음파반응 금 나노약물을 이용한 오토파지 항암치료 : 음파에 반응하는 금 나노입자를 함유한 오토파지 증가를 통한 항암치료를 연구

정용규 가천대학교 대학원 2022 국내석사

초음파역학 요법(sonodynamic therapy, SDT)와 음파반응물질(sonosensitizer)은 치료부위를 국소화 하고 독성 부작용을 최소화하는 장점을 가져 최근 다양한 유형의 종양치료에 사용되고 있는 첨단 기술이다. 그러나 초음파역학 요법(sonodynamic therapy, SDT)은 20kHz 이상의 초음파(ultrasound)에 의해 발생되며 가청주파수(audible frequency)를 질환치료에 이용하는 연구는 현재까지 보고된 바 없다. 본 연구에서 양전하를 띤 금 나노로드에 30Hz의 음파를 가해주면 서로 응집하여 입자의 크기가 커지는 것을 최초로 발견하였다. 이렇게 응집하는 현상을 이용하여 종양세포 내로 함입된 금 나노입자를 가청주파수에 반응시키고 궁극적으로 오토파지의 형성에 크게 영향을 줄 수 있음을 확인하였다. 음파에 의해 응집되는 나노 약물에 의한 오토파지의 증가는 종양세포에 나노 항암제의 증폭된 엔도좀(endosome)이입을 초래하여 효과적으로 세포사멸을 유도할 수 있다. 본 연구의 가청주파수 음파를 이용한다면 기존 초음파역학요법보다 환자들의 거부감이 적을 뿐만 아니라 복잡한 장비와 설비없이 좀 더 대중적으로 접근된 새로운 항암치료법을 제공할 수 있다.

Accelerating the evolution of bacteriophages targeting Acinetobacter baumannii in host range expansion and determination of its background genetic mechanisms

Vu Thao Nguyen Graduate School, Yonsei University 2024 국내박사

Prior to the discovery and widespread use of antibiotics, bacteriophages (phages) were a potential solution for preventing and treating bacterial infections (1, 2). With the emergence of antimicrobial resistance, there has been a renewed interest in phages (3). However, a major limitation to using phage as therapy is their narrow host range, limiting their effectiveness to specific bacterial strains of the same species (4). To address this limitation, phage training, such as Appelmans protocol, has been proposed as a strategy for expanding the host range of phages (5). Despite its potential, there are currently no reports of the protocol being applied to phages targeting Acinetobacter baumannii, an opportunistic pathogen responsible for a variety of hospitalacquired infections with high mortality rate (6). The aims of my dissertation were to apply the Appelmans protocol for expanding the host range of a phage cocktail targeting A. baumannii and evaluate the therapeutic potential of the expanded host range phages generated from the protocol. Additionally, the genetic mechanism underlying the protocol was also investigated to gain insights into its effectiveness. Chapter I provides a brief overview of Acinetobacter baumannii and highlights the significant global concern surrounding carbapenem-resistant A. baumannii (CRAB) strains. Furthermore, it introduces the fundamental background on bacteriophages and phage therapy, along with their limitations and discusses various approaches to overcome these challenges. Chapter II describes the application of the Appelmans protocol as a host range expansion method to broaden the host range of a phage cocktail targeting CRAB. The assessment of the host range expansion covered both the output cocktail and individual phage clones generated from the method. While the protocol consistently demonstrated the capability to expand the host range of the cocktail, it faced challenges when dealing with CRAB strains. Chapter III delves into the screening and characterization process used to identify potential therapeutic phages. The focus was on evaluating individual phage clones generated from the Appelmans protocol for therapeutic applications. Throughout the evaluation, key factors such as host range stability,lytic growth, transduction potential, and the absence of toxin genes were rigorously examined. However, it is noteworthy that the expanded host range phages obtained from this protocol exhibited limited stability, raising concerns about their suitability for therapeutic purposes. Chapter IV discusses the genetic insights into the Appelmans protocol’s mechanism, not only based on previous studies involving phages targeting different bacterial species but also when applied to the phage cocktail targeting CRAB. A bioinformatics workflow was established to analyze the ancestral of the expanded host range generated from the protocol and the finding revealed that they were recombinant derivatives of prophages induced from encountered bacterial strains. Favorable conditions and explanations for prophage induction during this protocol were proposed to support this genetic mechanism. In conclusion, this dissertation focused on the potential and challenges of a phage training method, especially, the Appelmans protocol in expanding the host range of phages for therapeutic applications. The findings emphasize the importance of understanding the genetic mechanisms underlying phage-host interactions to optimize phage therapy strategies for combating antibioticresistant bacterial infections. Parts of this dissertation are adapted from Vu TN, Clark JR, Jang E, D'Souza R, Nguyen LP, Pinto NA, et al. Appelmans protocol - A directed in vitro evolution enables induction and recombination of prophages with expanded host range. Virus Res. 2023;339:199272.

파지 디스플레이 라이브러리를 이용한 항 아데노바이러스 Knob scFv 제조

금재욱 서울대학교 대학원 2003 국내석사

Discovery of Specific Monoclonal Antibody for Cathepsin Z Contributing to Mesenchymal Glioblastoma

Kim Han-gil 국민대학교 일반대학원 2022 국내석사

Autophagy contributes to the insensitivity to gefitinib in ovarian cancer cells with wild-type EGFR

이지현 서울대학교 대학원 2020 국내석사

표피 성장인자 수용체는 (EGFR) 잦은 과발현과 과활성화로 인해 많은 상피암의 치료적 타겟이다. Gefitinib (ZD1839, Iressa)은 EGFR의 세포 내 티로신 키나아제 영역에 ATP와 경쟁적으로 결합하여 티로신 키나아제 기능을 억제하는 약제이다. Gefitinib의 단독 투여는 난소암을 포함한 야생형 EGFR을 가진 암 환자에게 생존 혜택을 제공하지 못했다. 그러나 야생형 EGFR을 가진 비소세포성 폐암 환자 일부에서 Gefitinib에 대한 항암 효과를 보인 임상 사례가 보고된 바 있다. 현재 EGFR 표적 치료와 관련한 연구는 선천적인 내성이나 후천적인 내성을 유발하는 기전을 규명하고, 병용 요법의 효과를 확인하는 것으로 활발히 진행되고 있다. 최근 여러 연구에서 EGFR이 키나아제 기능과 독립적으로, 오토파지를 (autophagy, 자가포식작용) 유도하여 EGFR 표적 치료법에 대한 내성에 관여한다는 것이 입증된 바 있다. 본 연구에서는 Gefitinib에 대한 불감증을 가진 난소암 세포에서 오토파지의 역할을 분석하였다. Gefitinib에 대해 불감증을 보이는 난소암 세포에서 오토파지의 역할을 평가하기 위해 난소암 세포주인 SKOV3에 Gefitinib을 처리하고 오토파지의 유동과 산성 소낭 세포소기관의 양을 측정하였다. Gefitinib 처리 시, 야생형 EGFR을 가진 난소암 세포에서 LC3-II로의 변환과 산성 소낭 세포소기관의 양이 증가하였다. 오토파지가 Gefitinib에 대한 불감증에 기여하는지 검증하기 위해 Wortmannin, Chloroquine (CQ) (오토파지 억제제), Rapamycin (오토파지 유도제)을 사용하여 세포 증식 양상을 확인하였다. 야생형 EGFR을 가진 난소암 세포에서 오토파지 억제제와 병용 처리는 세포 증식을 억제하여 Gefitinib에 대한 민감도를 유의적으로 향상시켰다. 또한 말라리아 치료제인 클로로킨을 (CQ) 사용하여 Gefitinib과 병용 처리 시, 세포 사멸을 유도하여 Gefitinib의 종양 억제 효과가 향상됨을 확인하였다. 결론적으로, 본 연구를 통해 Gefitinib에 의해 유도된 오토파지는 야생형 EGFR을 가진 난소암 세포에서 Gefitinib에 대한 불감증에 관여함을 확인하였다. 이러한 연구 결과는 대부분 야생형 EGFR을 가진 난소암 세포에서 오토파지 억제가 Gefitinib에 대한 반응성을 향상시키는 새로운 치료 전략이 될 수 있고, 말라리아 치료제인 클로로킨은 Gefitinib의 항암 효과를 높이는 잠재적 치료제가 될 수 있음을 제시한다. The epidermal growth factor receptor (EGFR) has been a therapeutic target for many epithelial cancers due to its frequent overexpression and hyperactivation. Gefitinib (ZD1839, Iressa), which is a tyrosine kinase inhibitor (TKI), is a small molecule that competes with the binding of ATP to the intracellular tyrosine kinase domain of EGFR. Clinical studies have suggested that treatment with gefitinib monotherapy did not provide survival benefits to patients with wild-type EGFR cancers including ovarian cancer. However, several cases have been reported that non-small-cell lung cancer (NSCLC) patients with wild-type EGFR responded to gefitinib. This study was conducted to elucidate underlying mechanisms contributing to the insensitivity to gefitinib in ovarian cancer cells. Recently, several studies have demonstrated that kinase-independent EGFR has a role in autophagy initiation and this was involved in the resistance to EGFR-targeted therapy. This study investigated the pro-survival role of autophagy induced in response to gefitinib in ovarian cancer cells. To evaluate the role of autophagy in gefitinib-insensitive ovarian cancer cells, ovarian cancer cells (SKOV3) were treated with gefitinib and autophagic flux was examined by expression of autophagy markers and acidic vesicle measurement. Treatment with gefitinib increased conversion to LC3-II and acidic vesicle formation in ovarian cancer cells with wild-type EGFR. To evaluate whether autophagy contributes to the insensitivity to gefitinib, wortmannin, chloroquine (autophagy inhibitor) and rapamycin (autophagy inducer) were used. Treatment with autophagy inhibitors significantly improved sensitivity to gefitinib in ovarian cancer cells suppressing cell proliferation. Using FDA-approved anti-malarial drug, chloroquine (CQ), we investigated the anti-cancer effect of the combination of gefitinib with CQ and its molecular mechanisms in ovarian cancer cells (SKOV3). We found that combining CQ improved the tumor-suppressive effect of gefitinib by inducing apoptosis. Taken together, our results suggest that induction of autophagy could be critical for the insensitivity to gefitinib in ovarian cancer cells. Our results suggest that inhibition of autophagy can be a novel therapeutic strategy potentiating gefitinib sensitivity of ovarian cancer. Therefore, CQ could be a potential therapeutic agent enhancing the anti-cancer effect of gefitinib in ovarian cancer cells.