트로폴로아이소퀴놀린화합물의 간결화한 전합성 및 갈락토오스 유래 바이오 기반 아디픽산 합성공정 개발

학위논문 (박사)-- 서울대학교 대학원 공과대학 화학생물공학부, 2017. 8. 김영규.This thesis comprises two chapters. Chapter 1 describes the concise total synthesis of tropoloisoquinolines via radical anionic coupling. Chapter 2 describes the process development of bio-based adipic acid from galactose by utilizing deoxydehydration process to remove oxygen contents. Chapter 1. Tropoloisoquinoline alkaloids isolated from a plant family, Menispermaceae, which exhibit significant cytotoxic activity against leukemia P388 cell line. Motivated by its high cytotoxicity and unique structure, more concise and divergent syntheses of tropoloisoquinolines were accomplished by using commercially available bromoisoquinolines, based on previously established synthetic strategy employing radical anionic coupling of phenolic nitronate. The key intermediate, phenolic nitronate, was prepared by palladium-catalyzed coupling reaction between phenols and isoquinolines and following Reissert-type nitromethylgroup addition. As a result, various tropoloisoquinoline alkaloids including pareitropone have been synthesized ii and these compounds should be useful for structure-activity relationship (SAR) study. In the analogs, the number of the methoxy groups has been controlled from 0 to 3 and tropone moiety modified to chlorinated tropone. In particular, chlorinated tropone could be used for further functionalization on the tropone moiety. Chapter 2. Adipic acid, one of the commercially important dicarboxylic acids, was efficiently prepared from galactaric acid via deoxydehydration (DODH) process. DODH reaction was applied to remove two pairs of vicinal diols in galactaric acid, with the use of oxo-rhenium catalyst affording corresponding a key intermediate, muconate, First, an efficient large scale one-pot process was established for the mass production on bio-based adipic acid. The desired bio-based adipic acid was achieved in one-pot process that is consists of 4-step reaction: 1) esterification, 2) rhenium-catalyzed DODH reaction, 3) palladium-catalyzed hydrogenation, 4) hydrolysis. With our facile and green synthetic one-pot process, adipic acid was obtained at overall yield of 64% in high purity in 18 g scale from galactaric acid. Second, recyclable green process for bio-based adipic acid was developed by using ionic liquid as a reaction media. Relatively rare and expensive rhenium catalyst was recycled dissolved in ionic liquid. With ionic liquid-mediated DODH reaction, bio-based adipic acid was synthesized in quantitative yield, and the recovered ionic liquid was reused more than 10 times, affording the desired key intermediate muconate with similar yields.Chapter I Concise total synthesis of tropoloisoquinolines Introduction 1 1.Tropoloisoquinoline alkaloids 1 2.Previous synthetic studies toward tropoloisoquinolin alkaloids 5 3.Radical anionic coupling 8 4. Previous total synthesis of pareitropone via radical anionic coupling 10 Result and Discussion 12 1.Concise reaction strategy for tropoloisoquinoline 12 2.Concise total synthesis of pareitropone and its analogs 16 3.Structure determination of chlorinated tropoloisoquinolins 26 Conclusion 29 Experimental Details 30 Chapter II Process development of bio-based adipic acid from galactose 45 Introduction 45 1.Global research trend in biomass 45 2.Bio-based adipic acid 47 3.Deoxydehydration(DODH) reaction 51 Result and Discussion 53 1. Efficient large scale one-pot process of bio-based adipic acid 53 1.1 Introduction 53 1.2 Result and Discussion 54 1.2.1 Adipic acid synthesis via DODH reaction from glucose 54 1.2.2 Adipic acid synthesis via DODH reaction from galactose 56 1.2.3 Large-scale DODH reaction 59 1.2.4 One-pot process for adipic acid 63 1.3 Exprimental Details 66 2. Recyclable green process for bio-based adipic acid using ionic liquid 69 2.1 Introduction 69 2.1.1 Rhenium catalyst recycling 69 2.1.2 Ionic liquids 70 2.2 Result and discussion 71 2.2.1 DODH reaction with various ionic liquid 71 2.2.2 Recycling of rhenium catalyst using ionic liquid 75 2.3 Experimental Details 81 Conclusion 84Docto

신장이식 환자에서 이식 후 혈당조절이 장기적 예후에 미치는 영향

학위논문 (석사)-- 서울대학교 대학원 : 임상의과학과, 2015. 2. 김연수.Introduction Diabetic nephropathy is the leading cause of end stage renal disease (ESRD). The number of kidney transplantations due to diabetic nephropathy is increasing and there is debate on glycemic control after kidney transplantation. In this study, I used a multi-center database to determine the relationship between post-transplant glycemic control and the outcomes of kidney transplantation in patients with diabetic nephropathy. Methods: I conducted a retrospective chart review of kidney transplant recipients(KTRs) with diabetic nephropathy from three tertiary hospitals to analyze the association between post-transplant glycemic control and the clinical outcomes of graft failure, including patient death and biopsy-proven acute rejection (BPAR). Among 3,538 KTRs, a total of 476 patients received kidney transplantation because of diabetic nephropathy. I assessed time-averaged glucose level and hemoglobin A1c (HbA1c) for 36 months after kidney transplantation. Results: Mean time-averaged glucose and HbA1c levels were 147 ± 46 mg/dl and 7.7 ± 1.5 %, respectively. The highest quartile of baseline glucose was related to poor graft outcomes and the 3rd quartile of time-averaged HbA1c was associated with significantly better graft outcomes than the 1st, 2nd or 4th quartiles. On the other hand, time averaged glucose levels were not significantly related to graft outcomes. There were no significant differences in the risk of BPAR across the 4 quartiles of glucose and HbA1c. Conclusions: Strict glycemic control post-transplantation is not necessary for successful outcomes but poor glycemic control is associated with poor graft outcomes. There was no significant relationship between post-transplant glycemic control and BPAR.Abstract...i Contents...iii List of tables and figures...iv Introduction ...1 Material and Methods...5 Results...8 Discussion...23 References...28 Abstract in Korean...33Maste

Diagnostic significance of serum HMGB1 in colorectal carcinomas

High mobility group box 1 protein (HMGB1), a nuclear protein, can be translocated to the cytoplasm and secreted in colon cancer cells. However, the diagnostic significance of HMGB1 has not been evaluated in colorectal carcinomas. For this purpose, we have screened the expression and secretion of HMGB1 in 10 colon cancer cell lines and 1 control cell line and found that HMGB1 was detected in the culture medium. To evaluate the diagnostic value of HMGB1, we performed an enzyme-linked immunosorbent assay to measure HMGB1 levels and compared them to carcinoembryonic antigen (CEA) levels in the serum samples of 219 colorectal carcinoma patients and 75 healthy control subjects. We found that the serum HMGB1 level was increased by 1.5-fold in patients with colorectal carcinoma compared to those in healthy controls. When HMGB1 and CEA levels were compared, HMGB1 had similar efficacy as CEA regarding cancer detection (the sensitivity was 20.1% for HMGB1 vs. 25.6% for CEA, and the specificity was 96% for HMGB1 vs. 90.7% for CEA). Moreover, the diagnostic accuracy of HMGB1 for stage I cancer was significantly higher than that of CEA (sensitivity: 41.2% vs. 5.9%; specificity: 96% vs. 90.7). When we combined HMGB1 and CEA, the overall diagnostic sensitivity was higher than that of CEA alone (42% vs. 25.6%), and the diagnostic sensitivity for stage I was also elevated (47% vs. 5.9%). However, the prognosis of patients was not related with serum HMGB1 concentrations. Our findings indicate that serum HMGB1 levels are increased in a subset of colorectal carcinomas, suggesting their potential utility as a supportive diagnostic marker for colorectal carcinomas.ope

조기 종결 코돈을 가진 돌연변이 유전자들과 nonsense-mediated mRNA decay 억제 후의 번역 억제 규명

Dept. of Medical Science/박사Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in cancers of high-microsatellite instability (MSI-H). Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). In this study, 12 novel genes were identified that are frequently mutated at their cMNR by blocking NMD in two MSI-H cancer cell lines. After blocking NMD, differentially-expressed genes were screened using DNA microarrays, and then database analysis was used to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. Among them, mutations at cMNR of 15 genes have not been previously reported in cancers. Mutations at cMNR of each of the 15 genes in 10 MSI-H cell lines and 21 MSI-H cancers were analyzed, and frequent mutations of 12 genes in MSI-H cell lines and cancers were found, but not in microsatellite stable (MSS) cancers. In addition, these mutated genes are degraded by NMD and protein expressions are down-regulated in MSI-H cancers. Although NMD is an efficient mechanism that down-regulate the PTC-containing mRNA at post-transcription level, there are several stressors to inhibit NMD in vivo. Thus, down-regulated transcripts by NMD can be recovered and might produce truncated proteins. To clarify whether truncated proteins are produced or not, β-globin modified vector system was generated which has been used as a conventional model construct to study NMD mechanism. When NMD was blocked using siRNA of hUPF1, three different drugs, and hypoxic condition, mutant proteins were rarely detected, although PTC-containing transcripts were sufficiently recovered. To verify this discrepancy, several possibilities including translation repression, rapid degradation, and insufficiency of recovered transcript, were checked by using polysome analysis, proteasomal inhibition by MG132, and quantitation of their mRNA expressions, respectively. These experiments indicated that the translations are repressed from PTC-containing transcripts which are recovered by NMD inhibition. In addition, eIF4A3 was involved in this translation repression of PTC-containing mRNAs. All of these results demonstrated that PTC-containing mRNA derived from frameshift mutation is inhibited in the generation of mutant protein production via NMD. Although mutant mRNA expression is recovered by NMD inhibition through cellular stressors, translation repression is developed, and therefore harmful cellular changes by truncated proteins are prevented.ope

Developing of ICU diaries for critical care patients

임상간호 전공본 연구는 중환자실 환자를 위해 중환자실 일지(ICU diary)와 관련한 근거를 분석하고 통합하는 과정을 통해 중환자실 실정에 적합한 입원일지를 개발한 방법론적 연구이다. 본 연구는 중환자실 환자를 위한 입원일지를 개발하기 위해 다음과 같은 과정을 거쳤다. 제 1단계로 중환자실 일지와 관련한 최신의 가이드라인 및 문헌 고찰을 통해 핵심질문요인을 도출하고, 이에 대해 중환자실 의료진 10인, 중환자 가족 10인을 대상으로 요구도 조사를 시행하여 중환자실 입원일지를 구성하는 핵심 항목을 결정하였다. 제 2단계로 중환자실 입원일지의 초안을 구성하였다. 제 3단계로 10인의 전문가 집단으로부터의 내용 타당도 검증을 시행하였다. 제 4단계로 중환자 간호사 5인, 중환자 가족 5인을 대상으로 사용자 타당도 검증 및 예비 조사를 시행하였다. 이러한 단계를 거쳐 최종 중환자실 입원일지 책자를 개발하였으며, 구체적인 연구의 결과는 다음과 같다. 1. 중환자실 입원일지와 관련한 최신의 가이드라인 및 문헌을 검색하여 최종 27개의 문헌을 선정하였다. 선정된 문헌을 검토하여 중환자실 환자를 대상으로 하는 입원 일지를 구성하기 위한 핵심 질문 요인을 도출하였다. 2. 문헌고찰을 통해 도출된 핵심질문 요인에 대해 중환자 가족 및 중환자 의료진을 대상으로 하는 요구도 조사를 통해 의견을 수렴하여 중환자실 입원일지를 구성하는 핵심 항목 및 내용을 결정하였다. 3. 중환자실 입원일지의 초안은 중환자실 입원일지 적용 전 사정, 구성 형식과 내용, 관리의 영역으로 구성되었다. 중환자실 입원일지 적용 전 사정에는 적용 대상자 선정 및 제외 기준과 사전 동의서 취득의 내용으로 구성되었다. 중환자실 입원일지 구성 형식과 내용 항목에는 크기, 작성 도구 등의 외적 형식과 입원일지의 페이지별 구성 내용에 대한 세부 지침, 표준화된 작성 주기, 작성 언어, 사진사용과 관련한 내용으로 구성되었다. 중환자실 입원일지 관리 항목에는 일지 관리 목록, 작성 지침, 보관 위치, 인수인계 방법의 내용으로 구성되었다. 4. 중환자실 입원일지 초안에 대한 내용 타당도 검증 결과 전체 문항의 평균 타당도는 .98로 나타났다. 각 문항별 타당도 중 중환자실 입원일지의 형식 항목의 ‘A5크기의 책을 이용하여, 직접 손 글씨로 작성한다.’는 문항은 CVI 0.8로 상대적으로 낮은 합의율을 보였고, 이를 제외한 모든 항목에서는 CVI가 1.0으로 높게 나타났다. 5. 중환자실 입원일지에 대한 사용자 타당도 검증 및 예비조사에서 일지의 외적 형식에 있어 일지의 크기는 A4의 크기가 편리하고 사용에 용이하다고 답변하였다. 날짜별 일지 중 의료진 작성란에는 중환자의 임상적 주요 사건, 중환자의 반응, 사용 중인 약물 등을 요약하여 작성할 수 있는 공통 서식이 필요하다고 답하였다. 방문객 작성란에는 중환자 가족이 제공하는 사진이나 편지, 스티커 등을 붙일 수 있도록 하는 칸이 별도로 마련되면 좋겠다고 답변하였다. 6. 전문가 집단의 내용 타당도와 사용자 타당도 검증 및 예비조사 과정을 통해 중환자실 입원일지 초안을 수정·보완하여 중환자실 입원일지 책자를 최종 완성하였다. 최종 중환자실 입원일지 책자는 표지, 환자관련 정보 페이지, 중환자실 입실 경과 페이지, 날짜별 일지(환자 본인, 방문객, 의료진 작성란), 부록(중환자실 일지 작성 지침, 동의서, 일지 관리 목록)으로 구성되었다. 본 연구에서 개발된 중환자실 입원일지는 임상현장에 적용되어 중환자가 중환자실에서의 경험을 인지하게 하여 장·단기적 정신적, 심리적 건강문제를 예방하고 감소시킬 수 있을 것으로 기대된다. 또한 중환자실 입원일지는 중환자 가족이 중환자 치료에 참여할 수 있는 저비용의 현실적으로 실행가능한 간호중재로서, 중환자 진료의 질 향상을 도모할 수 있을 것으로 기대된다.open석

Identification and Selective Degradation of Neopeptide-Containing Truncated Mutant Proteins in the Tumors with High Microsatellite Instability

PURPOSE: Frameshift mutations in coding mononucleotide repeats (cMNR) are common in tumors with high microsatellite instability (MSI-H). These mutations generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, mRNAs containing PTCs are degraded by nonsense-mediated mRNA decay (NMD). However, mRNAs containing PTCs located in the last exon are not subject to degradation by NMD (NMD-irrelevant). This study aimed to discover whether genes with frameshift mutations in the last exon generate truncated mutant proteins. EXPERIMENTAL DESIGN: We identified 66 genes containing cMNRs in the last exon by bioinformatic analysis. We found frequent insertion/deletion mutations in the cMNRs of 29 genes in 10 MSI-H cancer cell lines and in the cMNRs of 3 genes in 19 MSI-H cancer tissues. We selected 7 genes (TTK, TCF7L2, MARCKS, ASTE1, INO80E, CYHR1, and EBPL) for mutant mRNA expression analysis and 3 genes (TTK, TCF7L2, and MARCKS) for mutant protein expression analysis. RESULTS: The PTC-containing NMD-irrelevant mRNAs from mutated genes were not degraded. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and we failed to detect endogenous mutant MARCKS. By polysome analysis, we showed that mRNAs from genomic mutant MARCKS constructs are normally translated. After inhibiting 3 protein degradation pathways, we found that only inhibition of the proteasomal pathway facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS. CONCLUSIONS: Our findings indicate that cancer cells scavenge potentially harmful neopeptide-containing mutant proteins derived from NMD-irrelevant abnormal mRNAs via the ubiquitin-proteasome system, and these mutant proteins may be important substrates for tumor-specific antigens.ope

High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells

High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-ζ, λ, and ι) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-ζ by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-ζ in colon cancer tissues. Our findings suggest that PKC-ζ is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.ope

Identification of frequently mutated genes with relevance to nonsense mediated mRNA decay in the high microsatellite instability cancers.

Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in high-microsatellite instability (MSI-H) cancers. Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). We have identified novel genes that are frequently mutated at their cMNR by blocking NMD in two MSI-H cancer cell lines. After blocking NMD, we screened for differentially expressed genes using DNA microarrays, and then used database analysis to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. cMNR mutations have not been previously reported in MSI-H cancers for 15 of the 28 genes. We analyzed the cMNR mutation of each of the 15 genes in 10 MSI-H cell lines and 21 MSI-H cancers, and found frequent mutations of 12 genes in MSI-H cell lines and cancers, but not in microsatellite stable (MSS) cancers. Among these genes, the most frequently mutated in MSI-H cell lines were MLL3 (70%), PHACTR4 (70%), RUFY2 (50%) and TBC1D23 (50%). MLL3, which has already been implicated in cancer, had the highest mutation frequency in MSI-H cancers (48%). Our combined approach of NMD block, database search, and mutation analysis has identified a large number of genes mutated in their cMNR in MSI-H cancers. The identified mutations are expected to contribute to MSI-H tumorigenesis by causing an absence of gene expression or low gene dosage effects.ope