Multi-miRNA panel of tumor-derived extracellular vesicles as promising diagnostic biomarkers of early-stage breast cancer

Extracellular vesicles (EV) have been emerging as potential biomarkers for disease monitoring. In particular, tumor-derived EV (TDE) are known to carry oncogenic miRNA, so they can be used for diagnosis of early cancer by analyzing the expression levels of EV-miRNA circulating in the blood. Here, using our novel microfluidic device, we rapidly and selectively isolate cancerous EV expressing breast cancer-derived surface markers CD49f and EpCAM within 2 minutes. Based on seven candidates of miRNA nominated from The Cancer Genome Atlas (TCGA) database, the expression levels of miRNA in TDE were validated in a total of 82 individuals, including 62 breast cancer patients and 20 healthy controls. Among seven candidates, four miRNAs (miR-9, miR-16, miR-21, and miR-429) from the EV were highly elevated in early-stage breast cancer patients compared with healthy donors. The combination of significant miRNAs from specific EV has high sensitivities of 0.90, 0.86, 0.88, and 0.84 of the area under the receiver operating characteristic curve (AUC) in each subtype (luminal A, luminal B, HER-2, and triple-negative) of early-stage breast cancer. Our results suggest that the combination of four miRNA signatures of specific EV could serve as a sensitive and specific biomarker and enable early diagnosis of breast cancer using liquid biopsy.ope

Tumor-Specific miRNA Signatures in Combination with CA19-9 for Liquid Biopsy-Based Detection of PDAC

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most aggressive malignancies and has high mortality and poor survival rates. Therefore, there is an urgent need to discover non-invasive biomarkers for early detection before PDAC reaches the incurable stage. We hypothesized that liquid biopsy of PDAC-derived extracellular vesicles (PDEs) containing abundant microRNAs (miRNAs) could be used for early diagnosis of PDAC because they can be selectively enriched and because they are biologically stable. We isolated PDEs by immunocapture using magnetic beads, and we identified 13 miRNA candidates in 20 pancreatic cancer patients and 20 normal controls. We found that expression of five miRNAs, including miR-10b, miR-16, miR-155, miR-429, and miR-1290, was markedly higher in PDEs. Furthermore, the miRNA signatures along with serum carbohydrate antigen 19-9 (CA19-9) were optimized by logistic regression, and the miRNA signature and CA19-9 combination markers (CMs) were effective at differentiating PDAC patients from normal controls. As a result, the CMs represented a high sensitivity (AUC, 0.964; sensitivity, 100%; specificity, 80%) and a high specificity (AUC, 0.962; sensitivity, 85.71%; specificity, 100%). These findings suggest that five miRNAs expressed in PDEs and CA19-9 are valuable biomarkers for screening and diagnosis of pancreatic cancer by liquid biopsy.ope

중증 외상에서 병원 사망과 장애에 대한 현장 저산소증과 저혈압의 교호 작용

학위논문 (석사)-- 서울대학교 대학원 : 의과대학 임상의과학과, 2019. 2. 신상도.중증 외상은 대한민국에서 44세 이하 환자에서 사망 원인 1위를 차지하고 있다. 매년 30,000명 정도의 환자가 외상으로 인해서 사망하고 있으며, 많은 후유 장애를 남기게 된다. 중증 외상 환자에서 사망과 후유 장애를 줄이고 이를 조기에 평가하기 위해서 병원 전 단계인 사건 현장에서 구급대에 의해 측정 및 기록되는 변수들에 대한 분석이 필요함. 이중 현장에서 측정된 저산소증 여부가 사망률 및 후유 장애율에 영향을 미치는지에 대한 분석을 진행하였음. 또한 현장에서의 저혈압 여부에 따라서 이러한 저산소증의 영향이 증가하는지 함께 분석하였음. 2012년, 2013년 동안 구급대를 통해 전국 700여개 병원에 내원한 성인 중증 외상환자 중 17,406명의 구급기록 및 의무기록을 수집하여 분석을 진행하였음. 구급대에 의해 측정된 저산소증 여부(산소포화도 94% 미만)를 독립변수로 설정하였고, 중증 외상환자의 병원 내 사망률 및 후유장애율을 결과 변수로서 설정하였다. 후유장애 발생 여부는 Glasgow Outcome scale의 2점 이상의 감소로 정의하였다. 교란요인 보정을 위해 다변량 회귀분석 및 interaction 모델을 사용하여 저산소증 및 저혈압 여부와 중증 외상환자의 사망률, 후유 장애율의 오즈비와 95% 신뢰구간을 계산하였다. 2012-2013년 중증외상환자 17,406명이 분석되었음. 이 중 14.9%에 해당하는 2,598 명이 사망하였고 21.5%에 해당하는 3,292명에서 후유 장애가 발생하였다. 또한 중증외상환자 중 16.7%에 해당하는 2,922명의 환자에서 사고현장에서 측정시 산소포화도 94% 미만의 저산소증이 확인되었음. 중증 외상환자 중 저산소증이 확인된 환자의 사망률(35.7%)이 그렇지 않은 환자의 사망률(10.7%) 보다 높게 확인되었고 후유 장애 발생 비율도 각각 51.2%와 15.9%로 저산소증이 확인된 환자에서 더 높게 확인되었다. 중증외상환자에서 저산소증 여부의 환자 사망 위험에 대한 오즈비는 2.15(1.92-2.40) 이었음. 또한 저산소증 여부의 후유 장애 위험에 대한 오즈비는 1.97(1.75-2.21) 로 확인되었다. 중증외상환자에서 수축기혈압 90mmHg 미만의 저혈압이 동반되었을 경우 저산소증 여부의 환자 사망 위험에 대한 오즈비는 2.66(2.32-3.04)이었다. 또한 저산소증 여부의 후유 장애 위험에 대한 오즈비는 2.17(1.87-2.53)로 확인되었다. 결론적으로, 중증 외상 환자에서 저산소증 여부가 환자의 사망 위험 또는 후유 장애 위험을 높이며, 저혈압이 동반된 경우 그 위험도가 더욱 증가하므로, 구급단계에서 산소포화도 및 혈압 등의 생체징후를 확인하여 환자의 중증도를 평가하고 적합한 치료를 받을 수 있도록 하여야 함.Background It is unclear whether effect size of the hypoxia is different on in-hospital mortality and disability according to hypotension status in the field. Methods Adult severe trauma (ST) patients during 2012-2013 who were treated by emergency medical services (EMS) and had abnormal revised trauma scores in the field or who had positive trauma triage criteria were analyzed. Exposure was hypoxia (<94%) measured by EMS. End points were hospital mortality and disability defined as a Glasgow Outcome Scale that decreased by 2 points or more. Multivariable logistic regression with interaction model between hypoxia and hypotension was used for outcomes to calculate the adjusted odds ratios (AOR) with 95% confidence intervals (95%CIs) after adjusting for potential confounders. Results A total of 17,406 EMS-ST patients were analyzed. Of those, 2,598 (14.9%) died, and 3,292 (21.5%) were considered disabled at discharge. The total hypoxia group showed higher mortality and disability indices (35.7% and 51.2%) than the non-hypoxia group (10.7% and 15.9%), (each p-value <0.0001). The AOR of hypoxia was 2.15 (1.92-2.40) for mortality and was 1.97 (1.75-2.21) for disability. In the interaction model, AORs for mortality by hypoxia in the hypotensive and non-hypotensive groups were 2.66 (2.32-3.04) and 1.74 (1.61-1.87), respectively (P<0.0001 for interaction). The AORs for disability in the hypotensive and non-hypotensive groups were 2.17 (1.87-2.53) and 1.55 (1.42-1.69), respectively (P<0.0001 for interaction). Conclusions The effect of hypoxia was much greater in the hypotensive group than in the non-hypotensive group both in terms of mortality and disability.1. Background ………………………………………………………… 1 2. Methods ……………………………………………………………… 4 2-1) Study setting ………………………………………………… 4 2-2) Study design and data source …………………………… 6 2-3) Study population …………………………………………… 8 2-4) Data variables ………………………………………………… 8 2-5) Outcome measure ………………………………………… 10 2-6) Statistical analysis ………………………………………… 11 3. Results ……………………………………………………………… 13 3-1) Demographic findings …………………………………… 13 3-2) Main analysis ……………………………………………… 19 3-3) Interaction analysis ……………………………………… 19 3-4) Sensitivity analysis for different study population …20 4. Discussion ………………………………………………………… 27 5. Limitation ………………………………………………………… 33 6. Conclusion ………………………………………………………… 35 7. Disclosure ………………………………………………………… 36 8. Acknowledgment ………………………………………………… 36 9. Reference ………………………………………………………… 37 10. 국문 초록 ……………………………………………………… 42Maste

비균일 격자계에서의 파수확장 유한체적 기법의 개선에 관한 연구

The focus of the present study is to improve the numerical accuracy of wavenumber-extended finite volume scheme in non-uniform grid system. The basic idea of the present study is to optimize the dispersion-relation at a local cell postion. An optimization procedure is based on grid-optimized finite difference scheme in finite difference framework, and is extended to conservative approximation in finite volume framework. An optimization procedure at a local cell position obtains the numerical accuracy on wave propagation in non-uniform grid system. In addition, to eliminate excessive numerical dissipation in the continuous region, a distinguishing step is modified to predict the suitable approximation value in non-uniform grid system. A structural change of wavenumber-extended finite volume scheme is able to enhance the numerical accuracy of caculation of aeroacoustic problems in non-uniform grid system. An adoption of modified distinguishing step is able to implement e-MLP in non-uniform grid system. Through numerous test cases such as spherical wave propagation, shock/sine wave interaction, acoustic pulse scattering, moving vortex preservation, and shock vortex interaction problems are carried out in non-uniform grid system. The proposed scheme is proven to have better numerical accuracy than the original wavenumber-extended finite volume scheme.본 연구의 목표는 기존에 개발된 파수확장 유한체적 기법의 정확도를 개선하기 위함이다. 비균일 격자계에서 발생하는 수치적인 오차를 최소화 하기 위하여 본 연구에서는 각 특정 지점에서의 이산관계 보존에 근거하여 최적화를 수행하였다. 본 연구에서 적용된 최적화 기법은 유한차분법에 기초한 격자 최적화 이산관계보존법을 응용하여 이를 유한체적법에 적용하여 각 유한 체적의 셀의 경계면근사에 적용하였다. 이러한 최적화 기법은 모든 셀의 각 경계면에 대해서 이루어지며 이를 통해 비균일 격자상에서 전파문제의 정확도를 향상시켰다. 선형 영역에서의 제한자로 인해 불필요한 수치적인 감쇄를 배제하기 위해서 유동의 특성을 결정하는 검증 함수를 비균일 격자에 적합하도록 조정하였다. 기존 파수확장 유한 체적기법의 구조적인 변화를 통해서 비균일 격자계에서의 공력소음 문제에 대한 수치적 정확도를 향상시킬 수 있었으며, 또한 적절한 검증함수를 적용함으로 비균일 격자계에서도 개선된 다차원 제한자를 도입이 가능하였다. 이러한 수치적 기법의 개선점을 검증하기 위해서 구면파 방사, 충격파-사인파 상호작용, 음향파 전파 현상, 와류 보존 현상, 및 충격파-와류 상호작용의 문제와 같은 검증문제를 비균일 격자계상에서 해석하였다. 이를 통해 개선된 수치기법은 기존 파수확장 유한 체적 기법에 비해 보다 높은 정확성을 가짐을 확인할 수 있었다.Docto

Predicting Subway Passenger Flows By Spatio-temporal Modeling

학위논문 (석사)-- 서울대학교 대학원 자연과학대학 통계학과, 2017. 8. 임채영.지하철은 신속성과 편리함으로 인해 많은 사람들이 이용하는 교통수단이다. 통계적 모형화를 통해 시간대별 지하철 승차인원을 예측하고자 이 논문이 쓰 여졌다. 많은 종류의 데이터 중에서 위치정보를 포함한 공간 데이터가 주목을 받고 있으며 위치정보에 의한 의존성을 고려한 공간 통계 방법론이 발전하고 있다. 서울의 각 지하철역에 위치정보를 부여하고 공간적 의존성과 시간에 따른 의존성을 함께 고려한 시공간 모형화를 통해 지하철 승차인원을 예측하려 한다. 지하철 승차인원을 정규분포로 가정하고 몇 가지 공변량과 B-스플라인 곡선으로 평균구조를 구성한다. 공분산구조는 시간에 따른 의존성과 공간에 의한 의존성이 분리가능하다고 가정하여 지수모형을 적용하며 최종 모형의 모수는 최대우도 추정법으로 추정된다.1 서론 (Introduction) 1 1.1. 공간 데이터 (Spatial data) 1 1.2. 시공간확률과정 (Spatio-temporal Stohastic Process) 2 1.3. 연구 목표 2 2 데이터 설명 (data description) 4 2.1. 데이터 전처리 (data pre-processing) 5 2.2. 데이터 시각화 (data visualizing) 6 3 의존성의 탐색 (Exploring dependencies) 10 3.1. 배리오그램 (Variogram) 11 3.2. 배리오그램 구름 (Variogram cloud) 12 3.3. 경험적 배리오그램 (Empirical variogram) 13 3.4. 모수 배리오그램-공분산 모형 적합 (Parametric variogramcovariance model fitting) 16 4 시공간 크리깅모형 (Spatio-Temporal Kriging Model) 20 4.1. 시공간 가우시안 과정 (Gaussian Process) 20 4.2. 평균 구조 (Mean Structure) 21 4.3. 공분산 구조 (Covariance Structure) 22 4.4. 최대우도추정 (Maximum Likelihood Estimator) 23 4.5. 일반 크리깅 (Universal Kriging) 25 5 데이터 분석 (Data Analysis) 27 5.1. 예측 결과 27 6 결론 (Conclusion) 33Maste

대장균에서의 CRISPR/Cas9 시스템 및 라이보자임을 이용한 다중 게놈 편집 방법 구축 및 응용

학위논문 (박사)-- 서울대학교 대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2018. 8. 김병기.Engineering cellular metabolism for improved production of valuable products requires extensive modulation of bacterial genome to explore complex genetic spaces. In order to introduce genetic modifications for rebalancing the metabolic flux, target genes to be modulated should be determined, and the expression of target genes should be optimized. However, it is difficult to select the target genes precisely because it is very labor-intensive to identify all the effects of the expression changes of the all genes involved in the metabolic pathway on the production of the target product. In addition, even if several genes were selected to be modulated, it is difficult to optimize the metabolic pathway because the combination of modulation may cause detrimental effects on the strain. Therefore, in order to overcome the drawbacks described above, it is reasonable to conduct the metabolic engineering using a combinatorial approach. A combinatorial approach is to screen strain with the best phenotype among the various mutation library expected to improve the desired phenotype. Therefore, in order to generate the mutant library, the tool to introduce mutations at multiple loci is required. In this thesis, we established the multiple sgRNA generation strategy to enable the metabolic pathway optimization using the CRISPR/Cas system. The dCas9-ω, which fused the transcriptional activator domain to inactivated cas9 (dCas9), can activate or repress target gene expression depending on the design of the sgRNA. Therefore, if multiple sgRNAs can be generated, the expression of several genes can be modulated simultaneously without genetic modification. For this reason, the strategy to generate multiple sgRNAs was required, and we constructed a strategy to produce several sgRNAs from one primary transcript using the self-cleavage property of Rz. By combining multiple sgRNA production strategies with the dCas9-ω system, target genes that have a strong effect on phenotypic enhancement can be efficiently identified. After identification of target genes to be modulated, genetic engineering should be performed to alter the expression of those genes. Modulating in the expression level of a single gene to fine tune the metabolic flux often results in a change in the overall flux. Therefore, when optimizing the expression levels of target genes to achieve the desired phenotype through the metabolic engineering, the combinatorial approach is more reasonable than the sequential approach. To do this, it is necessary to be able to construct a library of sufficient size that incorporates genetic modifications to various locations within the chromosome. However, the multiplexing methods reported in the literature have limitations in producing mutant libraries for applying the combinatorial approach because the recombination efficiency is very low and negative selection is not efficient. Therefore, we have designed a system that allows a combinatorial approach by using the CRISPR/Cas system to introduce a mutation library for one site in one cycle and accumulate a mutation library for multiple sites by repeating this cycle. Therefore, we have constructed a plasmid capable of obtaining high CFU with high editing efficiency through the CRISPR/Cas system, and confirmed that a strain in which mutations were introduced into three different target genes could be generated with high efficiency.Chapter 1. Introduction 1 1.1 Genome editing with CRISPR/Cas system 2 1.1.1 CRISPR/Cas system 2 1.1.2 CRISPRi system 8 1.2 Multiple sgRNA generation strategy 12 1.2.1 Conventional methods to generate multiple sgRNAs. 12 1.2.2 Multiple sgRNA generation by using Rz 16 1.2.3 Methods to calculate the Rz efficiency 16 1.3 Multiplex genome editing . 21 1.3.1 MAGE . 21 1.3.2 CRMAGE 23 1.4 The scope of thesis. 23 Chapter 2. Materials and methods. 25 2.1 Bacterial strains and culture conditions 26 2.2 Plasmid construction 30 2.2.1 Design of transcripts containing HHRzs 30 2.2.2 Plasmid construction to express transcripts containing HHRzs 32 2.2.3 Construction of sgRNA transcribing plasmid 36 2.2.4 plasmids construction for CRISPR/Cas system . 42 2.3 Extraction of RNA from E. coli 47 2.4 In vitro transcription 47 2.5 Reverse transcription. 47 2.6 Quantification of transcript. 48 2.7 Circularized RT-PCR 48 2.8 Genome editing using CRISPR/Cas system . 49 2.9 Measurement of the endonuclease activity of Cas9 protein 51 2.10 Plasmid curing . 51 2.11 Lycopene measurement 51 Chapter 3. Development of Quenching-qPCR (Q-Q) assay for measuring absolute intracellular cleavage efficiency of Rz. 53 3.1 Development of calculation method of absolute cleavage efficiency of HHRz . 54 3.2 Quenching method to deactivate the in vitro cleavage activity of HHRz by using asDNA and anti-asDNA 57 3.3 The calculated intracellular cleavage efficiency of Rz was constant regardless of the degree of mRNA degradation. 61 3.4 Conclusion . 65 Chapter 4. Multiple engineering in Escherichia coli by using CRISPR/cas system couple with Rz . 67 4.1 Multiple sgRNA generating strategy by using Rz 68 4.2 In vivo cleavage assay of Rz . 71 4.3 Mature sgRNAs cleaved by Rz have function as a navigator of dCas9 73 4.4 The number of functional sgRNAs was increased up to five by transcribing a primary transcript using strong promoter. 76 4.5 Efficient multiplex genome engineering in Escherichia coli via CRISPRz. 79 4.6 Application of CRISPRi system in lycopene production . 82 4.7 Conclusion . 85 Chapter 5. Enhancement of the CRISPR/Cas9 editing by improving the homologous recombination efficiency 88 5.1 Attempts to enable multiple genome editing through improved HR efficiency have not been successful. 89 5.2 Verification of endonuclease activity of Cas9 of pCASRec. 96 5.3 Verification of HR efficiency of pCASRec 99 5.4 Optimization of experimental conditions using pCASRec: Temperature 101 5.5 Optimization of experimental conditions using pCASRec: Arabinose induction . 103 5.6 Sequential mutagenesis using pCASRec allows multiple editing 105 5.7 Conclusion . 108 Chapter 6. Overall Conclusion and Further Suggestions 110 6 Overall conclusion and further suggestions. 111 References . 114 Abstract in Korean . 121Docto

Diminished rostral anterior cingulate activity in response to threat-related events in posttraumatic stress disorder : (a)functional magnetic resonance imaging study

학위논문(석사) --서울대학교 대학원 :심리학과 임상·상담심리학 전공,2006.Maste

임피던스 지면에서의 광대역음장의 전파와 반사에 관한 수치적 연구

학위논문(석사)--서울대학교 대학원 :기계항공공학부,2006.Maste