Studies on prediction of SPS1 gene function and microRNA transcriptional regulatory element by bioinformatical analysis

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 생물정보학전공, 2012. 8. 이병재.Bioinformatics is an important area to analyze the massive biological data and predict the biological meanings using computational and statistical methods. Since the massive and highly qualified data have been accumulated by development of microarray technology, researches for finding biological meanings through predicting gene functions and transcriptional regulatory elements by using bioinformatical approaches are actively progressed. In these studies, we show the predicted and confirmed results for gene function and transcriptional regulatory element through two examples, selenophosphate synthetase 1 (SPS1) and microRNA genes. For example predicting gene function, we used SPS1 which functions are unknown yet. There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis. The function of SPS1, however, has not been elucidated. Using microarray data from obtained SPS1 knockdown, differentially expressed genes were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage (day 3). Interestingly, genes related to defense and amino acid metabolism were affected at the later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by SPS1 knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by SPS1 knockdown and the formation of megamitochondria, which is the major phenotypic change observed by SPS1 knockdown. These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities. For example for predicting transcriptional regulatory elements, we selected miRNA genes. miRNAs are important post-transcriptional regulators of various biological processes. Although our knowledge of miRNA expression and regulation has increased considerably in recent years, the regulatory elements for miRNA gene expression, especially for intergenic miRNAs, are not fully understood. We identified the differentially methylated regions (DMRs) occurring 1000 bp upstream from all miRNAs in human neuroglioma cells using microarrays and discovered a unique sequence motif C[N]6CT. This motif was preferentially located within 400 bp or from 800–1000 bp upstream of the intergenic miRNA start, corresponding to the highly methylated region. Interestingly, treatment of cells with a methyl transferase inhibitor (5-aza-2-deoxycytidine, DAC) significantly increased expression of miRNA genes with a high frequency of the C[N]6CT motif in DMRs. Statistical analysis showed that the frequency of the C[N]6CT motif in DMRs is highly correlated with intergenic miRNA gene expression, suggesting that C[N]6CT motifs associated with DNA methylation regions play a role as regulatory elements for intergenic miRNA gene expression.ABSTRACT ..................................................................................................... i TABLE OF CONTENTS ............................................................................... iv LIST OF FIGURES ....................................................................................... viii LIST OF TABLES ....................................................................................... x CHAPTER 1. LITERATURE REVIEW ....................................... 1 1. BIOINFORMATICAL APPROACHES FOR EXPRESSION DATA 2 1.1. DNA microarray technology ..................................................................... 2 1.2. Applications of microarray ........................................................................ 5 1.3. Microarray data analysis ........................................................................... 6 1.3.1. Standardization of microarray data ................................................. 6 1.3.2. Pre-processing of microarray data .................................................. 8 1.3.3. Normalization ................................................................................. 8 1.3.4. Statistical methods ........................................................................... 11 1.4. Cluster analysis ........................................................................................ 15 1.4.1. Hierarchical cluster ......................................................................... 15 1.4.2. Non-hierarchical cluster .................................................................. 16 1.4.2.1. k-means clustering ............................................................. 18 1.4.2.2. Self-organizing maps (SOM) clustering ............................ 18 1.5. Gene Ontology (GO) analysis .................................................................. 21 1.5.1. Foundation of GO ........................................................................... 21 1.5.2. Three categories of GO .................................................................... 21 1.5.3. Application of GO for functional genomics .................................... 24 2. SELENIUM BIOLOGY .......................................................................... 26 2.1. Selenium and human health ..................................................................... 26 2.2. Selenium metabolism ............................................................................... 29 2.3. Selenoproteins biosynthesis ..................................................................... 29 2.3.1. Mechanism of selenocysteine biosynthesis .................................... 33 2.3.2. Incorporation of selenocysteine into protein .................................. 35 2.3.3. Components of selenocysteine biosynthesis .................................. 37 2.4. Selenophosphate synthetase (SPS) ........................................................... 39 2.4.1. Structural characteristic of eukaryotic SPS .................................... 40 2.4.2. Functional characteristic of eukaryotic SPS .................................... 42 3. VITAMIN B6 ............................................................................................... 45 3.1. Physiological roles of vitamin B6 ............................................................. 45 3.2. Vitamin B6 biosynthesis ............................................................................ 47 4. MICRORNA ............................................................................................... 51 4.1. Biogenesis of microRNA ........................................................................ 51 4.2. Functions and regulation of microRNA ................................................... 54 4.2.1. MicroRNA-mediated gene regulation ............................................ 54 4.2.2. Epigenetic regulation of microRNA ............................................... 55 4.3. Promoters and microRNA ........................................................................ 56 4.3.1. MicroRNA transcription .................................................................. 56 4.3.2. Promoter regions ............................................................................. 56 4.3.3. Transcription factors ........................................................................ 59 5. EPIGENETICS ........................................................................................... 62 5.1. DNA methylation ..................................................................................... 62 5.2. Histone modification ................................................................................ 64 CHAPTER 2. DROSOHPILA SELENOPHOSPHATE SYNTHETASE 1 REGULATES VITAMIN B6 METABOLISM: PREDICTION AND CONFIRMATION ...................................... 68 1. INTRODUCTION ...................................................................................... 69 2. MATERIALS AND METHODS ............................................................. 73 2.1. Regents and other material ....................................................................... 73 2.2. SL2 cell culture and RNA interference ..................................................... 73 2.3. Microarray experiment ............................................................................. 73 2.4. Microarray data processing ...................................................................... 75 2.5. Temporal clustering .................................................................................. 77 2.6. Gene ontology analysis ............................................................................ 78 2.7. RT-PCR and quantitative real time RT-PCR ........................................... 78 2.8. Measurement of intracellular PLP concentration .................................... 79 2.9. Mitochondrial staining and confocal microscopy .................................... 80 3. RESULTS ..................................................................................................... 81 3.1. Identification of differentially expressed genes ....................................... 81 3.2. Functional distribution of differentially expressed genes ....................... 90 3.3. Construction the gene sets by temporal clustering ................................... 92 3.3.1. Clustering DEGs by SOM algorithm ............................................ 92 3.3.2. Functional distribution of six clusters ............................................ 92 3.3.3. Construction of three gene sets for GO analysis ............................. 95 3.4. Identification of statistically overrepresented biological processes ......... 96 3.5. Validation of expression by quantitative PCR ......................................... 100 3.6. Intracellular pyridoxal phosphate level was decreased by SPS1 knockdown 104 3.7. Inhibition of PLP biosynthesis and SPS1 knockdown showed similar expression patterns ................................................................................... 107 3.8. The reduction of intracellular PLP level inhibits cell growth and induces megamitochondrial formation .................................................................. 109 4. DISCUSSION .............................................................................................. 112 CHAPTER 3. IDENTIFICATION OF METHYLATION-DEPENDENT REGULATORY ELEMENTS FOR INTERGENIC MICRORNAS IN HUMAN H4 CELL ....... 118 1. INTRODUCTION ...................................................................................... 119 2. MATERIALS AND METHODS ............................................................. 122 2.1. Cell lines and culture ............................................................................... 122 2.2. Identification of miRNAs from sequence and annotation data ............... 122 2.3. Probe design ............................................................................................. 122 2.4. Microarray experiment ............................................................................ 124 2.5. Microarray data analysis .......................................................................... 124 2.6. Distribution analysis ................................................................................ 126 2.6.1. Predicted transcription factor binding sites (TFBSs) ...................... 126 2.6.2. Predicted transcription start sites (TSSs) ........................................ 127 2.6.3. Differentially methylated regions (DMRs) ..................................... 127 2.7. Motif analysis .......................................................................................... 127 2.7.1. Multiple alignments of DMRs and clustering ................................. 127 2.7.2. Identification of statistically significant motifs .............................. 128 2.8. Reverse transcription PCR reaction ......................................................... 128 2.9. Quantitative real-time PCR ...................................................................... 129 2.10. miRNA targets prediction ...................................................................... 129 2.11. Gene ontology analysis .......................................................................... 129 2.12. Analysis of bisulfite sequencing data ................................................... 130 3. RESULTS ..................................................................................................... 131 3.1. Identification of DMRs ............................................................................ 131 3.2. Distribution of DMRs, TFBSs and TSSs ................................................. 131 3.3. Clustering DMRs using phylogenetic method ......................................... 141 3.4. Prediction of sequence motifs from DMPs .............................................. 141 3.5. Effect of demethylation on intergenic miRNA expression ...................... 149 3.6. Correlations between the C[N]6CT motif in DMRs and intergenic miRNA expression ............................................................................................... 152 4. DISCUSSION .............................................................................................. 154 CHAPTER 4. CONCLUSIONS AND FUTHER REMARKS 157 1. CONCLUSTIONS .................................................................................... 158 2. FUTHER REMARKS .............................................................................. 159 REFERENCES ................................................................................................ 160 ABSTRACT IN KOREAN ............................................................................ 181Docto

All-optical full-adder using photonic crystal mode junction structure

학위논문 (석사)-- 서울대학교 대학원 : 전기. 컴퓨터공학부, 2011.8. 박남규.Maste

A study on dualism of the urban administration in Korea under the rule of Japanese imperialism

이 글은 식민지 근대화론에 대한 비판적 검토를 도시행정 차원에서 시도하고자 한다. 수탈론과 근대화론으로 대비되는 기존의 논의구도를 탈피하기 위해, 사회현상을 인간행위의 의도된 결과와 의도되지 않은 결과로 대별하여 분석하는 방법을 도입하였다. 부제(府制)라는 도시행정구역과 도시계획, 그리고 도시지역에 실시된 지방자치제도를 중심적으로 살펴본 결과, 일제 강점기의 도시행정제도가 근대적인 요소를 가지고 있었던 점은 인정되지만, 도시 공간이 민족 차별적으로 분화되고 대표성이 차별적으로 나타나는 식민지적 특징이 드러났다. 근대적인 도시행정제도로부터 대다수의 한국인을 소외시킨 것이 일제가 의도한 결과였다는 점에서 한국의 근대적 발전에 일제 통치가 기여했다는 주장은 반박되어야 한다고 본다. This article is to review the urban administration within the framework of criticizing the theory of colonial modernization. The new method is introduced to overcome the existing controversy of the theory. It makes social phenomena divided the intended results and the unintended results. Looking over urban administrative district, city planning, local autonomy institutions in urban area, it is admitted that there is some modern aspects in the urban administrative institution under the rule of Japanese imperialism. But the distinction of colonialism is showed. For example, urban space was divided with national discrimination and representative institution was not constructed in favor of the Korean. Because of these facts, it should be strongly disputed that the rule of Japanese imperialism contributed to modernization of Korea.본 논문은 한국학술진흥재단의 지원에 의한 것임(KRF-2004-073-AS2006

이온플레이팅법을 이용하여 제조한 (Ti1-xAlx)N 박막의 특성 및 Ti 중간층이 접착력에 미치는 영향

학위논문(석사)--서울대학교 대학원 :금속공학과,1997.Maste

Microwave field enhancement by deep subwavelength metallic gaps down to five nanometers

학위논문(박사)--서울대학교 대학원 :자연과학대학 물리·천문학부,2017. 2. 김대식.In this dissertation, I describe experimental demonstrations of strong field enhancements in microwave regime achieved by deep subwavelength metallic gaps down to 5 nanometers. I built a Ku band (12~18 GHz) free-space setup to measure transmittances and electric field enhancement factors of the samples. I prepared λ/400-width slot antennas by punching aluminum foils with a razor blade and the hand-made antennas exhibited good agreements to the previous studies of terahertz nanoantennas which have a similar aspect ratio of the incident wavelength and the widths of slots. To investigate deeper subwavelength regime, λ/10,000,000-width nanogaps were fabricated by high-throughput atomic layer lithography. I built three open-ended waveguide pair setups to cover Ku, K (18~26.5 GHz), and Ka (26.5~40 GHz) band spectra and observed giant electric field enhancement factors up to 5,000 with the nanogaps. I also performed terahertz time-domain spectroscopy with the same sample and confirmed a convergence to the microwave range. As a potential application of subwavelength microwave optics, I exhibited a λ/2000-width microwave switch based on insulator-metal transition. The aforementioned researches would open up a way to enhance nonlinearities and detection sensitivities of microwave and millimeter-wave optics applications and enable non-invasive molecule trappings and designed fluidic controls by light.Chapter 1. Introduction 1 Chapter 2. Preparation of subwavelength gaps 4 2.1 Perforation by razor blade 5 2.2 Photolithography 8 2.3 Focused ion beam 12 2.4 Atomic layer lithography 14 Chapter 3. Experimental setups 17 3.1 Horn antenna pair 18 3.2 Open-ended waveguide pair 21 3.3 Terahertz time-domain spectroscopy 24 Chapter 4. Microwave field enhancement by λ/400-width hand-made gaps 26 4.1 Anomalous band formation in antenna arrays 27 4.2 Selective enhanced transmittances of asymmetric antenna pairs 31 4.3 Effects of fabrication errors 33 4.4 Summary 35 Chapter 5. Microwave field enhancement by λ/10,000,000-width nanogaps 36 5.1 Field enhancement by λ/2,000-width gaps 37 5.1 Details on the nanogap sample 39 5.2 Microwave transmission measurements 42 5.3 Comparison with terahertz time-domain spectroscopy 44 5.4 Summary 50 Chapter 6. Conclusions 51 Bibliography 54 요약 (국문초록) 65 List of publications 66 Conferences 67Docto

(A) study on the relationship between stressful life events and statement of health in official workers

간호학교육/석사[한글] 현대인은 급격한 사회 경제적 발전으로 여러가지 상황에 예기치 못한 생활변화로 크건 작건 간에 일상생활에서 스트레스를 경험하게 되며, 최근에는 스트레스와 관련된 질병이 점차 증가되는 추세에 있다. 특히 사무직 근로자들의 업무상황에서 경험하는 스트레스는 피할 수 없는 현상으로 어느정도의 스트레스는 역할 수행에 도움을 주고 개인의 성장과, 생산성 증가를 가져오지만 스트레스가 감당하기 어려운 정도이거나 그 상태가 오래 지속되면 부적응 상태가 되어 신체적, 정신적, 정서적으로 탈진상태에 빠지에 된다. 이러한 소진을 경험하는 사무직 근로자들은 부정적인 자아개념 및 직업적 태도를 갖게 되며, 자신의 업무에 무관심해지고, 불만족하여 실수를 저지를 수 있어 결국 개인의 손실, 기업의 손실 뿐만 아니라 국가적인 손실을 초래하게 된다. 그러므로 본 연구는 사무직 근로자들의 건강상태에 영향을 미칠 수 있는 생활사건 스트레스 인지량을 간호중재의 근거로 삼고자 시도되었다. 연구대상은 서울시내 강남구에 소재하는 D그룹 사무직 근로자 중 여성을 제외한 30세 이상 60세 미만의 남자 사무직 근로자 150명을 대상으로 하였다. 자료수집 기간은 1992년 3월 23일부터 4월10일 까지 17일 동안 이었다. 연구도구는 이(1984)가 개발한 생활사건 스트레스 측정도구, 남(1995)의 간이 건강조사표 및 인구사회학적 특성으로 구성된 설문지를 사용하였으며 수집된 자료는 코딩 카드에 부호화 한 후 SPSS를 이용하여 전산처리하였다. 자료분석으로는 대상자의 인구사회학적 특성 및 생활사건을 경험한 대상자수를 실수와 백분율로 산출하였고, 대상자가 경험한 생활사건 스트레스 경험향은 각 생활사건에 대한 스트레스 인지량과 사건 경험횟수를 곱한 값으로 산출하였다. 인구사회학적 특성에 따른 생활사건 스트레스경험량과 건강상태의 차이는 t-test와 일원변량분석(ANOVA)를 이용하여 검증하였다. 연구결과를 요약하면 다음과 같다. 1) 대상자가 인지한 각 생활사건에 대한 스트레스 인지량과 평균값은 가능점수 0점-100점 범위에서 최고 84.15(자녀의 죽음), 최저 33.57(신앙생활의 시작)으로 나타났다. 2) 대상자가 경험한 스트레스양은 "가족의 구속"이 평균 630점으록 가장 높았고, "자녀의 학업중단" 508점, "불화로 인한 부부간의 별거" 325점 순으로 높았으며 전혀 경험하지 않은 항목은 7개 항목으로 이는 한국인에게는 맞지 않는 것으로 생각된다. 3) 대상자의 건강상태 호소율은 0점-57점 범위이고 가장 높은 빈도를 보인 항목은 "눈의 피로가 오거나 또는 충혈될 때가 자주 있습니까?"로 113명이 호소하였으며 가장 낮은 빈도를 나타낸 항목은 "갑자기 소리가 나면 몹시 놀라 벌떡 일어나거나 몸이 벌벌 떨립니까?"로 5명이었다. 4) 대상자의 생활사건 스트레스 경험량과 건강상태의 상관관계는 순상관관계(r=.2215, P<.01)를 나타내어 스트레스가 많을 수록 건강이 나쁜 것으로 나타났다. 5) 인구사회학적 특성에 따른 생활사건 스트레스 경험량의 차이검증에서는 결혼상태(t=3.00, P<.01), 수입(t=-2.17, P<.05)에서 통계적으로 유의한 차이가 나타났는데, 기혼자가 미혼자보다 월등히 높은 스트레스량을 나타냈고 수입이 많은 집단이 적은 집단보다 높은 스트레스량을 나타냈다. 6) 인구사회학적 특성에 따른 건강상태의 차이 검증에서는 연령(F=3.15, P<.05), 직위(F=3.25, P<.05)에서 통계적으로 유의하게 나타났는데 나이가 40세 에서 44세 사이에서 건강상태 호소점수가 가장 높았고 또한 직위별로는 과장직위에서 가장 높은 호소점수가 나타났다. 결론적으로 사무직 근로자는 직장에서 또 가정에서 스트레스를 끊임없이 받고 있고 이러한 스트레스는 건강상태와 상관관계가 있으며 생활사건중에서 특히 자녀, 부부 문제 등 인간관계에서 야기될 수 있는 문제들로 인해 많은 스트레스를 받고 있으므로 사무직 근로자가 그들의 스트레스에 적절히 대응할 수 있도록 가족이나 부부상담 등 전문적인 상담과 대인관계 기술 교육등의 간호전략을 수립하여 사무직 근로자의 정신 및 신체건강을 증진시킬 수 있는 방안을 강구하여야 할 것이다. [영문]Today, a modern person experiences stress in every day living because of many unexpected changes in society as a result of rapid scientific development. Stress from socioenvironmental factors has been defined as one of the causes of various diseases. In particular, office workers experience stress in the work place as a result of situations which are unavoidable. The purpose of this study was to examine the relationship between stress life events and health status in order to give direction to nursing interventions directed at improving the mental health of office workers. The subjects of the study were a convenience sample of 150 male office workers between the age of 30 to 60 years of age working in a D-group located in Gangnam in Seoul. The data were collected for 17 days from March 23 to April 10. 1991. The tools used to collect the data were "Lee's Stressful Life Events Scale(1984)", "Cornell-Medical Index Quetionaire Modified by Nam(1965)" and a socio-demographic characteristics questionnaire. Actual numbers and percentages were used in the analysis of the data, for the socio-demographic characteristics of the subjects. The experience of stressful life events and the total stress were analyzed using actual numbers, percentages, means and standard deviation, and t-test and ANOVA were used to test differences according to the socio-demographic characteristics between stress life events and total stress score. The results of the study were as follows; 1) The mean value for the stress level ranged from a high of 84.15 (Death of a children), to a low 33.57 (begining a religious life). 2) The total stress was highest with an average of 630 points for the item "family restriction". The second highest was for "school dropout by children" with a mean of 508 "living separately from spouse" ranked third at 32.5 points. There were 7 items which were not reported as having been experienced by the Korean people in the study. 3) The health status scores were distributed from zero to 57. The item with the highest score was "frequent tiredness and redness in eyes" with a score of 113. 4) The correlation coefficient for stressful life events with mental heath was r=.2215 (P<.01) indicating that increased stress is related to lower mental health. 5) The scio-demographic characteristics that showed a significant difference for the experience of stressful life events were satisfaction with marriage (t=-3.00, P<.01), and icome (t=-2.17, P<.05). The women with the highest stress scores were women who were married. 6) The socio-demographic characteristics that showed a significant difference for mental health status were age(F=3.15, P<.05) and occupation (F=3.25, P<.05). The mental health from men age 40 to 44 years were the highest. For specialty in occupation, the mental health socres for the head of a section were the highest of all the scores for occuation. In conclusion, stress in life for office workers is closely connected with mental health. Today, there is an increased stress in human relationships such as argument so that it is desitable to help office workers cope with their stress constructively through special counselling and training in improving human relations.prohibitio

Numerical analysis for thermally induced residual stressand effect on birefringence in injection-molded center-gateddisk

Maste

(An)Empirical analysis on the performance of South Korea`s city governments

학위논문(박사)--서울대학교 대학원 :정치학과,2003.Docto

Legislation of Citizen Participation in LocaI Governments

이 글은 지방자치 부활 이후 지방자치단체에서 이루어진 주민참여 입법현황을 살펴보고자 한 것이다 직접민주주의와 대의민주주의 가교역할을 할 것으로 기대되는 지방자치는 지난 10 여 년 동안 다양한 주민참여제도를 만들어 내었다 주민감사청구， 정보공개， 옴부즈만， 시민감독관， 민원법정 등 지방자치단체에 도입되고 있는 참여제도는 지방정부의 투명성을 높이는데 초점을 둠으로써 민주주의 발전과 삶의 질 향상에 기여하고 있는 것으로 생각된다 즉 한국에서 지방자치의 부활은 대의민주제뿐만 아니라 참여민주주의도 확장시키고 있는 것이다. 그러나 입법형식이 훈령위주로 되어 있다는 점에서 한국의 지방정치가 단체장에 의해 주도되고 있음을 보여주고 있다. 참여입법이 조례형식을 갖추어야 하는 것은 제도의 안정성을 확보한다는 점에서뿐만 아니라 지방의회를 활성화시킨다는 점에서도 중요하다. 또 참여입법 활동이 특정한 지역에 편중되어 있는 문제점도 나타나고 있는데， 이는 지역시민사회의 활성화가 불균등하게 이루어지고 있는 것과 연관된 것으로 보인다. 민주주의 작동과 시민사회의 성숙 간에는 불가분의 관계가 있음을 생각하게 한다

Effect of acute exercise on phosphorylation of ERK and expression of GLUT-1 in murine lung

학위논문(박사)--서울대학교 대학원 :체육교육과, 2006.Docto