. Introduction ·········································································· 33 3. Materials and Methods ···························································· 36 3.1. Bacterial strains, plasmids and culture conditions ······················· 36 3.2. Purification of overexpressed proteins ····································· 36 3.3. Ligand fishing experiments using metal affinity chromatography ····· 37 3.4. Bacterial two-hybrid (BACTH) assays ····································· 38 3.5. In virto assay for ppGpp hydrolase activity of SpoT ····················· 38 3.6. Detection of intracellular (p)ppGpp levels ································· 39 3.7. Determination of the in vivo phosphorylation state of HPr ············· 39 3.8. RNA isolation and qRT-PCR ················································ 40 4. Results ················································································ 44 4.1. (p)ppGpp hydrolase SpoT interacts with Rsd ····························· 44 4.1.1. Identify a factor that interacts with His-tagged Rsd in E. coli ······ 44 4.1.2. Confirmation of interaction between Rsd and SpoT ··············· 46 4.1.3. Interaction of Rsd with the TGS domain of SpoT ·················· 48 4.1.4. Specific interaction of Rsd with SpoT ······························· 50 4.2. Activation of the (p)ppGpp hydrolase activity of SpoT by Rsd ········· 55 4.2.1. The stimulatory effect of Rsd on the (p)ppGpp hydrolase activity of SpoT in vitro ··············· 55 4.2.2. Rsd stimulates (p)ppGpp hydrolase activity of SpoT in vivo ····· 59 4.2.3. Stimulatory effect of Rsd on the (p)ppGpp hydrolase activity of SpoT is depdendent on the TGS domain ········· 61 4.2.4. Activation of SpoT (p)ppGpp hydrolase activity is indepdendent of s70 activity of Rsd ··················· 65 4.3. (p)ppGpp hydrolase activity of SpoT is regulated by different carbon sources ········································ 69 4.3.1. Dephosphorylated HPr blocks the stimulatory effect of Rsd on the (p)ppGpp hydrolase activity of SpoT ······················· 69 4.3.2. Regulation of (p)ppGpp hydrolase activity of SpoT by Rsd is dependent on carbon sources ················ 71 4.4. Implication of Rsd during a carbon source downshift ···················· 78 4.4.1. Rsd regulates the stringent response during carbon source downshift ······································ 78 4.4.2. Rsd counterbalances RelA-mediated (p)ppGpp accumulation during a carbon source downshift ······· 79 5. Discussion ············································································· 85 6. References ············································································· 88 Chapter III. A pGpG-specific phosphodiesterase in Vibrio cholerae ········· 97 1. Abstract ················································································ 98 2. Introduction ·········································································· 99 3. Materials and Methods ··························································· 102 3.1. Bacterial strains, plasmids and culture conditions ······················ 102 3.2. Purification of overexpressed proteins ···································· 103 3.3. Size exclusion chromatography (SEC) ··································· 103 3.4. In vitro assay for pGpG hydrolyzing activity ···························· 103 3.5. Assay for pGpG hydrolyzing activity in cell lysates ···················· 104 3.6. Crystalization of the protein for structural determination ·············· 104 3.7. Structural determination and refinement ··································105 3.8. RNA isolation and qRT-PCR ··············································· 105 4. Results ················································································ 110 4.1. Characterization of VCA0593 in V. cholerae ···························· 110 4.1.1. Oligoribonuclease activity of Vibrio cholerae Orn ··············· 110 4.1.2. Characterization and purification of VCA0593 in V. cholerae ·· 114 4.1.3. PggH is highly conserve within Vibrio species ················ 114 4.2. pGpG-specific phosphodiesterase activity of PggH ········· 116 4.2.1. Characterization of pGpG hydrolytic activity of PggH ····· 116 4.2.2. Orn degrades various two to five nucleotides (nanoRNAs) including pGpG with its broad substrate specificity ·············· 116 4.2.3. PggH specifically degrade pGpG with its narrow substrate specificity ················································ 120 4.3. Structural basis for the pGpG-specific activity of PggH ········· 122 4.3.1. Structural determination of PggH ··································· 122 4.3.2. Identification of metal binding sites in DHH domain and GGGH motif in the DHHA1 domain ·········· 122 4.3.3. Structural comparison with the canonical NrnA protein ········· 123 4.3.4. Searching for the active site of PggH ······························· 123 4.4. V. cholerae PDE-B enzyme, PggH, is required to respond to oxidative stress ········································ 127 4.4.1. An pggH-deficine mutant is defective in hydrolyzing pGpG ··· 127 4.4.2. The expression level of pggH increases in the stationary phase of growth ··································· 127 4.4.3. PggH regulate the expression level of rpoS through modulating cellular c-di-GMP concentrations ·· 130 4.4.4. pGpG-specific activity of PggH is required for V. cholerae to respond to oxidative stress ················ 130 4.4.5. PggH regulates cellular c-di-GMP levels as a PDE-B enzyme under oxidative stress condition ········· 134 5. Discussion ············································································ 136 6. References ··········································································· 142 국문초록 ·············································································· 148Docto

심근 경색 모델에서의 심장 조직 증대를 위한 페인터블 탈세포 세포외기질 하이드로겔

학위논문(석사) -- 서울대학교대학원 : 공과대학 화학생물공학부(에너지환경 화학융합기술전공), 2023. 2. 황석연.Myocardial infarction (MI) is a cardiovascular disease that causes thinning of the ventricular wall, loss of cardiac function, and heart failure due to heart muscle necrosis occurred by decreasing blood flow to the coronary artery of the heart. Currently, studies using injectable biomaterials or patches that require additional treatment, such as sutures, limit MI recovery due to secondary damage. Here, paintable hydrogel containing heart-decellularized extracellular matrix (hdECM) can be conveniently painted as a patch onto the round beating heart without adverse liquid leakage. hdECM provides a heart-like environment to induce differentiation of cardiomyoblasts and induces blood vessels in necrotic tissues through cell infiltration of epithelial cells, thereby obtaining the effect of tissue regeneration This tyramine conjugated hyaluronic acid (HA_Tyr) based paintable hydrogel exhibits robust adhesion strength on wet surfaces by tyrosinase-catalyzed oxidative reaction, adequate swelling ratio, and sufficient mechanical strength to assist heart beating. In addition, in vivo, the hdECM-containing paintable hydrogel is maintained for 28 days with high stability and adhesiveness, reduces fibrosis and thinning of the ventricular wall in the MI region, and induces angiogenesis by hdECM, leading to effective myocardial infarction repair. Overall, the hdECM-containing paintable hydrogel, which is simply applied and has a cardiac tissue-specific environment, treats MI with adequate mechanical strength and angiogenic ability.심근경색증은 심장의 관상동맥으로 가는 혈류가 감소하여 발생하는 심근괴사로 인해 심실벽이 얇아지고 심장 기능이 상실되며 심부전이 발생하는 심혈관계 질환이다. 현재 널리 연구되는 주사 가능한 생체 재료는 주사바늘에 의한 2 차적인 조직 손상이 발생하며 치료용 패치의 경우 접착력의 부족으로 인해 봉합과정이 필요하고 이 과정 또한 2 차적인 손상을 유발한다. 이번 연구에서는 습윤한 조직 표면에서도 강한 접착성을 갖는 페인트 가능한 하이드로겔은 사용함으로써 박동하는 심장에 2 차적인 피해없이 편리하게 적용할 수 있고 안정적인 치료효과를 얻을 수 있었다. 심장 탈세포 기반 재료는 심장조직에 심장과 같은 미세환경을 제공하여 심근모세포의 분화를 유도하고 상피세포의 침투를 통해 괴사조직에 혈관을 유도하여 조직재생의 효과를 얻을 수 있었다. 타이라민 작용기가 부착된 히알루론산 기반의 페인터블 하이드로겔은 습윤한 심장 조직에도 강한 접착력을 나타내었고 적절한 팽윤율 및 심장 박동을 돕기 위한 충분한 기계적 강도를 가졌다. 또한 생체 내에서 심장 탈세포 기반 재료가 함유된 페인트 가능한 하이드로겔은 높은 안정성과 접착력으로 28 일 동안 유지되며 심근 경색 부위 심벽의 두께 감소와 섬유화를 감소시키고 혈관신생을 유도하여 효과적인 조직재생을 유도했다. 전반적으로, 페인팅이라는 간단한 방법으로 조직에 적용 가능하며 심장 조직에 특이적 환경을 갖는 심장 탈세포 기반 재료가 함유된 페인트 가능한 하이드로겔은 적절한 기계적 강도와 혈관신생 능력으로 심근 경색을 치료했다List of Figures and tables １ Chapter 1. The scientific background ２ 1.1. Myocardial infarction (MI) ２ 1.2. Decellularization ３ 1.2.1. Extracellular matrix (ECM) ３ 1.2.2. Decellularized Extracellular Matrix (dECM) ４ 1.3. Bio-functional hydrogels ５ 1.4. Streptomyces avermitilis-derived tyrosinase (SaTy) ７ 1.5 Research aims ８ Chapter 2. Experiment Section １０ 2.1. Materials １０ 2.2. Methods １０ 2.2.1. Decellularization of porcine heart １０ 2.2.2. Biochemical characterization of hdECM １１ 2.2.3. Synthesis of paintable hydrogel １１ 2.2.4. Synthesis and purification of SA_ty １２ 2.2.5. Measurement of adhesiveness １３ 2.2.6. Swelling behavior of HA_tyr hydrogel １４ 2.2.7. Compression properties of paintable hydrogel １４ 2.2.8. Rheological behavior of HA_Tyr hydrogel １４ 2.2.9. Degradation behavior of HA_Tyr hydrogel １５ 2.2.10. Live/Dead Assay １５ 2.2.11. MI Modeling in Rats and Hydrogel Painting １６ 2.2.12. Echocardiography １７ 2.2.13. Masson's Trichrome (MT) Staining １７ 2.2.14. Immunofluorescence (IF) Staining １８ Chapter 3. Results and Discussions １９ 3.1. Decellularization １９ 3.1.1. Optimize decellularization solution concentration １９ 3.1.2. Quantification of ECM components and DNA ２２ 3.2. Paintable hydrogel ２３ 3.2.1. Synthesis paintable hydrogel ２３ 3.2.2. Specific activity of SA_Ty ２５ 3.2.2. Optimize concentration of HA_Tyr hydrogel ２６ 3.2.3. Swelling property of paintable hydrogel ２９ 3.2.4. Mechanical strength of swelling hydrogel ３１ 3.2.5. Adhesiveness of paintable hydrogel ３２ 3.3. In vitro analysis ３６ 3.3.1. Cell viability ３６ 3.3.2. Cell differentiation ３８ Chapter 4. Conclustion ４９ References ５０ Abstract in Korean ５３석

Preparation and Characterization of Graphene Oxide Modified Membranes for Water Treatment

학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2015. 2. 이정학.Today a third of the global population lives in water shortage country, one of the pressing needs of people throughout the world is adequate supply of drinking water. To satisfy the demand for an enormous amount of water required by expanding global population, there have been much erudite discussions and practical attempts covering a wide scope including wastewater reuse as well as desalination. Among several technologies, in particular, membrane bioreactor (MBR) and reverse osmosis (RO) process have attracted much attention in the field of wastewater treatment and desalination, respectively, due to various strengths such as high quality of treated water and a small footprint. However, MBR have membrane fouling which is the major obstacle in maximizing their efficiency leading to short membrane lifetime and high operating costs. Also, for the RO process, low energy efficiency still remains unanswered as a serious challenge in RO process over the past few decades. In this study, it is demonstrated that the application of graphene oxide (GO) to membrane fabrication can be a novel strategy to overcome the aforementioned residual problems with each membrane process. In detail, GO was applied to fabrication of polysulfone (PSf) ultrafiltration (UF) membrane for the improvement of hydrophilicity and electrostatic repulsion characteristics, and the anti-biofouling capability of GO nanocomposite membrane was proved to be effective in MBR. Furthermore, addition of GO enhanced mechanical strength of GO nanocomposite membrane due to its high modulus and aspect ratio, which enabled the GO nanocomposite UF membrane to have mechanical strength comparable to existing support layer for commercial RO membrane and highly porous structure simultaneously. It is worth noting that RO membrane consisting of the PSf/GO nanocomposite support layer outperformed others including commercial membranes as well as the previously reported membranes in open literature. Also noteworthy is increasing the porosity of support layer could lead to improving the efficiency of RO membrane. To clarify the reason why porous structure of support layer induces higher water permeability of RO membrane, a non-intrusive experimental method was devised for representing the characteristics of the support layer as related to water flux.Table of Contents Abstract i List of Figures vii List of Tables xvi I. Introduction 1 I.1. Backgrounds 2 I.2. Objectives 4 II. Literature Review 7 II.1. Phase inversion in polymer system 8 II.1.1. Introduction 8 II.1.2. Nonsolvent induced phase separation method 12 II.1.3. Membrane structures prepared by nonsolvent induced phase separation: finger- and sponge-like structure 17 II.1.4. Factors affecting pore structure of membrane prepared by nonsolvent induced phase separation 21 II.2. Interfacial polymerization 28 II.2.1. History of reverse osmosis membrane 28 II.2.2. Fabrication of thin-film composite reverse osmosis membrane using interfacial polymerization 35 II.2.3. Recent trend of reverse osmosis membrane 39 II.3. Graphene oxide 48 II.3.1. Introduction 48 II.3.2. Synthesis of graphene oxide platelets 54 II.3.3. Preparation of graphene-based polymer nanocomposite: Non-covalent dispersion methods 58 II.3.4. Mechanical properties of graphene-based polymer nanocomposite 59 III. Graphene Oxide Nanoplatelets Composite Membrane with Hydrophilic and Antifouling Properties for Wastewater Treatment 61 III.1. Introduction 62 III.2. Experimental section 65 III.2.1. Materials 65 III.2.2. Preparation of graphene oxide and reduced graphene oxide solution. 66 III.2.3. Preparation of the membranes 68 III.2.4. Characterization 69 III.2.5. Pure water flux measurement 72 III.2.6. Microorganism attachment test 73 III.2.7. Membrane bioreactor operation 74 III.3. Results and discussion 78 III.3.1. Conformation of graphene oxide in polysulfone/graphene oxide membrane 78 III.3.2. Anti-biofouling activity of polysulfone/graphene oxide membrane 82 III.3.3. Hydrophilicity of polysulfone/graphene oxide membrane 88 III.3.4. Membrane pore size distribution and pore cross-sectional structure 91 III.3.5. Mechanical strength of polysulfone/graphene oxide membrane 99 III.3.6. Membrane bioreactor operation 101 III.4. Conclusions 104 IV. Impact of Support Layer on Thin-Film Composite Reverse Osmosis Membrane Performance 105 IV.1. Introduction 106 IV.2. Experimental section 108 IV.2.1. Fabrication of polysulfone porous support layer 108 IV.2.2. Fabrication of polyamide active layer by interfacial polymerization 109 IV.2.3. Characterization 110 IV.2.4. Reverse osmosis test 113 IV.2.5. Forward osmosis test 115 IV.3. Results and discussion 116 IV.3.1. Control of support layer structure using pore formation mechanism in phase inversion 116 IV.3.2. Correlation between mean surface pore size of support layer and active layer thickness of reverse osmosis membrane 119 IV.3.3. Correlation between active layer characteristics of reverse osmosis membrane and water flux 123 IV.3.4. Pressure drop and water transport behaviour in support layer during reverse osmosis operation 127 IV.3.5. Correlation between the characteristics of support layer and water flux of reverse osmosis membrane. 134 IV.3.6. Various characteristics of a thin-film composite membrane on water flux of reverse osmosis membrane 138 IV.4. Conclusions 142 V. Size-Controlled Graphene Oxide Enabling Thin-Film Composite Reverse Osmosis Membrane to Have Highly Porous Support Layer for High Performance 143 V.1. Introduction 144 V.2. Experimental section 148 V.2.1. Preparation of size-controlled graphene oxide platelets 148 V.2.2. Fabrication of size-controlled graphene oxide platelets composite reverse osmosis membrane 150 V.2.3. Characterization 151 V.2.4. Reverse osmosis test 153 V.3. Results and discussion 154 V.3.1. Size control of graphene oxide platelets by applying different mechanical energy input per volume 154 V.3.2. Structural integrities of graphene oxide platelets according to different sizes 156 V.3.3. Mechanical properties of polysulfone/graphene oxide nanocomposite membranes 158 V.3.4. Effective characteristics of graphene oxide platelets to improve mechanical properties of polymer nanocomposites 160 V.3.5. Influence of graphene oxide addition on structural characteristics of active and support layers 166 V.3.6. Performance of reverse membrane made of polysulfone/graphene oxide nanocomposites support layer 175 V.4. Conclusions 178 VI. Conclusions 180 Nomenclature 183 Greek letters 187 References 188 국문 초록 213Docto

A Study on Characteristics of International Bunkering Oil Price

학위논문 (석사)-- 서울대학교 대학원 공과대학 에너지시스템공학부, 2017. 8. 허은녕.본 연구는 원유 및 제품유에 대한 동조화, 지역화 및 인과관계에 대한 연구를 선박유(Bunkering Oil)의 가격 변동 특성으로 확장하였다. 전 세계 벙커링 수요의 60%를 싱가포르, 미국, UAE, 네덜란드와 한국이 담당하고 있다. 이들 지역에 위치한 벙커링 거점 항만간 경쟁으로 인한 선박유의 가격 변동 특성을 실증적으로 연구하였다. 전 세계 선박유 판매량 1, 2위 지역인 싱가포르와 로테르담의 두 가지 선박유와 네 가지 제품유를 연구 대상으로 하였다. 선박유는 HFO(Heavy Fuel Oil) 180 CST와 HFO(Heavy Fuel Oil) 380 CST이며 제품유는 납사(Naphta), 고급휘발유(Premium Gasoline), 항공유/등유(Jet/Kerosene), 경유(Gasoil/Diesel)이다. 자료의 분석 기간은 8년으로 2009년 1월부터 2016년 12월까지이며 월별(Monthly) 및 주간(Weekly) 데이터를 분석 하였다. 2008년 급격한 유가 하락 이후 새롭게 자리 잡은 가격 구조를 분석하고자 하였다. 연구 주제인 선박유에 대한 이해를 돕기 위해 해운 시장과 국제 벙커링 시장에 대한 소개를 하였다. 해운업계의 기존 연구사례로 벙커링 공급 최적가격 결정 요인과 벙커링 항만의 결정 제안 관련 논문을 정리 하였다. 원유 및 제품유 분야의 기존 연구 사례로는 원유 시장의 동조성 및 인과관계 관련 논문을 정리 하였다. 자료 분석은 시계열 자료의 안정성 확인, 시차 확인 공적분 검정 및 인과관계 확인의 순서로 진행하였다. 분석 결과를 종합하자면 다음의 세 가지로 설명할 수 있다. 첫 번째 가설은 선박유 시장 가격과 타 석유 제품 시장 가격이 상호 연동 되는가이다. 각 시장에서 2개의 선박유와 4개의 제품유와 동조성 여부를 검증하였다. 월별 시계열 자료의 안정성 확인, 공적분 검정 등을 진행하였다. 공적분 검정결과 공적분이 모두 존재하여 오차수정모형(VECM)을 통해 가격 간의 장기적, 단기적인 인과관계를 확인하였다. 그랜져 인과관계(Granger causality) 분석결과 싱가포르와 로테르담 모두에서 HFO 180 CST와 HFO 380 CST 가격과 Premium Gasoline 가격은 상호적인 인과관계가 있음을 확인하였다. 싱가포르에서는 HFO 180 CST의 가격이 Naphta, Jet/Kerosene, Gasoil/Diesel의 가격에 영향을 주고 있으며, HFO 380 CST 가격이 Naphta 가격에 영향을 주고 있었다. 다만 HFO 380 CST 가격과 Jet/Kerosene, Gasoil/Diesel 가격과의 인과관계는 확인 할 수 없었다. 로테르담 지역에서는 HFO 180 CST 가격이 Naphta 가격에 영향을 주고 있으며 Jet/Kerosene, Gasoil/Diesel 가격과는 인과관계가 확인되지 않았다. HFO 380 CST 가격은 HFO 180 CST 가격과 동일하게 Naphta가격에는 영향을 미치나 Jet/Kerosene, Gasoil/Diesel 가격에는 영향이 없었다. 선박유 가격은 제품유 가격과 연동되는 경향이 있다. 연동 되는 경우 선박유가 영향변수로 작용하고 있었다. 두 번째 가설은 두 개의 선박유인 HFO180 CST 가격과 HFO 380 가격이 장기적으로 같이 움직이는가이다. 월간 자료와 주간 자료를 대상으로 Johansen(1991) 공적분 검정을 진행하였고 모두 공적분이 있음을 확인 할 수 있었다. 그러나 오차수정모형(VECM) 검정 결과 월간자료에서는 의미 있는 관측 값을 확인하기 힘들었다. 주간 자료를 분석할 결과 싱가포르와 로테르담 모두에서 HFO 380 CST가격이 HFO 180 CST의 영향을 주고 있음을 확인 할 수 있었다. 세 번째 가설은 싱가포르와 로테르담 두 시장은 선박유 거래에서 같이 움직이는가이다. 싱가포르와 로테르담의 월간 및 주간 HFO 180 CST 가격과 HFO 380 CST가격 관계에 대한 Johansen(1991) 공적분 검정 결과 모두 공적분이 있었다. 이에 오차수정모형(VECM)으로 인과관계 분석을 진행하였다. 월간단위 자료 에서는 싱가포르의 HFO 380 CST 가격이 로테르담의 HFO 180 CST 가격에 영향을 미치고 있었으며 HFO 180 CST 가격에 대해서는 두 시장 간의 인과관계가 없는 것으로 나타났다. 주간 자료 분석의 경우에는 싱가포르 HFO 180 CST 가격이 로테르담의 HFO 180 CST 가격에 영향을 주고 있었다. 그러나 HFO 380 CST 가격에 대해서는 두 시장 간에 인과관계를 확인 할 수 없었다. 싱가포르의 선박유 가격이 로테르담의 선박유 가격에 영향을 주고 있었다. 본 연구 결과는 친환경 선박 연료의 사회기반기설 구축 정책 검토 과정에서 기존 선박유 시장의 가격적인 경쟁관계를 파악하는데 참고할 수 있는 기초자료로 사용이 가능할 것이다.제 1 장 서 론 1 제 1 절 연구의 배경 및 목적 1 제 2 절 국제해운 및 선박유 시장 3 1. 국제 해운 동향 3 2. 선박의 분류 6 3. 선박유 시장의 소개 15 제 3 절 논문의 구성 26 제 2 장 기존 연구의 검토 28 제 1 절 선박유 시장의 기존 연구 사례 28 1. 선박유 최적 공급 계약에 관한 연구 28 2. 벙커링 항만 선택에 관한 연구 33 제 2 절 제품유 시장의 기존 연구 사례 36 제 3 장 연구 방법론 및 분석자료 39 제 1 절 단위근 검정 방법 39 제 2 절 공적분 검정 방법 43 제 3 절 Granger 인과관계 검정 방법 45 제 4 절 분석자료 48 제 4 장 분석 결과 57 제 1 절 가설1 : 선박유 시장 가격과 타 석유 제품 시장 가격이 상호 연동되어 있는가 57 제 2 절 가설2 : 2개의 선박유인 HFO180과 HFO380이 장기적으로 같이 움직이는가 67 제 3 절 가설3 : 싱가포르와 로테르람 두 시장은 선박유 거래에서 같이 움직이는가 71 제 5 장 결과 요약 76 제 1 절 Granger 인과관계 검정 결과 종합 76 제 2 절 연구 결과 요약 및 한계점 80 참 고 문 헌 83 Appendix 87 Abstract 97Maste

신용평가사와 등급감시의 경제적 기능

학위논문 (석사)-- 서울대학교 대학원 : 경영학과, 2015. 2. 안태식.Despite increasing importance, the economic function of watchlist of Korean credit rating agency (CRA) has not been studied profoundly yet. Two explanatory lines exist for the reason behind watchlist placement: information delivery vs. implicit contract. Information delivery argument suggests that CRAs place watchlist to meet investors demand for accurate and stable rating information. Implicit contract argument suggests that CRAs place watchlist to give a firm chance to recover from deteriorated credit quality. I find that watchlist placement is more strongly associated with the information delivery argument. Albeit not strong, evidence also indicates that CRAs do consider fundamental quality of a firm and that accounting quality matters in watchlist placement process. Overall, findings suggest that a CRA in Korea has enhanced its economic role as an information intermediary using watchlist.1. Introduction 1 2. Hypothesis Development 6 3. Sample and Research Design 11 3.1 Data and Sample selection 11 3.2 Research Design 12 4. Results 20 4.1 Descriptive Statistics 20 4.2 The determinant of watchlist placement 22 4.3 Market reaction to watch-preceded downgrade and direct downgrade 24 4.4 Post-downgrade recovery effect 26 5. Conclusion 26 Reference 29 TABLE 1 Descriptive Statistics 32 TABLE 2 Pearson Correlation Matrix 34 TABLE 3 The determinants of watchlist placement 36 TABLE 4 Univariate test of market reaction between watch-preceded downgrade and direct downgrade 38 TABLE 5 Multivariate test of market reaction between watch-preceded downgrade and direct downgrade 39 TABLE 6 Comparison of post-downgrade recovery effect 41 Appendix 42Maste

요철이 설치된 종횡비가 큰 사각덕트에서의 유동과 열전달의 특성

학위논문(석사)--서울대학교 대학원 :기계항공공학부,2003.Maste

Characterization of dielectrophoretic force for the structural shapes of window and in microfluidic chip

의공학과/석사본 논문에서는 미세유체칩 내에 특정 모양의 Trap window를 설치하여 유전영 동 힘의 특성을 정밀하게 분석, 연구 하였으며, 더 나아가 동일 미세유체 칩 내에서 세포의 특성을 분석, 실시간 관찰 할 수 있는 기술에 대해 연구하였다. 먼저 다양한 Trap window를 가진 IDT 전극이 어떠한 유전 영동 힘을 가지는지 에 대해서 수치해석적인 방법과 실제 실험을 통해서 어떤 움직임을 보이는지에 대해 연구하며 최종 적으로 한 개의 모델을 선택한다. 이 후 앞서 만든 미세유 체칩 내에서 세포 실험을 진행한다. 세포는 외부의 환경 변화에 따라서 그 모양이 실시간으로 다변화하며 세포내 이온 또한 출입을 자유롭게 하는데, 본 연구에서 이러한 세포의 변화는 미세유 체칩 내에서 유전 영동 기술을 이용하여 세포 외부 환경조건의 변화에 따른 물 리적 특성 변화, 세포막의 효율 변화와 같은 생리적인 변화까지 실시간으로 관 찰 하게 된다. 이 기술은 세포의 물리적인 특성을 할 수 있고, 특히 외부 환경 변화에 따른 세포의 상태 변화를 실시간으로 알 수 있다는 점에서 이후 이러한 Lab-on-a-chip 시스템을 활용 해서 각각의 생체미소입자의 특성 분석 뿐만 아 니라 이후 약물 스크리닝과 같은 연구를 보다 효과적으로 진행 할 수 있을 것 으로 기대된다.ope

영한 기계 번역을 위한 문장의 구조 분석에 관한 연구

학위논문(석사)--서울대학교 대학원 :전기.컴퓨터공학부,2001.Maste

정보화마을, 지역정보화 추진정도를 중심으로

학위논문(석사)--서울대학교 대학원 :행정학과,2012. 2. 박상인.본 연구의 목적은 지방자치단체 웹사이트의 이용도를 실질적으로 측정하고, 이에 영향을 미치는 요인이 무엇인지 알아보고자 하는 것이다. 이러한 목적을 달성하기 위해 2006년 234개의 기초지자체의 모든 웹사이트의 실제 이용도를 인터넷 패널의 클릭스트림(clickstream) 데이터를 활용하여 분석하였다. 클릭스트림 데이터는 웹사이트의 실제 이용자라는 측면에서 수요자의 입장에서 접근한 것이다. 따라서 자기보고방식을 따르는 설문조사 데이터보다 실제 이용자의 행태에 대해 신뢰성 높은 정보를 지니고 있다는 장점이 있다. 본 연구에서는 클릭스트림 데이터 중 총체류시간(Total Time Spent), 방문횟수(Visit), 페이지뷰(Page View)를 웹사이트 이용도로 활용하였다. 설명변수로는 정보화마을 유무와 지방자치단체 전자정부추진정도 그리고 수도권 여부를 선정하여 살펴보았다. 지방자치단체 웹사이트는 지방자치시대에 지방행정의 서비스제공에 있어서 유용한 도구이자 구심점 그리고 대민 접점의 역할을 지닌다. 이러한 중복의 역할을 수행하는 지방자치단체 웹사이트의 이용에 대한 분석에 있어서 정보화마을이라는 지역적 특성을 지니고 지역의 네트워크 형성이 지니는 의미를 살펴보고자 하였다. 그리고 실제 공급측면에서의 전자정부추진정도로서의 정보화 투자가 지방자치단체 웹사이트 이용도에 얼마나 역할을 하고 있는지 살펴보고자 하였다. 이러한 연구를 통해 지방자치단체 웹사이트의 이용을 활성화하고 궁극적으로는 전자지방정부의 발전에 유익한 시사점을 도출하는 것이 이 연구의 목적이다. 본 연구를 수행한 결과 및 시사점은 다음과 같다. 실증연구를 수행한 결과, 먼저 수도권인가 비수도권인가 하는 점은 이용도에 큰 영향을 미치지 않는 것으로 나타났다. 전자정부추진정도를 나타내는 평가지표들 중에서는 정보화예산신규투자비율이 유의미하게 나타났는데, 즉, 정보화 업무에의 신규투자 예산을 늘리는 것이 지자체 웹사이트 이용도를 높인다는 사실을 확인할 수 있었다. 이는 단순히 기존 사업의 확장 뿐 아니라 항상 새로운 정보화 개선의 노력이 필요함을 의미한다. 이에 따라 지역정보화의 신규사업을 위한 비전의 형성과 관련 기관과의 협조를 통한 예산의 확보 노력이 필요할 것이다. 따라서 기관장 또는 CIO 등의 리더십에 따른 책임성 높은 행정운영이 담보되어야 할 것이다. 그리고 인구와 공무원수가 많을수록 그리고 평균연령이 높을수록 이용도가 높다는 것을 알 수 있었다. 마지막으로 정보화마을이 있는 기초지자체의 경우 지자체 웹사이트 이용도가 그렇지 않은 경우보다 더 높다는 사실을 확인할 수 있었다. 이러한 사실에서 볼 때, 고연령층이 저연령층보다 지자체 웹사이트 이용도가 높다는 사실은 지자체 웹사이트의 컨텐츠 및 서비스가 어떤 방향으로 제공되어야 하는지에 대해 유용한 정보가 될 수 있다. 즉, 단순히 웹사이트 개설에만 초점을 두는 것이 아니라 고연령층에게도 접근이 용이하도록 홈페이지의 운영이 필요함을 의미한다. 그리고 지속적인 정보화교육을 통해 좀 더 효율적인 정보화 성과가 나타날 수 있도록 해야 할 것이다. 그리고 정보화마을을 운영하는 경우 지자체 웹사이트 이용도가 높게 나타난다는 사실을 통해 정보화마을이 전자지방정부의 활성화에 기여함을 확인할 수 있다. 단순히 지금까지는 정보화마을 사업 자체에만 초점을 맞추고 실제 그 운영을 전자정부 측면에서는 살펴보는 연구가 존재하지 않았다. 그러나 지역정보화에 있어서 정보화마을은 지역네트워크의 중심으로 자리 잡고 있음을 확인하였고, 이를 토대로 본 연구에서는 정보화마을 운영이 전자지방정부의 행정서비스 제공 평가요소로서 고려될 수 있는 중요한 요인일 수 있음을 보여주는 것이라 하겠다. 앞으로 정보화마을에 초점을 둔다면 수요자 중심의 정보화마을 운영을 위한 서비스제공의 방향에 대한 준거가 될 수 있을 것이다. 이러한 연구 결과를 통해 지방자치단체의 웹사이트 이용에 영향을 미치는 요인들을 확인할 수 있으며, 앞으로 지방전자정부의 발전방향에 대한 단초를 제공할 수 있을 것으로 기대해 본다.This thesis empirically examines the actual extent of utilization of websites for local governments and discover the influencing factors. The reason why websites for local governments are significant is that play a roll as effective tools to offer public service. Moreover, they are contact points between the public and local e-government. The purpose of this study is to revitalize utilization of such websites and to present useful implications for the early settlement and development of e-government. This study was based on the analysis of clickstream data of 2006 collected from the panel. The panel consisted of the users of websites for 234 elementary local governments. The clickstream data are more objective compared to those from questionnaires since they contain accurate and detailed information about the habits of internet users. In addition, they gave consequence to this study compared to the previous studies because they were made out of user-oriented approach. This study assumed that Total Time Spent, Number of Visits and Page View reflect the extent of websites utilization. The descriptive variables are the existence of information village, the extent of how much the local entity focused on the e-government business. The implications of this study are as follows. First, the distance from the capital to the local entity was not such influential factor to the utilization of websites. As generally expected, the ratio of new investment for informatization of the local government was proven to be the meaningful valuation indices among those for measuring how much a local entity focuses on the e-government business. And, the population and the number of civil servants and the average age of the residents positively correlated with the utilization of local governments' websites. One of the remarkable results is that old people visit and use local governments' websites more than younger ones. This suggests that websites for the local governments should target the relatively old-aged people when they provide contents and electronic service through the websites. Moreover, the effect of information village on the utilization of local governments' websites means that the evaluation method of informatization of the local government should contains the information village. Through focusing the information village, more studies about development of local e-government can provide consumer oriented contents and services.Maste

절제 불가능한 담낭 선암 환자에서 고식적 항암화학요법 전후 종양 표지자의 변화와 예후의 관계

학위논문 (석사)-- 서울대학교 대학원 : 의학과 내과학전공, 2016. 2. 김용태.Background: The prognostic value of tumor markers such as CEA, CA 19-9 in gallbladder cancer are poorly understood. Previous studies used the spot measure of CEA, CA 19-9 as predictive factors but not its change during therapeutic measures. This study aimed to evaluate the relation between the change in serum CEA, CA 19-9 during palliative chemotherapy and survival in patients with unresectable gallbladder cancer. Methods: A total of 123 patients with unresectable gallbladder cancer pathologically confirmed were enrolled. Pre and post chemotherapy (2 cycles) CEA and CA 19-9 were used in analysis. The change in tumor markers were defined as the pre & post ratio of each variables. CEAchange = CEApost/CEApre, CA19-9change = CA199post/CA199pre, combined tumor marker as COMBchange = CEAchange × CA19-9change (= CEApost/CEApre*CA199post/CA199pre). Survival was compared using variable cutoffs of tumor marker change variables. Results: Patients with decreased tumor marker variables (CEA, CA19-9, CEA×CA19-9) had a better PFS and OS than those with increased tumor markers variables (PFS with 5.9 vs 2.3 months, 5.6 vs 2.2 months, 5.3 vs 2.1 months respectively, and OS with 11.4 vs 6.2 months, 9.6 vs 6.6 months, 8.9 vs 6.2 months respectively). The pre & post CA19-9 ratio had the most highest AUC value to predict 3 month progression and 1 year all-cause mortality. Patients with decreased CA19-9 value under 40% of the initial value had even better PFS (7.3 months) and OS (11.4 months) and patients with CA19-9 value increased over 200% of the initial value had an even worse PFS (2.2 months) and OS (6.5 months). Increased CA19-9 value during chemotherapy and initial CEA value over 4ng/mL were independent factors of progression with an HR of 2.34 (p<0.001) and HR of 1.67 (p=0.019) respectively, and were also independent factors of all-cause mortality with an HR of 1.57 (p=0.029) and HR of 2.21 (p<0.001) respectively Conclusion: CA 19-9 kinetics is a reliable factor predicting survival in patients with unresectable gallbladder cancer receiving palliative chemotherapy Keywords: Gallbladder cancer, CA 19-9, CEAIntroduction 1 Methods 3 Results 6 Discussion 10 References 13 국문초록 32Maste