Temperature and physical property control for polymerization reactors

학위논문(박사)--서울대학교 대학원 :화학공학과,1996.Docto

Construction of efficient expression vector system using cis-acting elements for Lactococcus lactis

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최윤재.Many approaches have been attempted to improve the heterologous protein in various bacteria. Specifically, the plasmid-based expression system has been used to achieve the recombinant protein production as an easy and useful tool to manipulate. There are three strategies to improve the expression level of recombinant protein. Such as 1) introduction of high copies plasmid-based expression vector system, 2) construction of gene multimerization cassette as a insert and ligation with the backbone vector, and 3) search of new strong promoter. However there are several limitations for these strategies in that it is hard to replicate the DNA, is too large to transformed which causes genetic instability, and is hard to predict promoter strength. In this study, I modified the promoter region and tested on the promoter strength. In addition, I tried to introduce another cis-acting elements such as a transcriptional terminator and RBS (ribosome binding site) to improve the expression of recombinant protein. Lactococcus lactis subsp. lactis IL1403 is widely used in the dairy and animal industries, and it is also studied for a live oral vaccine product to elicit mucosal immune response. The translational elongation factor Tu (tuf) gene is a house-keeping gene, and tuf promoter is characterized as a strong promoter in IL1403. In this study, tuf promoter was modified to test the efficiency of protein expression using the luciferase gene as a reporter. Firstly two terminators, TrrnB and TpepN were tested for the luciferase gene expression efficiency. TpepN terminator showed better performance in luciferase expression. Next, series of tuf promoter modification were attempted. In bacteria, RNA polymerases and several sigma factors recognized and recruited approximately -35 and -10 region upstream from the transcription start site. The core region including -35 and -10 hexamers in tuf promoter (119 bp) was amplified and series of modified tuf promoters were constructed using PCR with partial complementary reverse primer. There PCR products (#1) were cloned into the promoterless pIL.Ptuf.Luc(X) vector. Luciferase activity of t2, t4, t6 and t7 were higher than control tuf promoter. Especially t2 and t4 showed better performance, thus selected for next experiment. It is well known that the sequence between RBS and start codon (ATG) are important for protein translation efficiency. Thus, I modified original sequence of this region, 'CATTTTTCAT' to 'AATTTTTAAA' to give more AT-rich. This modification was combined with selected modified tuf promoter to give a series of new tuf promoter cassette. The transformed IL1403s containing modified promoter (#2) were assayed for luciferase activity. Derivative of t2 and t4, t2-1 and t4-1 showed better performance. Combined all the modified clones, luciferase activity was compared. t4-1 showed much higher activity compared to the t4, indicating the sequence between RBS and start codon is important for protein expression. To confirm this results, luciferase expression was analyzed on SDS-PAGE and western blot assay. Luciferase bands (61 kDa) was not detectable in SDS-PAGE, but in western blot, clones with t2, t4, t4-1 showed stronger signal compared to original tuf one. In conclusion, this study revealed that introduction of modified strong promoter and additional cis-acting elements can improve the protein expression in IL1403. And this strategy has a prospect to improve recombinant protein expression. Since pIL252 is a low copy plasmid-based expression system, high copies-based plasmids are needed to increase recombinant protein expression.Summary I Contents IV List of Tables and Figures VII List of Abbreviations IⅩ I. Introduction 1 II. Review of Literature 3 1. Lactic Acid Bacteria (LAB) 3 1) Lactic Acid Bacteria 3 2) LAB as probiotics 4 3) Lactococcus lactis 7 2. Recombinant protein expression 8 1) Plasmid-based expression system 8 2) Recombinant protein expression strategy 8 3. Cis-acting elements 9 1) Promoter 9 2) Cis-acting elements 10 4. The tuf gene 11 III. Materials and Methods 12 1. Bacterial cultivation 12 1) Culture medium 12 2) Cultivation and harvest of bacterial cells 12 2. Transformation of bacteria 13 1) Preparation of IL1403 competent cells 13 2) Transformation of bacterial cells 13 3. DNA works 14 1) Plasmid 14 2) Preparation of plasmid DNA 14 3) Enzyme treatment 17 4) PCR reaction 17 5) PCR purification 17 6) Analysis of nucleotide sequences 18 4. Luciferase assay 20 1) Growth phase-dependent luciferase expression 20 2) Detection of chemiluminescence 20 5. Protein works 21 1) Protein extraction from LAB cells 21 2) Quantification of proteins 21 3) SDS-PAGE and western blot assay 21 4) Intensity measurement of protein bands 22 6. In vitro characterization 23 1) Growth of LAB cells 23 2) pH measurement of LAB cultures 23 IV. Results and Discussion 24 1. Introduction of reporter gene with terminator 24 1) Cloning of luciferase gene 24 2) Vector construction 25 2. Validation of reporter gene and terminator 26 1) Growth characteristics 26 2) Luciferase assay 27 3. Modification of tuf promoter (#1) 28 1) Amplification of short fragments 28 2) Construction of repeated fragments 29 3) Elongation of ribosome binding site (RBS) and enzyme sites 30 4) Introduction of modified promoter (#1) 31 4. Modified promoter (#1) activity assay 35 1) Growth characteristics (#1) 35 2) Luciferase assay (#1) 35 5. Construction of remodified promoter (#2) 37 1) Core promoter cassette preparation from modified promoter (#1) 37 2) Elongation of RBS and modified downstream sequences 38 6. Remodified promoter (#2) activity assay 41 1) Luciferase assay (#2) 41 2) Final luciferase assay 42 7. Confirmation of Protein expression 44 1) SDS-PAGE and western blot assay 44 V. Literature Cited 46 VI. Summary in Korean 58 VII. Acknowledgement 61Maste

다공성물질에서의 자연대류현상에 대한 열역학적 국소평형상태 가정의 고찰

학위논문(석사)--서울대학교 대학원 :기계공학과,2000.Maste

(The) study of the actual condition of visiting nurse projects in public health centers in Seoul

지역사회 간호학과/석사[한글] 방문간호사업의 기본 정책방향은 국민 누구나 포괄적인 보건의료의 형평성과 동등한 접근도를 유지하여 질적인 삶을 유지하게 하며 복지사회를 실현하고자 함이다. 본 연구는 보건소 방문간호사업의 조직, 인력, 예산, 준비도, 업무를 살펴보고 이를 기초로 효율적인 방문간호사업을 제시하고자 시도하였다. 연구방법으로는 방문간호사업의 방향설정에 관한 문헌고찰과 국내 기관의 통계자료를 사용하였고 또한 서울시 25개 보건소 방문간호사업 담당자 221명을 모집단으로 하여 우편을 이용하여 설문조사하였다. 자료수집 기간은 1996년 11월 2일 부터 1996년 11월 16일까지였으며 분석에 사용된 연구대상자 수는 23개 보건소 184명이었다. 본 연구의 주요 결과는 다음과 같다. 1. 조직 현황 서울시는 총 25개의 보건소가 있으며 크게 나누어 지역보건과를 두고 독립적으로 방문간호사업을 담당하는 8개구 보건소와 그렇지 않은 17개구 보건소로 나누어 지며 현존 조직으로는 방문간호사업을 수행하는데 한계가 있다고 할 수 있다. 2. 인력현황 서울시 보건소의 현원은 95년 현재 총 2,020명이며 이중 간호직이 647명으로 32%를 차지하고 있으나 그 직급은 상대적으로 낮음을 알 수 있다. 본 논문에서 조사한 184명을 대상으로 응답자의 일반적 특성을 살펴 보면 보건관리자는평균 46.5세, 방문간호사는 평균 35.7세로 나타났으며 교육수준은 전문대 졸업자가 가장 많은 부분을 차지하고 있었다. 특별교육과정중 CPHN과 가정간호사 교육은 대부분이 이수 하지 않은 것으로 나타났다. 이들의 직급은 보건관리자는 6급, 방문간호사는 8급이 가장 많은 것으로 조사되었으며 5급 이상 응답자 6명 중 2명만 간호직으로 조사되어 의사결정권이 미약함을 알 수 있었다. 현부서 근무기간은 방문간호사의 경우 평균 1.5년으로 이동이 잦음을 알 수 있었으며 임상경력은 평균 5년 이상으로 질적 서비스의 제공을 위해 바람직한 구성이라고 볼 수 있다. 3. 예산 현황 방문간호사의 예산에 대한 만족도는 전반적으로 낮은 것으로 나타났으며 차량지원 및 시설 및 장비에 대해서도 불만족하다는 응답이 높았다. 또한 예산편성에서 보건관리자는 신축성 있게 예산 편성이 이루어진다고 응답한 반면 방문간호사는 예산 편성이 신축적이 지 못하다고 응답하여 상반된 견해를 보였다. 4. 방문간호사업에 대한 방문간호사의 준비도 방문간호사업에 대한 준비도에서는 방문간호사업에 대한 교육의 기회가 보다 폭 넓게 이루어 져야 함을 알 수 있었으며 이들이 원하는 교육방법으로는 보건관리자는 세미나와 단기연수를, 방문간호사는 단기연수와 정규대학 /대학원을 선호하는 것으로 나타났다. 5. 업무현황 서울특별시의 방문간호사업은 생활보호대상자와 저소득 주민을 대상으로 환자관리, 건강관리, 연계처리, 순회진료, 보건교육을 하고 있었다. 방문간호사업의 업무 현황을 살펴보면 비교적 잘 되고 있는 것으로 나타났으며 특히 환자에 대한 추후관리와 주민을 대상으로 하는 특수사업에서 잘되고 있다고 응답하였다. 그러나 방문간호사업에 대한 주민 참여, 타 기관과의 연계망 형성, 전산화에서는 원활하게 이루어지고 있지 않다고 응답하여 개선이 요구되었다. 6. 방문간호사업에 영향을 미치는 요인 1) 지역보건과의 유무와 타 업무병행과의 관련성 지역보건과는 서울시가 방문간호사업을 실시하기 위한 일환으로 조직 개편하여 신설된 과로서 지역보건과가 없는 곳에서 타 업무를 더 많이 병행하고 있는것으로 나타났다. 2) 보건관리자의 관심도와 방문간호사업과의 관련성 방문간호사업에 대한 보건관리자의 관심도는 높은 것으로 나타났다. 또한 보건관리자의 관심도가 높을수록 예산에 대한 만족여부, 방문간호사업에 대한 평가, 주민을 대상으로 하는 특수사업에서 통계학적으로 유의하게 관련성이 높았다. 이상과 같은 결과에 기초하여 다음과 같이 제언한다. 첫째, 방문간호사업은 보건인력의 적극적인 지역사회 활동을 전제로 하는 것이므로 기존 인력을 개발하여 질적인 사업을 수행하게 하여야 할 것이며 근무하면서 자격을 취득할수 있는 제도적 보완이 정책에서 고려되어야 할 것이다. 둘째, 지역사회 기초진단, 관련부서와의 협조 및 타 기관과의 연계망 형성, 타 전문직종과의 협력, 환자의 조기발견 및 대상자에 대한 추후관리, 주민의 방문간호사업 참여 및방문간호사업에 대한 평가, 지역·인구·면적을 고려한 대상인구 분담 등의 방문간호사업 업무를 개선하여야 할 것이다. 셋째, 지역보건과를 신설하여야 할 것이며 관리자의 관심도가 방문간호사업에 중요하게영향을 미치므로 담당 과장급 이상의 관리자는 동일 직종에 의해서 업무 지시·감독을 받아야 할 것이다. 이 연구에서 제시된 결과들에 비추어 방문간호사업을 효율적으로 하기 위해서는 전문인력의 양성, 조직의 재개편, 예산의 확보를 통한 시설 및 장비의 보완, 업무지침개발 등의준비가 필요함을 알 수 있었으며 앞으로 이 연구는 방문간호사업의 적합한 방안을 모색하는데 참고자료로 제시되었으면 한다. [영문] This study aims to comprehensively grasp the trend of National health policy on the exploitation of medical and health resources and to find out what implications the current budget policy and its effect would give for setting up and enforcing the health policy hereafter. For these purposes, I did a diachronic analysis of the budget under the jurisdiction of the Health and Welfare Affairs, which has been invested with the aim of increasing the number of sickbed of private hospitals as well as of strengthening the function of both private hospitals and public health organizations for the recent five years. And I, also, compared the change in the sickbed number of each of medical facilities and its sickbed occupancy rate with the increased sickbed number through the Special financial resources from the national treasury loans and those from the farming and fishing villagers' tax. The results are as follows: In the first place, the increasing rate of the budget of general account under the jurisdiction of the Health and Welfare Affairs(which is 19.5% in 1996) is higher than that of National budget, and the component ratio of the Special financial resources from the national treasury loans and the farming and fishing villagers' tax to National budget has risen steadily every year. According to the annual change in National financial support for the purpose of exploiting medical and health resources, the budget of general account to improve the facilities of public health organizations and to strengthen their function was abolished in 1994, when the Special financial resources from the farming and fishing villagers' tax were newly- set up. But it was recompiled again with the view of supporting the Health Centers in some urban areas in 1996. Also, in 1996 and 1997, the budget for building private dementia hospitals was drawn up. The budget of the Special financial resources of the national treasury loans was made up mainly to increase the sickbeds of the private hospitals and in 1997, the another budget items were newly drawn up for the construction and extension of special purpose hospitals and the conversion of mental nursing homes into mental nursing hospitals. The 45.9% of the budget of the Special financial resources from the farming and fishing villagers' tax was assigned to public health organizations and the rest, 54.1%, to private hospitals. Secondly, in all hospitals, except such as tuberculous, leprous, and dental hospital, the rate of the sickbed number increased by financing to the total increased sickbed number for the last three years is 80.5%. The 31.1% of the former was given out to six large cities and the 33.6% of it was alloted to Seoul, Inchon and Kyoungki Area and Southern Kyoungsang Area. And the annual change in the allotment of the total sickbed number to each of cities and counties shows that there was more serious regional unbalance in 1996(urban area: 93.3%; county area: 6.7%) than in 1993. Moreover, the 82.3% of the sickbed number increased by financing was in urban area and the 17.7% of it was in county area. Finally, in hospitals and general hospitals, the rate of the sickbed number increased by financing to the increased sickbed number for the last three years in each of cities and Provinces is as follows: 445.7% in Southern Cheonla Province; 321.0% in Northern Kyoungsang Province; 199.7% in Kwangju Extensive City; 196.8% in Northern Chungchong Province. Although they are all over 100%, the 1996 sickbed occupancy rate of these areas was lower than the average. The annual average rate of the whole country and six large cities has diminished. In Southern Kyoungsang Province, Kyoungki Province and Pusan Extensive City, the component rate of the sickbed number of mental hospitals increased by financing is higher than any other areas. And also the occupancy rate is relatively high in comparison with other hospitals. In Chinese medicine hospitals, the 55.3% of the sickbeds increased by financing was alloted to six large cities and the 43.5% was to Seoul, Inchon and Kyoungki Area. But the 1996 sickbed occupancy rate of this Area is lower than the national average rate. The average sickbed occupancy rate of Chinese medicine hospitals, compared with that of other kind of hospitals, was reduced to a very high degree. As we can see in the above results, the project of the Special financial resources from national treasury loans and from the farming and fishing villagers' tax (which was originally intended to achieve the equity of medical services through increasing the sickbed number of private hospitals and reinforcing the function of both private hospitals and public health organizations), in effect, has resulted in the aggravation of the regional maldistribution primarily because the project increased the sickbed without considering its efficiency. Conclusively, to improve the equity of the sickbed number of private hospitals and the efficiency in running the sickbeds of private hospitals, I believe that it's necessary to review the current financial policy critically and to establish the directionality of the future financial policy.restrictio

Vascularized bone grafts based on gelatin-heparin conjugated cryogels

학위논문 (석사)-- 서울대학교 대학원 : 공과대학 화학생물공학부, 2018. 2. Nathaniel Hwang.A cryogel is a one of the scaffolds which have high porosity with interconnected macropores providing cell compatible microenvironment. In addition, cryogels can be utilized in minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a vascular endothelial growth factor (VEGF) and fibroblasts carrier for the treatment of ischemic hind limb mouse model. Our gelatin/heparin cryogel showed a mesh-like network dependent on the gelatin concentration, having a different mechanical properties in swelling ratio, interconnected porosity and elasticity. In addition, integrating heparin into cryogel allowed controlled release of VEGF for a long period, leading to a therapeutic effect demonstrated in in vitro endothelial cell angiogenesis study. Also, its sponge-like properties enabled cryogels to be applied as an injectable system with desirable maintenance of cells and proteins after injection. And, as gelatin is one of the natural polymers that have a high biocompatibility, gelatin/heparin cryogel showed high cell loading efficiency and viability without any other treatment. As a result, overall in this study, we optimized the combination of the concentration of gelatin and heparin in aspect of sustainable release of VEGF and injectability. Finally, optimized gelatin/heparin cryogel integrated with VEGF and NIH-3T3 fibroblasts was applied to in vivo ischemic hind limb model and demonstrated its angiogenic potential by improving neovascularization into the cryogel. As utilizing our gelatin/heparin cryogels with human mesenchymal stem cell (hMSCs), we expect to see the therapeutic effect for bone remodeling.1. Introduction 1 2. Experimental section 3 2.1 Preparation of gelatin-heparin cryogels 3 2.2 Characterization of gelatin-heparin cryogels 4 2.3 Rheology test 5 2.4 Heparin detection via alcian blue assay 6 2.5 Synthesis of RITC-heparin conjugated cryogel 6 2.6 Heparin mediated protein release kinetics 7 2.7 HUVEC migration and tube formation assay 7 2.8 Injecatability test 8 2.9 Preparation of cell seeded scaffolds 9 2.10 PicoGreen and live/dead assay 9 2.11 In vivo ischemic hinlimb mouse model 10 2.12 OCT embedding 10 2.13 Histology & immunostaining 11 3. Results and discussion 13 3.1 Fabrication of gelatin/heparin cryogel 13 3.2 Characterization of gelatin/heparin cryogels 14 3.3 Rheological properties of gelatin/heparin cryogels 16 3.4 Heparin conjugated microarchitecture of gelatin/heparin cryogels according to the heparin concentration 17 3.5 Heparin mediated VEGF release kinetics of gelatin/heparin cryogels according to the heparin concentration 18 3.6 In vitro angiogenic response of HUVEC on VEGF dependent on heparin concentration of gelatin/heparin cryogels 20 3.7 Injectability of gelatin/heparin cryogels 21 3.8 Injectable gelatin/heparin cryogel as an effective carrier of NIH-3T3 fibroblasts 23 3.9 Effect of gelatin/heparin cryogels containing VEGF or/and NIH-3T3 fibroblasts on in vivo ischemic hind limb mouse model for neovascularization 24 4. Conclusion 28 REFERENCES 39 요약 (국문초록) 47Maste