DSpace Community:https://oasis.postech.ac.kr/handle/2014.oak/4632024-03-28T02:27:21Z2024-03-28T02:27:21ZDesigner cell refactoring and organelle remodeling for highly enhanced terpene production손소희https://oasis.postech.ac.kr/handle/2014.oak/1182192023-08-31T16:30:32Z2023-01-01T00:00:00ZTitle: Designer cell refactoring and organelle remodeling for highly enhanced terpene production
Authors: 손소희
Abstract: As an engineering discipline for biological systems, the synthetic biology aims to design microbial cell factories towards efficient production of high-value products. The redesigned microorganisms permit a directed modification of metabolic pathways to rewire cellular metabolism for the synthesis of pharmaceuticals, chemicals, fuels, and other valuable products. However, the engineered metabolic pathways present several challenges, including unfavorable regulatory responses, suboptimal physicochemical environments, loss of intermediates to competing pathways, and metabolite toxicity. In achieving optimal metabolic reactions or supporting unique physiological functions, harnessing subcellular organelles would be a promising strategy to address current limitations and future possibilities. Indeed, many researchers have devised the strategies that enhance the limited synthetic capacity of cells by remodeling cell functions to manipulate an intrinsic organelle-specific activity, shape, and capacity (e.g., size, number, and volume) and introducing new functionality to the cells for the production of natural and even non-natural products.
Here, we present two research projects for employing synthetic biology to design and refactor the cell with new functionality for the enhanced synthesis of high-value products. In the first study, we redesign lipid droplets (LDs) which is not a simple blob of fat but a highly dynamic organelle capable of regulating lipid metabolism, storage, and transportation. In guiding LDs to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids (e.g., terpene such as squalene and zeaxanthin) with biogenetic similarity but structural dissimilarity, we experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and β-carotene. Among medicinal terpenes, flexible squalene showed dramatically increased intracellular storage, by ~3100%, due to LD enlargement, while rigid zeaxanthin and β-carotene benefited from LD. In conclusion, we verify that intracellular storage of structurally flexible squalene significantly increases with LD volume expansion, but that of rigid zeaxanthin and β-carotene is enhanced through LD surface broadening. Importantly, this LD engineering strategy can be applied to the storage of non-saponifiable lipids and to other types of lipid transport systems and even metabolic systems.
In the second research project, by utilizing well-characterized signal tags which can accurately deliver proteins to intracellular organelles or extracellular milieu, we construct synthetic pathway for a metabolite trafficking system with a novel functionality that enables hydrophobic impermeable-product secretion by yeast cells. For this study, we systematically couple a supernatant binding protein, which is cytosolic lipid-binding protein as a large and hydrophobic medicinal terpene carrier, with an export signal peptide. By the novel metabolite trafficking system, terpenes are successfully secreted out of the cells (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). This sustainable metabolite secretion holds a great potential for mass production of high-value chemicals in an efficient and continuous manner by continuedly extracting the bio-based chemicals from extracellular medium without cell disruption. Thus, we implemented a semi-continuous fermentation to prove that our metabolite trafficking system could be used to produce and secrete the target metabolite in a continuous and efficient manner (~670 mg/L for squalene for 15-day semi-continuous culture). To the best of our knowledge, this is the most efficient cognate pathway for selective metabolite secretion in microorganisms, thus enabling the intracellularly-accumulated target compounds to pass through otherwise impermeable membranes.
Description: Doctor2023-01-01T00:00:00ZStudies on mechanism of extracellular vesicle production by membrane transport of calcium ion under shear stress강혜진https://oasis.postech.ac.kr/handle/2014.oak/1182222023-08-31T16:30:37Z2023-01-01T00:00:00ZTitle: Studies on mechanism of extracellular vesicle production by membrane transport of calcium ion under shear stress
Authors: 강혜진
Abstract: Extracellular vesicles (EVs) are nano-sized vesicles that cells release into the extracellular space. They contain materials from the host cell (RNAs, proteins and lipids). The host cell materials deliver the information to recipient cells as a cell communicator. EVs derived from mesenchymal stem cells have genetic materials derived from them, and thus have therapeutic efficacy in various disease models. However, the amount of EVs released from cells is too small to be useful as therapeutic agents.
In this thesis, mechanisms of high production of EVs under shear stress are demonstrated. Shear stress is controlled by flow in a flat-plate bioreactor and shaking culture. The yield of EVs released under shear stress was up to seven times higher than from static culture. The mechanism of high production of EVs under shear stress studied in relation to intracellular concentration of calcium ions. These ions are transferred through cell membranes into cells cultured under shear stress, and intracellular Ca2+. The expression of proteins involved in EV biogenesis is higher in EVs released under the condition of increased intracellular Ca2+ than under static condition. EV production is induced by the condition of increased intracellular Ca2+ under shear stress. To verify the function of EVs released under shear stress, their therapeutic efficacy of EVs was investigated on an acute kidney-injury model in vitro and in vivo. The acute kidney injury model regenerated in vitro and in vivo, and EVs released under shear stress had therapeutic efficacy and maintained function.
This thesis is a study of the increased release of EVs under shear stress, the mechanism of enhanced production of EVs, and the function of EVs about therapeutic efficacy. Therefore, this study is expected to contribute to research on therapeutic agents and various applications that use EVs.
Description: Doctor2023-01-01T00:00:00ZSynthesis and Characterization of Double Perovskite Nanocrystals with Controlled Electronic Structure최두원https://oasis.postech.ac.kr/handle/2014.oak/1183652023-08-31T16:34:06Z2023-01-01T00:00:00ZTitle: Synthesis and Characterization of Double Perovskite Nanocrystals with Controlled Electronic Structure
Authors: 최두원
Abstract: Lead halide perovskites (LHPs) are potent optoelectronic materials such as light emitting diodes, photovoltaic cells, scintillator and etc., due to high quantum yield and charge carrier mobility which originate from their defect tolerant nature. However, LHPs suffer from the potential toxicity concerns and structural lability against heat, moisture and light. Thus, the development of LHPs of non-toxic composition, having structural stability has become highly active research area nowadays.
As an alternative to lead halide perovskite, various types of lead-free perovskites have been recently studied for optoelectronic materials. Especially, double perovskite (DP), showing elpasolite perovskite structure (A2M+M3+X6 where A = Cs, CH3NH3: M+ = Ag, Au, Tl: M3+ = In, Au, Bi: X = Cl, Br, I.), is rising. This thesis reports a synthesis of highly phase-pure Cs2NaBiX6 (X = Cl, Br) double perovskite (DP) nanocrystals (NCs). They show characteristic absorption features of sharp and discrete peaks mostly originated from the Bi3+ s–p transition (6s2 → 6s1p1) in [BiX6]3− units within the crystal lattice of elpasolite structure. Such unique optical properties are attributed to the non-bonding character of electropositive sodium and electronically-isolated [BiX6]3− units in crystals. The shape of Cs2NaBiX6 NCs could be quantitatively controlled by adjusting the reaction temperature. Reaction temperatures above 180 °C favors development of cuboctahedral (CO) shape, whereas development of cuboidal (CB) shape is favored below 170 °C. CB NCs can be subsequently converted to CO NCs by heating to 200 °C. The CO NCs promote growth of heterostructure adducts on the (111) facets: these adducts could be post-eliminated by etching. Mn-doped Cs2NaBiCl6 NCs are synthesized: they show efficient energy transfers from the NC host to the dopants. The synthesis and shape control of Cs2NaBiX6 NCs and Mn-doped Cs2NaBiCl6 NCs could expand a new type of lead-free DP NCs applicable to optoelectronic applications.
Also, we conduct the research to utilize Na based double perovskite materials having a discrete electronic structure as a scintillator. To improve scintillation efficient, we focused on thermalization of hot carriers. A more quantized electronic structure causes the photoelectrons to slowly lose energy and induces the excited photoelectrons to create many excitons, resulting in a more efficient scintillation effect. Here, we reports Cs2MTbCl6 (M = Na, K) double perovskite nanocrystals. As we hypothesized, Cs2MTbCl6 NCs shows enhanced scintillation effect compare to CsPbBr3 NCs which are conventionally used as scintillator candidates. We expect that Cs2MTbCl6 NCs could potentially be applied to X-ray scintillation/detection and fluorophores.
Description: Master2023-01-01T00:00:00ZHigh-resolution Inkjet Bioprinting of in vitro Human Alveolar Barrier Construct강다윤https://oasis.postech.ac.kr/handle/2014.oak/1173302023-04-07T16:35:27Z2022-01-01T00:00:00ZTitle: High-resolution Inkjet Bioprinting of in vitro Human Alveolar Barrier Construct
Authors: 강다윤
Abstract: 조직공학 분야에서 바이오 프린팅 기술은 복잡한 구조의 3차원 조직 모사체를 제작하는데 사용할 수 있는 적합한 기술로 평가받고 있다. 바이오 프린팅 기술 중 하나인 잉크젯 바이오 프린팅은 피코리터 단위의 액적을 원할 때에만 토출하는 것이 가능하기 때문에 보다 높은 정밀한 세포 배치를 가능하게 한다. 따라서 섬세하고 복잡한 조직의 특성을 구성하는데 가장 적합한 기술로써 잉크젯 프린팅을 제시할 수 있다. 본 논문에서 모사하고자 하는 폐포 장벽은 매우 얇고 복잡한 조직이며, 기존의 전통적인 실험 방법 및 압출방식의 바이오 프린팅 기술로는 생체 모사도에 한계가 있어왔다. 따라서, 본 논문에서는 고 해상도의 잉크젯 바이오 프린팅을 이러한 한계를 극복할 수 있는 기술로 주목하였다. 본 논문에서는 잉크젯 바이오 프린팅을 이용하여 인공 폐포 모사체를 제작하고 이를 독성 평가, 질병 모델 제작 및 약물 평가에 활용하는 방법을 소개한다.
제 1장에서는 본 연구의 필요성과 전반적인 개요를 소개한다. 제 2장에서는 본 논문의 연구 배경과 관련된 문헌들을 다루고 있다. 2.2절에서는 조직공학 분야에서 사용되는 잉크젯 바이오 프린팅 기술의 개요에 대해서 설명한다. 2.3절에서는 잉크젯 바이오프린팅을 이용한 세포 미세 패턴화 및 하이드로젤 프린팅에 관한 구체적인 사례를 제시한다. 2.4절은 인체 폐의 생리학적 구조와 기능, 폐포 모델의 필요성에 논한다. 2.5절에서는 체외 폐포 모사 실험 모델에 관한 최근까지의 연구 결과들을 방법론에 따라 소개한다. 제 3장에서는 본 논문에서 사용된 재료 및 장비, 실험 방법에 대해 소개한다.
제 4장에서는 잉크젯 바이오 프린팅을 이용해 폐포 장벽 모사체의 제작 과정에 관한 내용을 포함한다. 잉크젯 프린팅 시 고려해야 할 프린팅 조건 및 공정 과정 최적화에 관한 내용부터 시작하여, 제작 과정에 대하여 설명한다. 제작된 조직의 구조 및 기능을 평가하여 잉크젯 프린팅 기반 폐포 장벽 모사체의 우수성을 입증하고 호흡기 바이러스 처리 후 항 바이러스 반응을 확인하는 활용 내용을 다룬다.
제 5장에서는 4장에서 수립된 잉크젯 프린팅 기반 폐포 장벽 모사체를 활용하여 미세먼지 입자의 유해성을 규명한 실험에 관해 소개한다. 다양한 농도 및 시간 변수에 해당하는 미세먼지 입자에 노출된 폐포 모사체 내 세포 사멸에 관해 확인하고, 구조 변화 관찰하는 과정이 포함된다. 폐포 주 기능인 계면활성제 분비 활성에 관해 평가하고, 세포 극성에 관해 확인 한 후 유전자 분석을 통해 실험 결과를 뒷받침한다.
제 6장에서는 잉크젯 프린팅 기반 폐포 장벽 모사체에 가습기 살균제 성분인 CMIT/MIT 처리 후 조직 생존율 평가 및 질병 현상을 확인한다. CMIT/MIT에 노출된 조직의 생존율을 확인하고 2차원 세포 생존율과 직접 비교하여 3차원 환경이 어떤 영향을 줄 수 있는지 확인한다. CMIT/MIT 처리 이후 염증반응 및 세포 신호 전달체계의 활성을 확인하고 상피-간엽전환 (EMT)의 바이오 마커에 관해서 조사한다. 마지막으로 폐 섬유화증의 바이오 마커 상향조절을 관찰하며 CMIT/MIT가 잠재적으로 폐 섬유화증을 비롯한 호흡기 질병의 원인이 될 수 있을 가능성에 대해 설명한다.
제 7장에서는 폐 섬유화증 질병 모델을 제작하고 치료 약물의 처리 후 효율성을 평가하는 내용을 포함한다. 잉크젯 기반의 폐포 장벽 모사체에 친 섬유화성 사이토카인을 처리한 후 세포 증식, 콜라겐 침착, 계면활성제 분비 등의 변화를 관찰함으로써 폐 섬유증이 성공적으로 유도됨을 확인하였다. 또한 EMT 및 폐 섬유화증 바이오마커의 상향 조절을 관찰한다. 이후 항 섬유화제 약물의 투여를 통해 폐 섬유화증의 완화를 확인함으로써 제작된 폐포 장벽 모사체가 새로운 질병 모델 및 신약 개발 플랫폼으로써 활용 될 수 있는 가능성을 제시한다.
제 8장에서는 제 1장에서 소개했던 연구 질문들에 대한 대답을 제시함으로 전체 연구 내용을 요약하고, 추후 발전 가능한 연구에 대해 소개하면서 논문을 마무리한다.; Bioprinting can bring technological advancements in the field of tissue engineering and regenerative medicine by reconstructing precise microenvironments. Among the different bioprinting approaches, piezoelectric inkjet bioprinting, which can eject ink drops at the picolitre level by drop-on-demand style, is mostly used in this thesis to produce a delicate and complex 3D alveolar barrier model. Fundamental printing factors, from jetting stability to cell viability, are evaluated systematically for method optimization. Therefore, alveolar cells of various types might be laminated to create a three-layer structure. A three-layered alveolar barrier model was created with unprecedented thickness using high-resolution alveolar cell deposition. The model structure, morphologies, and fundamental functions of lung tissue were demonstrated using constructed alveolar barrier constructions. Furthermore, the risk of hazardous inhalable dust particles and chemicals was demonstrated as revealed anomalies and alterations in the structure and genetic expression. Finally, the respiratory disease was induced on the constructed alveolar barrier model, and the treatment effectiveness of drugs was assessed. The all-inkjet-printed alveolar barrier model might be utilized as an alternative to traditional test models for pathological and pharmacological applications, as inkjet bioprinting allows for customization, scalable manufacture, and standardization of tissue models.
Description: Doctor2022-01-01T00:00:00Z