고속 다중변수 세포기반 분석을 위한 코드화된 미세입자를 이용한 지역화된 바이러스 기반의 유전자 전달

학위논문 (박사)-- 서울대학교 대학원 : 전기·컴퓨터공학부, 2015. 8. 권성훈.In this dissertation, I develop an adenoviral vector-immobilised patch-type encoded microparticle for high-throughput, high-content cellular assays and name this encoded viral micropatch. This technology spatially confines the adenoviral gene delivery to only the cells under the micropatch by simply pipetting a heterogeneous mixture of the two-dimensional (2D) shape-coded viral micropatches on monolayer-cultured cells. Distinct clusters of transduced cells are then created in correspondence with the randomly positioned micropatches and the delivered gene into the cells within each cluster can be identified using the shape of the micropatch. For this purpose, shape-coded polymer microparticles are fabricated by photolithography, and highly localized gene delivery is achieved by specifically immobilizing adenoviral vectors on the microparticles. This unique feature allows high-throughput compound screening by virtue of multiplexing in a well of a standard microplate and creates no restriction for fluorescence-based assay formats with high-content imagers. To highlight the capabilities of this technology, I demonstrate a multiplex G-protein coupled receptor (GPCR) internalization assay that requires compound treatments followed by fluorescence-based target tracking at the sub-cellular level. First, I develop the maskless lithography system supporting an automated step-and-repeat operation for the fabrication of microparticles with various 2D graphical codes. Using this system, I explore new applications of the encoded microparticles and lithography technique such as anti-counterfeiting of drugs, parallel loading of small volume liquid for multiplexed bioassays, and conformal phosphor coating for white light-emitting diodes (LEDs). For the development of the encoded viral micropatch, various shape-coded microparticles are fabricated with carboxyl groups on the surfaces for specific immobilization of adenoviral vectors. The chemical functionalization is achieved by the incorporation of acrylic acid to photocurable polymer solution. Then, two adenoviral vector immobilization methods are developed with this shape-coded microparticle. The first method is to directly link the carboxyl groups on the microparticle and the primary amine groups on the surface proteins of adenoviral vectors using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) plus N-hydroxysulfosuccinimide (Sulfo-NHS) crosslinking reaction. The immobilization of adenoviral vectors in this approach is confirmed by an immunofluorescence test and a scanning electron microscope (SEM) observation. The second method utilizes an avidin-biotin interaction. In this approach, both the microparticles and adenoviral vectors are biotinylated using amine-activated and amine-reactive biotin reagents, respectively. Then, they are linked by the mediation of avidin. The immobilized adenoviral vectors are well observed using a SEM. The localized viral gene delivery of two types of the encoded viral micropatches is evaluated by transducing a human osteosarcoma cell line (U-2 OS) cultured in a standard 96-well microtiter plate. The first type of the encoded viral micropatch fabricated via EDC/Sulfo-NHS reaction shows low rate of the localization of gene delivery due to an escape of non-specifically bound adenoviral vectors. However, the second type of the encoded viral micropatch fabricated utilizing avidin-biotin interaction offers highly localized gene delivery. This is owing to the viral receptor-independent transduction of the biotinylated adenoviral vector, which is further supported by the transduction experiment of an adenovirus receptor-deficient cell line. Finally, I demonstrate a multiplexed GPCR internalization assay based on the localized gene delivery with the encoded viral micropatches. The development of high-throughput cell-based GPCR functional assays is very important for screening large compound libraries in the drug discovery process and ligand-induced receptor internalization assays have broad applicability to various GPCR subfamilies among several GPCR assay formats. However, high-content imaging is required for fluorescence-based intracellular measurement of receptor internalization. To address this issue, I fabricate three types of encoded viral micropatches with adenoviral vectors bearing green fluorescence protein (GFP)-tagged GPCR genes. Then, the responses of multiple GPCRs against one ligand treatment is acquired in one reaction site by achieving simultaneous expression of multiple GPCRs with the fabricated viral micropatches in a cell monolayer cultured in a well of a 96-well plate. High-content analysis of this micropatch-based multiplexed assay shows comparable results in the receptor internalization with the conventional singlet assay using free adenoviral vectors while reducing the number of pipetting actions.Abstract i Contents v List of Figures viii List of Tables xvii Chapter 1 Introduction 1 1.1 Cell-based Assays in Drug Discovery 4 1.2 Image-based High-content Screening 7 1.3 Cell Microarray for High-throughput Screening 10 1.4 Main Concept: Encoded Viral Micropatch 12 Chapter 2 Development of Encoded Viral Micropatch 15 2.1 Introduction 16 2.2 Fabrication of Encoded Microparticles 19 2.2.1 Maskless Lithography System 19 2.2.2 Shape-coded Microparticles for Encoded Viral Micropatch 32 2.3 Immobilization of Viral Vectors 37 2.3.1 Recombinant Adenoviral Vector 37 2.3.2 Direct Targeting of Viral Capsid via Carbodiimide Crosslinker (Type 1 Encoded Viral Micropatch) 39 2.3.3 Indirect Targeting of Biotin-tethered Viral Capsid via Avidin (Type 2 Encoded Viral Micropatch) 44 2.4 Conclusion 52 Chapter 3 Localized Viral Gene Delivery 53 3.1 Introduction 54 3.2 Localized Gene Delivery with Type 1 Encoded Viral Micropatch 57 3.3 Localized Gene Delivery with Type 2 Encoded Viral Micropatch 60 3.3.1 Evaluation of the Localized Gene Delivery 60 3.3.2 Consideration of the Localized Gene Delivery 65 3.3.3 Transduction of an Adenoviral Receptor-deficient Cell Line 67 3.3.4 Transduction Efficiency of the Encoded Viral Micropatch 69 3.4 Conclusion 74 Chapter 4 Multiplex GPCR Internalization Assay 75 4.1 G Protein-coupled Receptor (GPCR) 77 4.2 Materials for the Assay 79 4.2.1 GPCR Adenoviral Vectors 79 4.2.2 Ligands 79 4.2.3 Cell Culture 79 4.3 Conventional GPCR Internalization Assay 80 4.3.1 Assay Procedure 80 4.3.2 Assay Result 83 4.4 Multiplex GPCR Internalization Assay 85 4.4.1 Preparation of Encoded Viral Micropatches 85 4.4.2 Assay Procedure 85 4.4.3 Assay Result 87 4.5 Conclusion 91 Conclusion 92 Bibliography 94 국문 초록 103Docto

Law and the Body in Joseon Korea: Statecraft and the Negotiation of Ideology

Once considered almost exclusively to be the domain of legal scholars, Joseon dynasty criminal law is recently attracting increasing attention from social, political and intellectual historians of Korea. Despite often reaching opposing conclusions on the characteristics of Joseon legal culture, historians and legal scholars share a strong focus on the dominating role of Confucian ideology. While acknowledging the importance of Confucianism for Joseon statecraft, this paper argues that in actual statecraft and the application of the law, this ideology was negotiated with the perceived needs of the state. The focus of analysis is the relationship between the judicial process—investigation, interrogation and punishment—and cosmological, ideological and cultural notions related to the body. The purpose is to show the tension between the state need to maintain the system and uphold social order (as defined by the state) and the need for the state itself to adhere to the basic principles of the ideology that underpinned this system. Addressing the role of law and punishment in statecraft, the analysis is based on a theoretical framework that combines a conflict-based understanding of society with one that is consensus-based. While on the one hand the violation of notions related to the body was the purport of punishment when dealing with the most severe crimes against the state and its ideology, we can also see how such notions influenced the discourses on penal benevolence, torture and exhumation, whilst partly constituting the reason why some forms of torture were prohibited

Autologous fibrin glue in peripheral nerve regeneration in vivo

치의학과/석사[한글] 말초 신경에 결손부가 생긴 경우 기능을 회복하기 위한 가장 좋은 방법은 자가 신경을 이식하여 수복하는 것이다. 하지만 자가 신경 이식은 자가 신경의 채취를 위한 부가적인 수술과 이로 인한 공여 부위의 감각상실 등의 문제점이 있어서 이를 대체할 수 있는 방법들이 연구되어 왔다. 이들 방법 중 신경성장인자를 이용하는 것이 말초 신경의 재생에 도움이 되는 것으로 알려져 있다. 본 연구에서는 많은 성장인자들을 함유하고 있는 autologous fibrin glue의 효과를 알아보고자 하였다. 백서의 종아리신경에서 15 mm의 신경 결손부를 형성하고 정맥 도관으로 수복한 후 도관 내부에 autologous fibrin glue를 주입하였다. 대조군에는 도관 내부에 생리식염수를 주입하였다. 8주 후 조직형태학적인 검사와 전기생리검사를 통하여 신경 재생을 평가하였다. 실험군에서 기시 잠시가 대조군보다 빠르게 측정되었고 진폭은 대조군보다 약 2.5배 더 크게 측정되었다. 신경 섬유의 개수는 실험군에서 대조군보다 훨씬 많았고 평균 직경도 대조군보다 더 컸다. autologous fibrin glue를 주입한 실험군에서 더 우수한 신경 재생이 관찰되었다. 본 연구를 통하여 autologous fibrin glue가 함유하고 있는 풍부한 성장인자들이 말초신경의 재생을 효율적으로 촉진하는 것으로 사료된다. [영문]The activity of several growth factors on peripheral nerve regeneration has been reported. Autologous fibrin glue contains a large number of platelets, which release significant quantities of growth factors. In order to understand the role of autologous fibrin glue in peripheral nerve regeneration, a 15-mm rabbit peroneal nerve defect was repaired using a vein graft filled with the autologous fibrin glue. Axonal regeneration was examined using histological and electrophysiological methods. The extent of axonal regeneration was superior when treated with the autologous fibrin glue. Our data suggest that fibrin nets formed by fibrinogen, in combination with growth factors present in autologous fibrin glue, might effctively promote peripheral nerve regeneration in nerve defects.ope