The study on transcription－replication conflicts induced by perturbation of replication timing control

학위논문(석사) -- 서울대학교대학원 : 사범대학 과학교육과(생물전공), 2022. 8. 이준규.전사-복제 충돌(TRCs)은 복제기구와 RNA 중합효소가 S기에 충돌할 때 발생한다. 비록 전사와 복제가 TRCs의 발생을 최소화하기 위해 조절되지만, TRCs는 정상세포에서 필연적으로 발생하며 특히 암세포에서는 더욱 많이 증가한다. 그러므로 S기에 TRCs를 적절하게 해소하는 것은 유전체의 안정성을 유지하는 데 매우 중요하다. 본 연구실의 선행연구에는 RecQL4-Orc4 융합단백질이 발현되는 즉, pre-RC에 RecQL4 단백질을 결합시킨 인간 골육종 세포에서 후기 복제 원점의 복제가 S기 초기에 활성화되어 TRCs일 가능성이 큰 복제 스트레스가 유발되며, 이 복제 스트레스에 대한 세포 반응에 TOPBP1이 필요함을 확인하였다. 본 연구에서는 복제 시기 조절 변화에 의해 발생하는 복제 스트레스가 TRCs인지 확인하기 위해서, 전사 억제제 처리시 복제 스트레스가 감소하는지 여부와 전사기구와 복제기구의 충돌이 실제로 발생하는지를 조사하였다. 그 결과 면역 형광 염색법을 통해 R-loop와 FANCD2의 foci가 전사 억제제 처리에 의해 감소하는 것을 확인하였다. DNA:RNA hybrid 구조를 가지고 있는 R-loop는 RNA 중합효소가 멈춘 곳에 형성되며 FANCD2는 정지된 복제분기점에 결합한다. 또한, 복제기구의 핵심 인자인 PCNA 항체와 RNA 중합효소Ⅱ의 C-말단 인산화 부위 특이적 항체를 사용하여 근접 결합 분석을 진행한 결과, 복제 시기 조절 변화에 의해 PLA foci가 증가하는 것을 확인하였다. 한편 이 때 발생하는 전사-복제 충돌 반응에서 TOPBP1의 역할과 작용 기전을 규명하기 위해서 TOPBP1 BRCT 도메인의 일부가 결실된 다양한 TOPBP1 단백질들을 발현시킨 다음 전사-복제 충돌 반응을 분석하였다. 그 결과, FANCD2 foci를 형성하는 데에는 ATR 활성화 도메인과 BRCT7-8 도메인을 포함하는 TOPBP1의 C말단만 있어도 충분하지만, 전사-복제 충돌에 의해 유발되는 DNA 이중 가닥 절단을 억제하기 위해서는 C말단과 더불어 BRCT3, 6 도메인이 필요함을 확인하였다. TOPBP1과 FANCD2의 상호작용이 TRCs가 발생했을 때 더욱 증가하는 것을 확인했다. 이러한 결과로부터 RecQL4-Orc4 융합단백질을 발현하여 후기 복제 원점의 조기 활성화를 유도한 세포에서 실제로 전사-복제 충돌이 일어나며, ATR 활성화 이외에도 TOPBP1이 직접적으로 관여하는 전사-복제 충돌에 대한 세포 반응 경로가 존재함을 알 수 있다. 또한, 본 연구에서 Doxycycline 처리로 손쉽게 전사-복제 충돌이 유도되는 안정적인 인간 세포주는 향후 인간세포에서 일어나는 TRCs 반응을 연구하는 모델 시스템으로서 유용하게 활용될 수 있을 것으로 기대된다.Transcription-Replication conflicts (TRCs) occur when replication machinery collides with RNA polymerase in S phase. Although transcription and replication are coordinated to minimizes TRCs, TRCs inevitably occur in normal cells and increase significantly in cancer cells. Therefore, proper resolution of TRCs in S phase is critical for the maintenance of genome integrity. In previous studies in this lab, it was confirmed that the late replication origin was activated in early S-phase in human osteosarcoma cancer cells that tethered RecQL4 on the pre-RC by expressing RecQL4-Orc4 fusion protein, leading to replication stress which is likely to TRCs and TOPBP1 was needed in the cell response to this replication stress. In this study, to confirm that the replication stress induced by perturbation of the replication timing control is TRCs, we investigated whether the replication stress is reduced when transcription inhibitor is treated and whether a collision between transcription machinery and replication machinery actually occurs. Immunofluorescence staining analysis showed that foci of R-loop, a loop containing DNA-RNA hybrids produced by stalling of RNA polymerases, and FANCD2, a protein binding to stalled replication fork, were decreased by transcription inhibition. In addition, as a result of using the PLA using an antibody specific to the C-terminal phosphorylation site of RNA polymeraseⅡ and PCNA antibody, which is a key factor in the replication machinery, it was confirmed that PLA foci were increased by perturbation of the replication timing control. Meanwhile, in order to identify the role and mechanism of TOPBP1 in the transcription-replication conflicts response, various TOPBP1 proteins in which some BRCT domains are deleted were expressed and then the TRCs response was analyzed. It was confirmed that only the C-terminus of TOPBP1 containing the ATR activation domain and the BRCT7-8 domain is sufficient to rescue FANCD2 foci, but the BRCT3 and BRCT6 domains as well as the C-terminus are required to reduce DNA double-strand breaks(DSBs) which is induced by TRCs. The interaction of TOPBP1 and FANCD2 was also increased when TRCs occurred. From these results, it was confirmed that TRCs actually occurs in cells in which early activation of late replication origin was induced by expressing RecQL4-Orc4 fusion protein and in addition to ATR activation, there is a cellular response pathway to the transcription-replication conflicts in which TOPBP1 is directly involved. In addition, it is expected that the stable human cell line, in which transcription-replication conflict is easily induced by treatment with Doxycycline in this study, will be usefully utilized as a model system to study the TRCs response in human cells in the future.제 1 장 서 론 1 제 1 절 이론적 배경 1 제 2 절 연구의 필요성 및 연구 목표 17 제 2 장 연구 방법 및 재료 23 제 1 절 세포 배양 23 제 2 절 플라스미드 24 제 3 절 프라이머 27 제 4 절 siRNA 형질주입 27 제 5 절 플라스미드 형질주입 29 제 6 절 웨스턴 블롯 30 제 7 절 공동 면역침강 31 제 8 절 면역 형광 염색법 33 제 9 절 근접 결합 분석 35 제 10 절 항체 38 제 3 장 연구 결과 39 제 1 절 복제 시기 조절 변화에 의해 발생하는 전사－복제 충돌(TRCs)의 확인 39 제 2 절 세포 내 TRCs 반응에 관여하는 ATR 비의존적 TOPBP1 도메인 탐색 47 제 3 절 TOPBP1과 FANCD2의 상호작용 탐색 52 제 4 장 논의 56 참고문헌 60 Abstract 80석

효율적인 정수 기반 동형 암호

학위논문 (박사)-- 서울대학교 대학원 : 수리과학부, 2015. 2. 천정희.Fully homomorphic encryption allows a worker to perform additions and multiplications on encrypted plaintext values without decryption. The first construction of a fully homomorphic scheme (FHE) based on ideal lattices was described by Gentry in 2009. Since Gentry's breakthrough result, many improvements have been made, introducing new variants, improving efficiency, and providing new features. The most FHE schemes still have very large ciphertexts (millions of bits for a single ciphertext). This presents a considerable bottleneck in practical deployments. To improve the efficiency of FHE schemes, especially ciphertext size, we can consider the following two observations. One is to improve the ratio of plaintext and ciphertext by packing many messages in one ciphertext and the other is to reduce the size of FHE-ciphertext by combining FHE with existing public-key encryption. In the dissertation, we study on construction of efficient FHE over the integers. First, we propose a new variant DGHV fully homomorphic encryption to extend message space. Using Chinese remainder theorem, our scheme reduces the overheads (ratio of ciphertext computation and plaintext computation) from $\tilde{O}(\lambda^4)$ to $\tilde{O}(\lambda)$. We reduce the security of our Somewhat Homomorphic Encryption scheme to a decisional version of Approximate GCD problem (DACD). To reduce the ciphertext size, we propose a hybrid scheme that combines public key encryption (PKE) and somewhat homomorphic encryption (SHE). In this model, messages are encrypted with a PKE and computations on encrypted data are carried out using SHE or FHE after homomorphic decryption. Our approach is suitable for cloud computing environments since it has small bandwidth, low storage requirement, and supports efficient computing on encrypted data. We also give alternative approach to reduce the FHE ciphertext size. Some of recent SHE schemes possess two properties, the public key compression and the key switching. By combining them, we propose a hybrid encryption scheme in which a block of messages is encrypted by symmetric version of the SHE and its secret key is encrypted by the (asymmetric) SHE. The ciphertext under the symmetric key encryption is compressed by using the public key compression technique and we convert the ciphertext into asymmetric encryption to enable homomorphic computations using key switching technique.Contents Abstract 1 Introduction 1 1.1 A Brief Overview of this Thesis 3 2 CRT-based FHE over the Integers 8 2.1 Preliminaries 12 2.2 Our Somewhat Homomorphic Encryption Scheme 14 2.2.1 Parameters 14 2.2.2 The Construction 15 2.2.3 Correctness 17 2.3 Security 19 2.4 FullyHomomorphicEncryption 27 2.4.1 BitMessageSpace 28 2.4.2 LargeMessageSpace 29 2.5 Discussion 35 2.5.1 SecureLargeIntegerArithmetic 35 2.5.2 Public key compression 35 3 A Hybrid Scheme of PKE and SHE 37 3.1 Preliminaries 39 3.1.1 HardProblems 40 3.1.2 Homomorphic Encryption Schemes 41 3.2 Encrypt with PKE and Compute with SHE 43 3.2.1 A Hybrid Scheme of PKE and SHE 44 3.2.2 Additive Homomorphic Encryptions for PKE in the HybridScheme 48 3.2.3 Multiplicative Homomorphic Encryptions for PKE in theHybridScheme 51 3.3 Homomorphic Evaluation of Exponentiation 56 3.3.1 Improved Exponentiation using Vector Decomposition 56 3.3.2 Improve the Bootstrapping without Squashing 59 3.4 Discussions 62 3.4.1 ApplicationModel 62 3.4.2 Advantages 63 3.5 Generic Conversion of SHE from Private-Key to Public-Key 68 4 A Hybrid Asymmetric Homomorphic Encryption 70 4.1 Preliminaries 72 4.2 A Hybrid Approach to Asymmetric FHE with Compressed Ciphertext 73 4.2.1 MainTools 73 4.2.2 Hybrid Encryption with Compressed Ciphertexts 76 4.3 ConcreteHybridConstructions 77 4.3.1 Hybrid Encryptions based on DGHV and Its Variants 77 4.3.2 Hybrid Encryptions based on LWE 87 4.4 Discussion 93 4.4.1 Comparison to Other Approaches 93 4.4.2 Other Fully Homomorphic Encryptions 94 5 Conclusion 95 Abstract (in Korean) 105 Acknowledgement (in Korean) 106Docto

LWE 문제 기반 공개키 암호 및 commitment 스킴의 효율적인 인스턴스화

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 수리과학부, 2018. 2. 천정희.The Learning with Errors (LWE) problem has been used as a underlying problem of a variety of cryptographic schemes. It makes possible constructing advanced solutions like fully homomorphic encryption, multi linear map as well as basic primitives like key-exchange, public-key encryption, signature. Recently, developments in quantum computing have triggered interest in constructing practical cryptographic schemes. In this thesis, we propose efficient post-quantum public-key encryption and commitment schemes based on a variant LWE, named as spLWE. We also suggest related zero-knowledge proofs and LWE-based threshold cryptosystems as an application of the proposed schemes. In order to achieve these results, it is essential investigating the hardness about the variant LWE problem, spLWE. We describe its theoretical, and concrete hardness from a careful analysis.1.Introduction 1 2.Preliminaries 5 2.1 Notations 5 2.2 Cryptographic notions 5 2.2.1 Key Encapsulation Mechanism 5 2.2.2 Commitment Scheme 6 2.2.3 Zero-Knowledge Proofs and Sigma-Protocols 7 2.3 Lattices 9 2.4 Discrete Gaussian Distribution 11 2.5 Computational Problems 12 2.5.1 SVP 12 2.5.2 LWE and Its Variants 12 2.6 Known Attacks for LWE 13 2.6.1 The Distinguishing Attack 14 2.6.2 The Decoding Attack 15 3.LWE with Sparse Secret, spLWE 16 3.1 History 16 3.2 Theoratical Hardness 17 3.2.1 A Reduction from LWE to spLWE 18 3.3 Concrete Hardness 21 3.3.1 Dual Attack (distinguish version) 21 3.3.2 Dual Attack (search version) 23 3.3.3 Modifed Embedding Attack 25 3.3.4 Improving Lattice Attacks for spLWE 26 4.LWE-based Public-Key Encryptions 29 4.1 History 29 4.2 spLWE-based Instantiations 31 4.2.1 Our Key Encapsulation Mechanism 31 4.2.2 Our KEM-Based Encryption Scheme 33 4.2.3 Security 35 4.2.4 Correctness 36 4.3 Implementation 37 4.3.1 Parameter Selection 38 4.3.2 Implementation Result 39 5.LWE-based Commitments and Zero-Knowledge Proofs 41 5.1 History 42 5.2 spLWE-based Instantiations 43 5.2.1 Our spLWE-based Commitments 44 5.2.2 Proof for Opening Information 47 5.3 Application to LWE-based Threshold Crytosystems 50 5.3.1 Zero-Knowledge Proofs of Knowledge for Threshold Decryption 50 5.3.2 Actively Secure Threshold Cryptosystems 58 6.Conclusions 63Docto

Unilateral standing leg tremor as the initial manifestation of Parkinson disease

Background: The aim of this study was to analyze the different forms of leg tremors exhibited while standing in patients with Parkinson disease (PD), and to determine if the type of leg tremor exhibited is indicative of prognosis or treatment response in PD patients. Methods: We studied the clinical characteristics of five PD patients (all women; mean age, 59 years, range, 53-64 years) with unilateral standing leg tremor as the initial manifestation of PD, including their electrophysiological findings and the results of long-term followup. Results: For each patient, parkinsonism either existed at the time of onset of the initial symptoms or developed later. Patient responses to drugs were generally good, but one patient showed a poor response to drugs, even though she had only a low frequency leg tremor. For two patients whom we could observe during the 10-year follow-up period, neither the leg tremor nor parkinsonism was aggravated. Conclusions: There are two forms of unilateral standing leg tremor in PD. One form is high frequency, similar to the primary orthostatic tremor. The other is low frequency and similar to the parkinsonian resting tremor. Based on these observations, it appears that progression might be slow if PD patients have standing leg tremor as the initial manifestationope

Clinicopathologic Analysis of Gastrointestinal Stromal Tumors of the Colon and Rectum

Purpose: This study’s aim is to investigate the clinicopathologic characteristics of colorectal gastrointestinal stromal tumors (GISTs) and to evaluate the result of those tumors. Methods: We retrospectively reviewed 22 patients who had been diagnosed with primary colorectal GISTs and who had undergone a surgical resection between October 1996 and July 2008. Results: Colorectal GISTs accounted for 0.28% of all colorectal malignancies and 7.7% of all GISTs. Rectal GISTs (19, 86.4%) were more common than colonic GISTs (3, 13.6%). According to the National Institute of Health’s (NIH) grading system, there were 1 (4.5%) very low, 5 (22.7%) low, 4 (18.2%) intermediate, and 12 (54.6%) high-risk tumors. The disease recurred in 7 patients (1 with intermediate risk and 6 with high risk). Recurrence sites were the liver (42.9%), the peritoneum (71.5%), and the lymph nodes (14.3%). Adjuvant imatinib therapy and/or radiation therapy were done for patients with microscopically positive margins of resection and high risk, of which one experienced a recurrence at 95 months after surgery. The five-year recurrence rates were 0% in the very-low-grade and low-grade groups, 33.3% in the intermediate-grade group, and 37.5% in the high-grade group. The five-year overall survival rates were 100% in the very-low-grade and low-grade groups, 66.7% in the intermediate-grade group, and 62.5% in the high-grade group. Conclusion: Poor prognosis of colorectal GISTs was closely related to the tumor’s histologic grade and size. Integrating surgery, molecular therapy, and radiation therapy might improve outcomes, but further study with more cases is needed.ope

Duplicated Inferior Vena Cava Recognized during Laparotomy

Duplicated inferior vena cava (IVC) is a congenital anomaly seen rarely in the general population. Patients with IVC variants usually do not present any symptoms and are found incidentally in many cases. However, physicians are urged to recognize the presence of such anomalies during diagnostic or invasive procedures as these variants of blood vessel systems can impose substantial implications in certain clinical situations. Subsequently, information about IVC variants may become critical if surgical injuries or predisposing conditions act as life-threatening risks to patients during medical procedures. We present a case of duplicated IVC in a 68˗year˗old female patient with rectal cancer where an IVC anomaly was found during surgical resection of her tumor. From our experience, we emphasize the importance of having the knowledge of IVC variations in patients undergoing invasive surgical procedures which may involve large vessels.ope

Robotic Anterior Resection for Sigmoid Colon Cancer: Short-term Outcome of a Pilot Study

Purpose: The DaVinci system is new emerging device for performing colorectal surgery. However, in the era of laparoscopic sigmoid colon cancer surgery, there are few previous reports on using the DaVinci system for sigmoid colon cancer. Therefore, the aim of this study is to evaluate the safety and feasibility of using the DaVici system for anterior resection in patients with sigmoid colon cancer, as compared with conventional laparoscopic anterior resection. Methods: Between March 2007 and Jun 2007, 7 sigmoid colon cancer patients underwent robotic anterior resection using the da Vinci Surgical system, and 9 patients underwent conventional laparoscopic anterior resection. The patients’ characteristics, the perioperative clinical results and the pathologic details were prospectively collected and compared between the two groups. Results: The patient characteristics were not significantly different between the two groups. The mean operation time were 205.9±17.6 in the robotic group and 102.4±25.0 in the laparoscopic group (p=0.001). The change of the hemoglobin level, the number of days until peristalsis and the average length of stay were not different between the groups. Also, the pathologic details were not different between the groups. There were no complications or conversion in the both groups. Conclusion: Our data demonstrates that robotic anterior resection is feasible and effective for sigmoid colon cancer patients. However, we could not find better outcomes for robotic anterior resection as compared with conventional laparoscopic anterior resectionope

Comparison between the Initial 25 Cases and the Last 25 Cases of Laparoscopic Colorectal Resection during a Learning Period and According to the Clinicopathologic Outcomes

Purpose: The present study was designed to investigate the clinicopathologic results of performing laparoscopic colorectal resection during a learning curve period. Methods: A prospective analysis of 50 consecutive patients who underwent elective laparoscopic colorectal surgery was conducted between April 2006 and September 2006. We monitored the learning curve of one surgeon. The perioperative clinical results, complications and pathologic details were evaluated prospectively. The 50 patients were divided into two chronological groups (the 25 early cases and the 25 late cases). Statistical analysis between the two groups was performed to evaluate the different outcomes and with taking into account the progressively increasing experience. Results: A total of 45 cases had colorectal cancer. The operative procedure was executed by the standard laparoscopic technique and according to the tumor location, with proper lymph node dissection. Curative resection was performed for the all malignant cases. The remaining 5 cases all involved benign disease. The overall complication and conversion rates were 12% and 6%, respectively. All the complications were treated conservatively. The pathologic outcomes for the malignant cases were acceptable, with no differences being manifested between the two groups. The complication rate and the length of stay were decreased in the late group. Conclusion: Laparoscopic colorectal resection can be performed safely and effectively. The clinicopathologic outcomes were acceptable even though the cases of the present study were done during the learning curve period of a surgeon.ope

Intersphincteric Resection and Coloanal Anstomosis for Very Low Lying Rectal Cancer

Purpose: Ultralow anterior resection and coloanal anastomosis (hand-sewn) has commonly been used for preserving the anal sphincter in patients with low-lying distal rectal cancer. Preoperative chemoradiation therapy is a contributing factor to preserve the anal sphincter. Intersphincteric resection has been introduced and has begun to be applied to distal rectal cancer for anal sphincter preservation. The aim of this study was to report on patients who underwent intersphincteric resection and coloanal anastomosis for very low-lying rectal cancer. Methods: Intersphincteric resection was performed in 21 patients with very low-lying rectal cancer (within 4 cm from the anal verge) between December 2004 and May 2008. All patients received colonic J pouch anal anastomosis and loop ileostomy. The patients were selected prospectively and followed up for the function of bowel movement and recurrence. Results: Mean tumor distance from anal verge was 2.8 cm (range 2∼4 cm). No postoperative mortality was encountered. One patient developed ischemic colitis of colonic J-pouch after high doses of tomotherapy. Subsequently he received abdominoperineal resection and permanent colostomy. One patient underwent diverting colostomy for severe incontinence after ileostomy takedown. The other cases reported good anorectal function such as frequency of bowel movement and fecal incontinence. There were two local recurrences during a mean follow-up period of 11.6 months. Conclusion: Based on a single surgeon’s experiences, postoperative morbidity and anorectal function after intersphincteric resection with coloanal anastomosis seems acceptable.ope

Intestinal endometriosis mimicking carcinoma of rectum and sigmoid colon: a report of five cases

Among women with intestinal endometriosis, the sigmoid colon and rectum are the most commonly involved areas. Sometimes, the differential diagnosis of colorectal endometriosis from carcinoma of the colon and rectum is difficult due to similar colonoscopic and radiologic findings. From October 2002 to September 2007, we performed five operations with curative intent for rectal and sigmoid colon cancer that revealed intestinal endometriosis. Colonoscopic and radiologic findings were suggestive of carcinoma of rectum and sigmoid colon, such as rectal cancer, sigmoid colon cancer and gastrointestinal stromal tumor (GIST). Anterior resection was performed in two patients, low anterior resection was performed in one patient and laparoscopic low anterior resection was done in two patients. We suggest to consider also intestinal endometriosis in reproductive women presenting with gastrointestinal symptoms and an intestinal mass of unknown origin.ope