Control of colloidal stability and bioavailability of lipid nanoparticles for oral delivery of food bioactives

반충진 서울대학교 대학원 2016 국내박사

Lipid carrier system capable of the controlled release for encapsulated bioactive materials has attracted an interest for the bioavailability increase and the targeted delivery of the bioactives in many industrial fields (foods, cosmetics, and pharmaceutics) for a long time. However, there was still no system as a perfect solution having both efficient functionality and economic feasibility. Lipid nanoparticle (LNP) system, including solid lipid nanoparticle and nanostructured lipid carrier, was invented as a novel strategy for substitution of conventional lipid carrier systems such as emulsion and liposome, with a little modification (the use of solid lipids) from the emulsion. LNPs have various merits for using physiological lipids, protecting from the outside stress, enhancing the oral bioavailability, modulating the release profile of core materials, and enabling the bulk production. Accordingly, despite many efforts of food scientists for applying LNPs to foods, it was not adopted in foods yet due to unsolved problems in terms of colloidal or storage stability. In this research, the LNP production process was optimized to enhance the stability, and flavonoid-loaded LNPs were developed to improve the bioaccessibility of the flavonoids based on the optimum process, then the uptake pattern of LNP-incorporated curcumins into the blood was controlled on the basis of modulating the lipid-water interfacial property. In detail, 6 min postsonication during the cooling process after the size reduction step of melted lipid droplets can diffuse self-assembled/solo emulsifiers onto the LNP surface, and the addition of 30 wt % oil into the solid lipid phase ameliorated the LNP colloidal stability resulting from the crystallinity reduction of solid lipid matrix. Additionally, under the simulated in vitro gastrointestinal tract (GIT), bioaccessibility values of quercetin, naringenin, and hesperetin encapsulated in LNPs prepared using 3.5 wt % fully hydrogen canola oil, 1.5 wt % squalene, 1.083 wt % soybean lecithin, and 0.583 wt % Tween 20 were increased 11.71-, 5.03-, 4.76-fold than those of the native-formed flavonoids, respectively. Lastly, because the mimicked GIT hydrolysis of LNPs covered with various PEGylated emulsifiers was controlled by the LNP designs in aspects of the PEG length, the emulsifier concentration, and the lipid type, the plasma residence of curcumin encapsulated in the PEGylated LNPs would be successfully extended or shortened as the designs under the in vivo rat model for oral administration. In summary, these results suggest that LNP systems developed in this study can satisfy enough an expectation of manufacturers and customers as a food-grade lipid delivery system. In conclusion, this study could serve as a basis for further research that aims to develop delivery systems for foods and pharmaceutics.

Studies on the molecular mechanism and function of non-autophagic LC3 lipidation under Golgi stress

강재민 서울대학교 대학원 2025 국내박사

Lipidated ATG8/LC3 proteins are recruited to single membrane compartments, such as endosomes and lysosomes, as well as double-membraned autophagosomes to support their functions. Although recent studies have shown that LC3 lipidation occurs on the Golgi apparatus membranes following several Golgi-damaging conditions, molecular mechanism and biological function of Golgi-LC3 lipidation remain unknown. Here, I established DLK1 overexpression as a new strategy for studying Golgi-specific LC3 lipidation. Combining this with Golgi-damaging reagents, niclosamide and AMDE-1, I unravel the mechanism and role of Golgi-LC3 lipidation. I found that upon DLK1 overexpression, the trans-Golgi network is lipidated by LC3 via ATG12-ATG5-ATG16L1 complex, two ubiquitin-like conjugation systems in autophagy. Upstream autophagy regulators including ULK1/2 and FIP200 are dispensable for DLK1-induced LC3 lipidation. I confirmed that post-Golgi trafficking blockade is the primary cause of Golgi-LC3 lipidation under DLK1 overexpression and niclosamide/AMDE-1 incubation. Golgi-LC3 lipidation requires ATG16L1 recruitment to the Golgi apparatus which is mediated through interaction between ATG16L1 WD40 domain and V-ATPase. During post-Golgi trafficking inhibition, TFE3, one of the key transcriptional regulators of the Golgi stress response, is translocated to the nucleus and upregulates Golgi stress response target genes. Defects in LC3 lipidation impair TFE3 nuclear translocation specifically under Golgi dysfuction, leading to dysregulation of the Golgi stress response. Taken together, this study demonstrates the mechanism and novel function of Golgi-LC3 lipidation in the Golgi stress response. ATG8/LC3단백질은 지질화되어 이중막 구조의 자가포식소체 뿐만 아니라 엔도솜과 라이소좀 등의 단일막 구조의 막에 위치하여 이러한 막 구조 소기관의 기능을 지원할 수 있다. 최신 연구에서 여러 종류의 골지체 손상을 유발하는 조건들에서 LC3 지질화가 골지체 막에서도 발생하는 현상들이 보고되었으나 골지체-LC3지질화의 분자 기전과 생물학적 기능은 아직 밝혀지지 않았다. 본 연구에서는 골지체 특이적인 LC3 지질화의 새로운 연구 방법으로 DLK1 과발현 조건을 정립하였다. DLK1 과발현 조건에 더해 골지체 손상을 유발하는 두 종류의 화합물인 niclosamide와 AMDE-1을 이용하여 골지체-LC3 지질화의 기전과 역할을 밝혔다. DLK1 과발현은 자가포식작용에서 유비퀴틴 유사체 결합 체계로 기능하는 ATG12-ATG5-ATG16L1 복합체를 통해 trans-Golgi network에서의 LC3 단백질의 지질화를 유도한다. 자가포식작용 경로의 상위 조절자인 ULK1/2와 FIP200는 DLK1에 의한 LC3 지질화에 관여하지 않는다. 본 연구에서 골지체 이후 단백질 수송 과정의 억제가DLK1 과발현 조건뿐만 아니라 niclosamide/AMDE-1에 의해 유도되는 골지체-LC3 지질화의 공통적인 원인임을 밝혔다. 골지체-LC3 지질화는 ATG16L1의 WD40 영역과V 형 ATP 가수분해효소의 상호작용을 통해 ATG16L1의 골지체로 이동이 선행되어야 한다. 골지체 이후 단백질 수송 과정의 억제는 골지 스트레스 반응의 주요 전사 조절자인 TFE3를 핵으로 이동시켜 골지 스트레스 반응의 대상 유전자들의 발현을 증가시킨다. LC3 지질화가 되지 못할 경우 골지체 기능장애에 의해 TFE3 가 핵으로 이동하는 현상이 저해되어 골지 스트레스 반응이 정상적으로 작동되지 못한다. 종합적으로 본 연구는 골지체-LC3 지질화의 분자 기전과 골지 스트레스 반응에서의 새로운 기능을 밝혔다.

Screening of genes involved in Lipid Droplet morphology and inheritance in Saccharomyces cerevisiae

Yuseon Kim 광주과학기술원 2025 국내석사

Lipid droplets (LDs) are conserved organelles that store neutral lipids and are essential for energy metabolism, lipid homeostasis, and stress responses. Although their formation and metabolic regulation have been extensively studied, the mechanisms that govern their inheritance and morphological integrity during cell division are poorly understood. To identify non-essential genes involved in LD inheritance and morphology, I conducted a fluorescence microscopy-based screen using Saccharomyces cerevisiae, examining 400 single gene deletion strains. LDs were visualized by BODIPY 493/503 staining, and their phenotypes were classified as inheritance defects based on LD presence in daughter cells, or as morphological abnormalities based on changes in droplet number, size, or shape. Among the screened strains, deletion of LDB16, a component of the seipin complex, resulted in a clear LD inheritance defect and an increase in LD size. In this mutant strain, 52% of daughter cells failed to receive LDs from mother cells, in contrast to 14% in the daughter cells of the wild-type strain. In addition, a number of mutant strains exhibited diverse abnormalities in LD morphology. LDs with ring-like morphology were observed in spo7Δ and nem1Δ mutants, while enlarged LDs were seen in tgl3Δ. Several mutants, including loa1Δ, arv1Δ, srx1Δ, cla4Δ, cnm67Δ, sla2Δ, sto1Δ, and chc1Δ, showed increases in LD number. These findings provide important insights into the genetic regulation of lipid droplet behavior. In yeast, Sey1p is the primary mediator of homotypic ER membrane fusion, whereas Dnm1p is well known for its role in mitochondrial and peroxisomal fission. However, the potential interplay between these machineries in ER network maintenance remains unclear. Here, I investigated the relationship between Dnm1p and Sey1p in Saccharomyces cerevisiae. Co-immunoprecipitation experiments demonstrated a physical association between Dnm1p and Sey1p, suggesting a possible connection between ER fusion and fission processes. Functional assays revealed that deletion of DNM1 resulted in inconsistent effects on in vitro ER microsome fusion activity, with both increased and decreased fusion observed in different experiments. Furthermore, in Sey1p-dependent proteoliposome fusion assays, the addition of Dnm1-MBP, as well as all control conditions—including boiled Dnm1-MBP, MBP alone, and TEV protease—led to a marked reduction in fusion compared to Sey1p alone, indicating non-specific inhibition rather than a direct regulatory effect of Dnm1p. These findings highlight the complexity of ER network regulation and indicate that while Dnm1p and Sey1p physically interact, Dnm1p does not play a considerable role in regulating Sey1p-mediated ER membrane fusion in vitro. To fully elucidate the functional significance of the Dnm1p-Sey1p interaction, future studies should employ optimized in vitro fusion assays and in vivo approaches to dissect the molecular mechanisms underlying ER dynamics.

Advancing Cationic Micelle Lipid Nanoparticles and Extracellular Vesicles as Vectors for Hydrophobic Anticancer Drug Delivery

Fisher, William University of California, Santa Barbara ProQuest D 2025 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Lipid nanoparticles are promising delivery vehicles for hydrophobic anti-cancer drugs that enable targeted accumulation of drug at specific tissues and reduced systemic side effects. However, their clinical success is limited by challenges in drug loading, targeting in vivo, and endosomal entrapment. Developing next generation lipid nanoparticles that overcome these challenges is critical to realize their full potential to transform cancer chemotherapy.This doctoral work establishes a protocol for isolation of extracellular vesicles (EVs) and expands their ability to load the hydrophobic cancer chemotherapy drug paclitaxel (PTX) for drug delivery applications. EVs are nanoscale, cell-secreted vesicles that facilitate intercellular communication. Exosomes are a subset of EVs with 30 to 150 nm diameters that have high potential as drug delivery vectors because they show low immunogenicity and cell-specific cytosolic delivery of their contents. A serial centrifugation and differential ultracentrifugation protocol was used to isolate vesicles from prostate and melanoma cancer cells that had protein enrichment, diameters, and high membrane rigidity consistent with exosomes. Despite achieving high yields of EVs for both cell types, we observed inefficient loading of PTX in isolated EVs, which restricts their therapeutic application. To overcome this, we adapted a Forster resonance energy transfer (FRET) based lipid mixing assay to study methods for fusion of EVs with synthetic, PTX-loaded liposomes to create hybrid PTX delivery vesicles. We discovered that acidic conditions enhanced the fusion of EVs with bare synthetic liposomes while avoiding contaminating depletants and preserving EV membrane proteins. Remarkably, acidic conditions also induced clustering of EVs with themselves. These findings reveal a previously unexplored protein-lipid or lipid-lipid component to EV content release and enables minimally perturbative modification of EV contents towards a hybrid drug delivery vesicle.In parallel, we investigated the capacity of cationic liposomes (CLs) and cationic lipid nanoparticles (CLNPs) with novel membrane lipids and micellar structures to load and deliver PTX to cancer cells in vitro and in vivo. We found that lipid tails containing two cis double bonds enhanced the PTX loading of CLs by nearly 3-fold over conventional single cis double bond tails used in clinical stage CLs currently (e.g. EndoTAG-1TM), while maintaining or enhancing their cytotoxic efficacy against cancer cells. With respect to lipid headgroups, prior work revealed that incorporation of polyethylene glycol (PEG)-conjugated lipids (PEGylation) above a threshold membrane content drives the formation of disc-shaped micelles (nanodiscs), a novel structure for CLNPs with fluid-phase membranes, and improves cellular uptake and cytotoxic efficacy of CLs. Building on this, we demonstrated that CLs with PEG-lipid contents above this threshold show 10 to 35-fold greater tumor accumulation than those with sub-threshold PEG-lipid contents in vivo. This led us to explore using the novel multivalent lipid MVL5 in PTX-loaded CLNPs following the observation that CLNPs containing 50 mol% MVL5 form particle populations consisting of entirely nanodiscs, with rod and spherical micelles forming at 75 mol% or greater MVL5. Unexpectedly, a nearly 2-fold improvement in PTX solubility was observed for micellar MVL5 CLNPs over CLs based on EndoTAG-1TM containing the univalent cationic lipid DOTAP. This enhanced solubility translated to improved cytotoxic efficacy at high PTX content in vitro, with PEGylation driving further improvement. Finally, we found that steric stabilization of sub-200 nm diameter particles by PEGylation significantly improves MVL5 CLNP cell uptake and penetration depth. This supports a model where the rate limiting steps of PTX delivery by CLNPs are diffusion of endocytic vesicles containing CLNPs across the actin mesh near the cell surface, combined with hopping of PTX from endosomal vesicle membranes to nearby microtubules. By identifying and enhancing physicochemical properties of CLNPs critical to their performance as PTX delivery vehicles, these findings provide actionable steps which would improve the cost, safety, and efficacy of CLNPs as hydrophobic drug delivery vehicles in clinical applications.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy

주혜민 서울대학교 대학원 2023 국내석사

Metabolic engineering of the tumor microenvironment has emerged as a new strategy for tumor therapy. Lactate dehydrogenase A (LDHA) is a prominent target for metabolic engineering. Here, we designed a cationic lipid nanoparticle formulation for LDHA gene editing. The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein (GFP). The delivery efficiency was affected by the ratios of three components: a cationic lipid, cholesterol or its derivative, and a fusogenic lipid. The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA, yielding the lipoplex, pCas9-sgLDHA/F3. The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells. Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 resulted in editing of the LDHA gene and increased the pH of the culture medium. pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture. In vivo, combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice. This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy. 대사공학(metabolic engineering)을 이용한 종양 미세환경(tumor microenvironment) 조절은 최근 새로운 항암치료 전략으로 부상하고 있다. 젖산탈수소효소 A(Lactate dehydrogenase A, LDHA)는 대사공학에서 중요한 표적으로 작용한다. LDHA의 기능을 억제하여 종양세포의 젖산 생산을 감소시킬 경우, 종양미세환경의 산성이 약화되어 T세포에 의한 종양세포 공격이 활성화된다. 이러한 결과를 목표로 하여, 이 연구에서는 종양의 LDHA 유전자를 편집하기 위한 양이온성 지질 나노입자(cationic lipid nanoparticle) 제제를 개발하였다. 우선 플라스미드 DNA(plasmid DNA, pDNA)를 높은 효율로 전달하기 위한 최적의 지질 조성을 스크리닝하였다. 양이온성 지질(cationic lipid), 콜레스테롤 및 그 유도체, 그리고 융해성 지질(fusogenic lipid)의 비율을 달리 하여 여러가지 조성의 지질나노입자를 구성하였다. 그 다음, 녹색형광단백질(GFP)을 암호화하는 pDNA와 지질나노입자의 복합체를 형성하여 이를 배양 세포에 전달하였다. 발현된 GFP의 형광 세기를 비교함으로써 최적의 지질 조성으로 ‘F3’ 조성을 선정하였다. 이어서 F3 지질나노입자를 CRISPR/Cas9 암호화 pDNA를 전달하는 데에 사용하였다. 먼저 F3 지질나노입자로 CRISPR/Cas9 암호화 pDNA를 세포에 전달하였을 때 유전자 편집에 성공할 수 있는지 확인하였다. Cas9 단백질과 GFP 특이적 sgRNA(single-guide RNA)를 동시에 암호화하는 pDNA(pCas9-sgGFP)와 F3 지질나노입자를 합친 리포플렉스(lipoplex)인 pCas9-sgGFP/F3를 형성한 뒤, 이를 HeLa-GFP 세포에 처리하였다. GFP 유전자 편집과 단백질 발현 감소를 바탕으로, 전달한 CRISPR/Cas9이 정상적으로 작동함을 확인하였다. 다음으로는 LDHA를 겨냥한 CRISPR/Cas9을 암호화하는 pDNA(pCas9-sgLDHA)로 pCas9-sgLDHA/F3 리포플렉스를 형성하였다. 이 pCas9-sgLDHA/F3를 B16F10 종양세포에 처리한 결과, LDHA 유전자가 편집되었으며 결과적으로 세포배양 배지의 pH가 상승하였다. 또한 B16F10 종양세포를 배양했던 배지 속에서 T세포를 배양한 실험에서, pCas9-sgLDHA/F3를 처리한 종양세포의 배지에서 이를 처리하지 않은 경우에 비해 T세포의 인터페론 감마(interferon-gamma) 및 그랜자임 B(granzyme B) 생성이 활성화되었다. In vivo 마우스 종양 모델에서 pCas9-sgLDHA/F3를 면역관문억제제(immune checkpoint inhibitor)인 항 PD-L1 항체(anti-PD-L1 antibody)와 병용투여한 결과, 상승적인 종양억제효과와 생존기간 연장효과가 나타났다. 이 연구의 결과는 지질나노입자를 이용한 종양미세환경의 대사공학적 조절을 면역관문억제제와 병용한 새로운 종양억제 치료전략의 가능성을 보여준다.

Application of Functional Lipid Nanoplatforms for Diagnosis and Therapy

장수진 과학기술연합대학원대학교 한국생명공학연구원(KRIBB) 2023 국내박사

지질 기반 나노구조체는 우수한 생체적합성, 구조적 다양성, 그리고 자기조립 특성을 나타내어 진단 및 치료 응용 분야에서 매력적인 플랫 폼으로 자리잡고 있다. 본 학위논문은 비색 바이오센싱과 생물학적 페 이로드의 효율적 전달을 위한 다기능성 지질 나노플랫폼을 개발하였다. 진단 응용 분야에서, 폴리디아세틸렌(PDA)을 두 가지 시스템으로 구 현하였다. 음료 내 암페타민형 각성제와 같은 불법 약물의 신속한 육 안 검출을 가능하게 하는 전기방사 나노섬유, 그리고 식품 부패 과정 에서 발생하는 휘발성 바이오제닉 아민을 모니터링하기 위한 3차원 센 서를 제공하는 리포좀-알지네이트 하이드로젤 복합체이다. 치료 응용 분야에서, 정밀한 크기 제어와 높은 캡슐화 효율을 갖춘 균일한 mRNA 탑재 지질 나노입자(LNP)를 합성하기 위한 첨단 미세유체 플 랫폼을 구축하였으며, 효율적인 세포 내 전달과 기능적 유전자 발현을 입증하였고 siRNA, 플라스미드 DNA, 단백질, 저분자 약물을 포함한 다양한 생물학적 페이로드를 캡슐화할 수 있는 다재다능함을 보여 광 범위한 치료 응용에 활용 가능함을 확인하였다. 본 연구는 지질 나노 플랫폼을 진단과 치료 응용을 연결하는 다목적 시스템으로 확립하였으 며, 첨단 바이오센싱 기술과 정밀 나노의학을 위한 기반을 제공한다. 주요단어(Key words): 지질 나노플랫폼, 폴리다이아세틸렌, 비색 바이 오센싱, 지질 나노운반체, 약물 전달 Lipid-based nanostructures exhibit superior biocompatibility, structural versatility, and self-assembly properties, establishing them as compelling platforms for diagnostic and therapeutic applications. This dissertation develops multifunctional lipid nanoplatforms for colorimetric biosensing and efficient delivery of biological payloads. For diagnostic applications, polydiacetylene (PDA) was engineered into two distinct systems: electrospun nanofibers enabling rapid, visual detection of illicit drugs such as amphetamine-type stimulants in beverages, and liposome-alginate hydrogel composites providing three-dimensional sensors for monitoring volatile biogenic amines during food spoilage. For therapeutic applications, an advanced microfluidic platform was established to synthesize uniform mRNA-loaded lipid nanoparticles (LNPs) with precise size control and high encapsulation efficiency, demonstrating efficient intracellular delivery and functional gene expression with versatility for encapsulating diverse biological payloads including siRNA, plasmid DNA, proteins, and small molecule drugs for broad therapeutic applications. This work establishes lipid nanoplatforms as versatile systems bridging diagnostic and therapeutic applications, providing a foundation for advanced biosensing technologies and precision nanomedicine. Key words: Lipid nanoplatform, Polydiacetylene (PDA), Colorimetric biosensing, Lipid nanocarrier, Drug delivery

매립지 침출수를 이용한 미세조류 배양과 레이저 조사를 통한 지질 추출

김명균 경북대학교 대학원 2014 국내박사

In this study, the possibility of culturing microalgae in landfill leachate and elimination capacities of nutrients such as total phosphorus and total nitrogen in leachate according to growth of microalgae was identified. Because Scenedesmus sp. has several advantages such as being less contaminated by other microorganisms and having a potential of propagation in the water containing high concentration of organic materials and heavy metals, it was selected as target microalgae. For identifying the production possibility of biodiesel using biomass obtained from Scenedesmus sp. cultured in landfill leachate, lipid extraction method with laser and absorbent was developed and its optimum operation control was also sought. In addition, this study compared lipid extraction efficiency between laser extraction method under the optimum condition as derived from the experiment and existing extraction method, and also analyzed FAME content according to reaction temperature and input of catalyst for optimizing trans-esterification reaction of extracted lipid. The following results were obtained. 1. In order to test Scenedesmus sp. cultivation ability in various concentrations of landfill leachate, the microalgae's growth rate was observed. Without any alteration to the pH, the microalgae required a lag phase of about four days, and after 8∼10 days, the growth rate began to decline. When the pH was maintained at 7, the microalgae were able to grow for more than 8 days. The reason for this being, when the pH exceeded 9.3, ionized ammonia was converted into NH3, and the ammonia acts as toxic material. This showed that Scenedesmus sp. does have the potential to grow in landfill leachate, as long as the pH is maintained under 9.3. When analyzing the removal rate of TP in concentrations of landfill leachate of 10%, 30%, 50% and 100%, the results were 69.2%, 63.3%, 58.1% and 24.1% respectively. The removal rates for T-N for the same concentrations of landfill leachate were 81.7%, 71.5%, 67.3% and 26.7% respectively. 2. Scenedesmus sp. used as a target microalgae in this study showed lower total lipid content(13.2±0.73%(w/w, d.b.)) than the result(23% average lipid content of green algae) reported by literatures on the lipid content of microalgae. Research proves that lipid content in microalgae tends to increase when microalgae is faced with high stress environments such as the lack of nutrients in culture medium. Thus, it is considered that low lipid content of Scenedesmus sp. in this study is because microalgae store less fat due to an abundance of nutrient in landfill leachate. As a result of GC analysis for fatty acid composition, it showed that Scenedesmus sp. in this study has suitable composition of fatty acid(C14∼C20) which is necessary for the biodiesel production, and the order of main fatty acids was C 18:2 > C16:2 > C16:0. As unsaturated fatty acid content(68%) was higher than that of saturated fatty acid(23%), it seems that biodiesel made of lipid extracted from Scenedesmus sp. in this study has the characteristics of a good flow property at low temperature and a poor oxidation stability. 3. As a result of experiments for optimizing lipid extraction method using laser and label, 75 Wh/ℓ of consumption energy showed maximum extraction efficiency(81.8%) in the case of laser extraction method. When conducting microwave treatment for ten minutes as pre-treatment, extraction efficiency was improved up to 90.8%. In the case of laser and label extraction method, 50 Wh/ℓ of consumption energy with activated carbon showed maximum extraction efficiency (93.8%) which is higher than that of the soxhlet method (79.6%). 4. When compared to fatty acid content of extracted lipid and fatty acid extraction efficiency by each lipid extraction method to identify the possibility of transforming extracted lipid into FAME, total fatty acid contents of extracted lipid by each extraction method were respectively laser and label extraction method 853.7 mg/g oil, soxhlet extraction method 825.4 mg/g oil and laser extraction method 811.4 mg/g oil. Laser and label extraction method had also the highest value (80.1%) in extraction efficiency of fatty acid, and laser extraction method(73.7%) was more efficient than soxhlet extraction method (65.7%). It means that laser and label extraction method might have a higher production rate of biodiesel than soxhlet method as there are more unnecessary fatty acids contents(34.3%) which is not able to transform into biodiesel in lipid extracted by soxhlet extraction method. 5. In esterification reaction to reduce high acid value of lipid extracted from microalgae, optimum input of catalyst was 1.0%(w/w) which showed the highest removal rate of free fatty acid. Removal rate was lower, as input of catalyst was increased. It is likely that oil and fat content was burned because of excessive catalyst input. 6. In order to optimize trans-esterification reaction, FAME content by reaction temperature and catalytic amount was analyzed. It was identified that FAME content generally increased by increase of reaction temperature and catalytic amount. However, FAME content decreased with reaction temperature of 75℃. It indicates that transformation rate of FAME is decreased because methanol is vaporized if heating temperature is more than 65℃ in transesterification reaction when methanol reacts to extracted lipid with fixed ratio under base catalyst(KOH). In input amount of catalyst, it was identified that FAME content increases as input amount increases. Although FAME content was the highest (94.2%) at 1.5(%, w/w) of input amount, the difference when compared with 1(%, w/w) of input amount, was imperceptible.

한국인 혈청과 두개강내 동맥조직 지질성분의 비교분석

김영돈 경북대학교 대학원 2009 국내박사

A few studies have been done until recently on intracranial arterial lipid composition compared with serum lipid level and composition. Furthermore, no article has been presented so far on the lipid analyses of living human being’s arterial tissue which was taken during a surgical procedure. In view of many recent papers presented, there is a big difference between western and oriental diet, so there would be a significant difference in lipid composition of intracranial artery of oriental people and American Caucasians and American-Africans. The author analyzed the lipid composition of intracranial arteries of Koreans who suffered from cerebral aneurysm and other intracranial diseases. This prospective study compared lipid composition of American Negroes, Caucasians and Nigerian Africans presented in the literatures(20), it shows that Koreans have a much lower amount of cholesterol in intracranial arterial lipid composition. The results of this study analogize that the severity and extent of cerebral arterial atherosclerosis would be far less prominent in Koreans than American Negroes, Caucasians and Nigerian Africans. The author could find that there was a significant correlation between the ratio of serum triglyceride to total cholesterol and the ratio of triglyceride to total cholesterol in intracranial arteries, but could not find out that the increasing levels of intracranial arterial lipid composition were in proportion to increasing those of serum lipids. The level of serum triglyceride would play a more important role on atherosclerosis of small caliber arterial tissue than that of total cholesterol. The further studies on more detailed investigation about lipid composition of arterial tissue of different races should be followed in a large randomized patient population.

Lipidomic analysis of saliva, feces and serum from patients with lung cancer by nanoflow UHPLC-ESI-MS/MS

황보영 Graduate School, Yonsei University 2023 국내석사

Lipidomic analysis of feces and saliva has great potential; however, lipid profiling for lung cancer patients has mainly focused on plasma and tissue. Until now, no significant changes in fecal and salivary lipid profiles have been observed. Therefore, in this study, lipid qualitative and quantitative profiling of feces, saliva, and plasma samples from lung cancer patients were performed using nUHPLC-ESI-Tandem MS. First, the overall molecular structures of lipids in feces, saliva, and plasma were identified. Then, targeted lipid quantification were conducted and lipids that showed significant changes were selected for statistical analysis. In fecal samples, a total of 206 lipid species (phospholipids: 45, sphingolipids: 44, glycerolipids: 110, sterol lipids: 7) were identified. In saliva samples, a total of 634 lipid species (phospholipids: 415, sphingolipids: 66, glycerolipids: 150, sterol lipids: 3) were identified. In plasma samples, a total of 408 lipid species (phospholipids: 241, sphingolipids: 25, glycerolipids: 133, sterol lipids: 9) were identified. After targeted lipid quantification, fecal, saliva, and plasma lipid profiles were compared between the control group and lung cancer patients. Statistical analysis using Student's t-test, PCA, and other methods were performed to identify lipids that showed significant differences. Through ROC analysis, a total of 11, 27, and 17 lipids were selected as potential biomarker candidates in feces, saliva, and plasma, respectively. This study revealed that the lipid changes in saliva were similar to those in plasma. Moreover, the changes in saliva lipids were more pronounced compared to plasma lipids. The results suggest that more lipid species were selected as potential biomarkers in saliva compared to plasma. Salivary lipids showed a greater degree of change compared to plasma lipids, indicating the potential use of salivary lipids as early diagnostic indicators for lung cancer. 폐암은 전 세계적으로 성별 구분 없이 발생률과 사망률이 가장 높은 치명적인 질병으로 알려져 있다. 현대에 이르러 폐암 진단 및 치료 전략이 발전했지만 폐암 환자의 예후는 좋지 않은데, 이는 조기 진단 시기를 놓쳐 근치적 수술이 어려운 상태로 발견되기 때문이다. 현재까지 널리 알려진 폐암 진단 방법들은 큰 비용이 따르고 침습적인 진단 시료 채취 방법을 이용하여 환자가 순응하기 어렵다는 문제점으로 가지고 있다. 따라서 기존 진단 방법보다 비용적으로 저렴하고 쉽게 이용할 수 있는 타액과 대변과 같은 비침습적 시료에서 새로운 바이오마커 및 진단 도구의 개발이 필요한 실정이다. 대변과 타액 속 지질 분석은 큰 잠재력을 가지고 있음에도 불구하고 폐암 환자에 대한 지질체 분석은 주로 혈장과 조직에 대해서 이루어졌다. 현재까지 대변 및 타액 지질 변화는 관찰되어 지지 않았다. 따라서 본 연구에서는 nUHPLC-ESI-Tandem MS를 이용하여 폐암 환자의 대변, 타액 및 혈장에서 지질 정성 및 정량 프로파일링을 진행하였다. 먼저 대변, 타액 및 혈장 지질 분자 구조의 전체적인 식별을 진행하고, 표적 지질 정량화를 진행한 후 상당한 변화를 보이는 지질을 선택하여 통계적으로 분석을 진행하였다. 대변 시료의 경우 총 206종의 지질체 (phospholipid: 45종, sphingolipid: 44종, glycerolipid: 110종, sterol lipid: 7종)을 확인하였고, 타액 시료의 경우 634종의 지질체 (phospholipid: 415종, sphingolipid: 66종, glycerolipid: 150종, sterol lipid: 3종)을 확인하였으며, 혈장의 경우 총 408종의 지질체 (phospholipid: 241종, sphingolipid: 25종, glycerolipid: 133종, sterol lipid: 9종)을 확인하였다. 식별된 지질체들에 대한 표적 지질 정량 분석을 통해 정상군과 폐암 환자군의 대변, 타액 및 혈장 지질 비교 분석을 진행하였고, 스튜던트 t 검정, PCA 등 통계 처리를 하여 의미 있는 차이를 보이는 지질을 선별하였다. 이들은 ROC 평가를 통해 폐암 진단용 바이오마커 후보군으로서 성능을 평가하였고, 최종적으로 대변, 타액, 혈장에서 각각 11종, 27종, 17종을 선별하였다. 본연구를 통해 폐암 환자의 타액과 혈장 지질종은 유사한 변화를 보이는 것을 확인할 수 있으며, 혈장 지질의 변화에 비해 타액 지질의 변화가 더 큰 변화를 보이는 것을 확인할 수 있다. 결과적으로 더 많은 지질종이 폐암 진단용 바이오마커 후보군으로 선정되었다. 타액 지질은 기존의 혈장 지질 보다 더 많은 바이오마커 후보군을 보여주어 폐암 초기 진단 지표로서의 지질 이용 가능성을 제시할 수 있을 것이다.

Physiological and molecular characterization of lipid-associated genes in higher plants

박기열 Graduate School, Yonsei University 2018 국내박사

Lipids are essential cellular components in living organisms. They not only provide the building blocks for cell membranes and fuels for energy productions, but also have roles as regulators in various cellular processes, such as gravitropism, cell proliferation and elongation, anther dehiscence, stomatal closure, senescence, and response to both abiotic and biotic stress. The lipid-derived signal transduction pathways are modulated by wide range of lipid molecules including phospholipid, glycerolipids, sphingolipids, free fatty acids, oxylipins, sterols, and their derivatives. These lipids and their derivatives are processed and coltrolled by numerous lipid-associated proteins including lipase, lipid kinases and phosphatases, -glucosidases, and non-enzymatic lipid-binding proteins. Therefore, the investigation of cellular and molecular functions of these lipid-associated proteins is necessary to understanding of the fine-tuned signaling pathways in higher plants. In the first chapter of the thesis, we assessed agricultural properties of CaPLA1 in rice (Oryza sativa L.). CaPLA1-overexpressed transgenic rice (Ubi:CaPLA1) showed more increased root:shoot mass ratio than wild-type rice and thereby enhance biomass was observed in Ubi:CaPLA1 plants at both greenhouse and paddy field conditions. The leaf sheath analysis and qRT-PCR data revealed that rate of cell proliferation was up-regulated in Ubi:CaPLA1. The transgenic rice produced more reproductive organs including tillers, panicles, braches per panicles than wild-type, all of which resulted in grain yield better. Transcriptome analysis suggests that gene clusters that are involved in cell proliferation, lipid metabolism, redox state, isoprenoid biosynthesis were markedly changed in Ubi:CaPLA1 plants. Correlated with the transcriptome data, reduced membrane peroxidation state was observed in Ubi:CaPLA1 plants as determined by measuring levels of malondialdehyde, conjugated diene, and peroxidase activity. In addition, changed transcript levels of genes involved in isoprenoid biosynthesis in CaPLA1-overexpressing rice were confirmed by qRT-PCR and 3-hydroxy-3-methyl-glutaryl-CoA reductase activity. Taken together, we suggest that overexpression of CaPLA1 affects increased grain yield and growth by the change of gene activities involved in cell proliferation, lipid metabolism, membrane peroxidation state, and isoprenoid biosynthesis. In the second chapter of the thesis, we elucidated the cellular functions of three SRP homologs in post-germinative growth and the drought tolerance responses in Arabidopsis. SRP-overexpressors showed more increased post-germinative growth significantly better tolerance to dehydration than wild-type plants. The over-expression of SRPs resulted in increased number and size LDs in seedlings as opposed to loss-of-function mutant seedling, which were exhibited the smaller and lesser LDs. SRPs appeared polymerization properties in vivo and in vitro. SRPs were sufficient and synergistic to each other to produce LD and was involved in the formation step of LDs from ER in planta. In addition, Responsive To Desiccation 20 (RD20), a well-known stress-responsive LD-localizing peroxygenase localized to LDs in the presence of SRPs and peroxygenase activity of RD20 was correlated with proper localization of RD20 to LDs. Our data suggest that higher plant-specific SRPs play dual roles as positive factors in post-germinative growth and the dehydration tolerance by regulating of LD biogenesis. In the third chapter of the thesis, we investigated cellular functions of cytosolic RING E3 Ub-ligase encoded by AtAIRP5. In Arabidopsis, airp5 mutant plants showed in ABA-insensitive germination and stomatal closure phenotype compared to wild-type plants while AtAIRP5-overexpressing transgenic plants exhibited ABA-hypersensitive phenotypes. This ABA-positive function of AtAIRP5 was also observed in drought stress treatment. AtAIRP5 overexpressors were significantly tolerant to drought stress, as opposed to atairp5, which was markedly susceptible. The qRT-PCR analysis revealed that the transcriptional change of ABA-related stress gene cluster including ABA-mediated transcription factors and ABA-downstream genes was correlated with expression of AtAIRP5. GLL22/23, the interacting partners of AtAIRP5 were identified by yeast two-hybrid, pull-down, and BiFC assay. Both GLL22/23 were successfully polyubiquitinated by AtAIRP5 in vitro. The expressions of GLL22/23 allow recruiting one of the -glucosidase, PYK10 in planta. Our data suggest that AtAIRP5 is the positive regulator of ABA signaling by regulating -glucosidase activity. In conclusion, this thesis is the studies on physiological and molecular characterization of lipid-associated genes in higher plants. CaPLA1 is the positive regulator of cell and tissue growth by modulating cell redox state and isoprenoid biosynthesis. In Arabidopsis, SRP homologs consist of 3 genes, and we characterized that SRPs confers both post-germinative growth and drought stress response via regulating biogenesis LDs. AtAIRP5, the C3H2C3-type RING E3 Ub-ligase, is involved in ABA-dependent abiotic stress response by means of regulating both GLL22/23, size regulators of -glucosidase. These studies will help us one step closer to understanding the various cellular mechanisms which are mediated by lipid-associated proteins in higher plants. 지질은 생명체의 필수적인 구성성분이다. 지질은 세포막의 구성성분과 대사를 위한 에너지 원천으로 쓰일 뿐만 아니라, 중력인지, 세포분열과 신장, 약의 열개 (dehiscence), 기공개폐, 노화, 생물학적 스트레스, 비생물학적 스트레스와 같은 다양한 세포반응을 조절한다. 지질로부터 유래되는 신호전달 경로는 phospholipid, glycerolipids, sphingolipids, free fatty acids, oxylipins, sterols과 같은 다양한 지질과 지질 부산물들에 의해 조절된다. 이러한 지질과 부산물들의 생성과 조절에는 lipase, lipid kinases and phosphatases, -glucosidases와 같은 효소 혹은 비효소적 지질결합 단백질들이 관여한다. 따라서, 섬세하게 조절되는 식물의 다양한 신호 전달 경로를 이해하기 위해서는 세포수준, 분자수준에서의 지질-관여 단백질들의 기능 연구가 필요하다. 본 논문의 첫 장에서는, 고추의 CaPLA1 유전자를 벼에 과다발현 시켜 그 농업적 형질을 평가하고자 하였다. CaPLA1 과다발현 형질전환 벼는 야생형에 비해 지상부 대비 뿌리의 무게가 많이 나가는 것을 관찰하였고, 이로 인해 전체적인 생장이 촉진된 것을 온실과 실제 논에서 확인하였다 잎 세포 관찰과 세포주기 관련 표지 유전자의 발현 비교를 통해 CaPLA1이 벼의 세포주기의 양성조절자임을 확인하였다. 또한, 이러한 촉진생장의 결과로 CaPLA1 과다발현 벼의 수확량이 증가하였다. CaPLA1 과다발현 벼에서 세포 분열, 지질 대사, 세포 산화-환원, 이소프레노이드 생합성과 관련된 유전자군이 변화한 것을 전사체 분석을 통해 조사하였고, 효소 활성과 표지 산물의 정량을 통해 전사체 분석 결과를 재확인하였다. 이러한 결과들을 종합하였을 때, CaPLA1은 상기한 다양한 유전자군의 변화를 매개로 벼에서 촉진생장과 수확량 증가를 유도하는 것으로 생각한다. 본 논문의 중반부에서는, 발아 후 생장과 가뭄스트레스반응에 관여하는 지질방울결합 단백질인 애기장대의 세 개의 SRP 유전자군의 세포학적 기능을 규명하였다. SRP 과다발현 애기장대는 야생형에 비해 촉진생장 표현형과 함께 수분스트레스에 더 강한 표현형을 나타내는 것을, SRP 유전자의 기능을 소실한 돌연변이체는 생장저하, 수분스트레스에 민감한 표현형을 보이는 것은 확인하였다. 또한 어린 식물체에서 지질방울을 조사하였을 때, 과다발현체는 크고 많은 지질방울을, 반대로 돌연변이체는 작고 적은 지질방울을 함유함을 조사하였다. SRP 단백질들은 단일 중합체를 이룰 수 있는 기능을 가지며 지질방울의 표면에 위치함을 확인하였다. 더 나아가, SRP 단백질들은 협력적으로 세포 내에서 지질방울을 형성하는데 관여하며, 소포체로부터 지질방울이 떨어져 나오는 초기단계부터 지질방울표면에 결합하여 작용함을 관찰하였다. 비생물학적 스트레스에 반응하는 지질방울 결합단백질로 알려져 있는 RD20단백질과의 관계 연구를 통해, SRP에 의해 지질방울이 형성되어야만 RD20 단백질이 지질방울과 결합하여 효소활성을 나타낼 수 있음을 확인하였다. 이러한 결과를 통해, 고등식물에서만 발견되는 특이적인 SRP 유전자가 지질방울의 형성을 조절함으로써 생장과 수분스트레스 반응의 양성인자로 작용한다고 생각한다. 본 논문의 최종 장에서는, RING E3 Ub-ligase를 암호화하는 AtAIRP5의 세포 내 기능을 밝히고자 하였다. 애기장대에서 앱시스산에 둔감한 발아 표현형을 보이는 돌연변이체를 확보하였고 이 돌연변이체는 AtAIRP5 유전자가 소실됨을 확인하였다. 이 돌연변이는 발아뿐만 아니라 잎의 기공개폐에서도 앱시스산에 둔감한 표현형을 나타냈고, AtAIRP5 과다발현 애기장대를 제작하여 확인하였을 때, 돌연변이체와는 반대로 앱시스산에 민감한 표현형을 관찰하였다. 또한 앱시스산 표현형과 유사하게 AtAIRP5 과다발현체와 돌연변이체에서 앱시스산 관련 유전자군의 발현이 변화한 것을 확인하였다. 이러한 변화로 인해 AtAIRP5 과다발현체는 가뭄스트레스에 강한 표현형을, atairp5 돌연변이체는 약한 표현형을 나타냄을 확인하였다. 효모단백질잡종법을 통해 AtAIRP5의 결합단백질인 GLL22와 GLL23을 발견하였고, 다양한 분자생물학적, 세포생물학적 방법을 통해 이 결합을 재확인하였다. AtAIRP5가 GLL22/23을 유비퀴틴 수식화시키는 것을 확인하였고, 세포질에서 발현되는 AtAIRP5가 GLL22/23에 의해 ER-body로 추측되는 구형의 구조물로 이동(relocalization)됨을 확인하였다. GLL22/23은 ER-body 내에 존재하는 -glucosidase의 크기와 효소활성을 증가시키는 양성조절자로, AtAIRP5가 GLL22/23 의존적으로 ER-body의 -glucosidase와 같은 세포 내 위치를 나타냄을 확인하였다. 이러한 결과를 종합해 볼 때, AtAIRP5는 -glucosidase의 효소활성에 관여하는 GLL22/23을 단백질 수준에서 조절함으로 써 앱시스산 의존적 가뭄스트레스에 반응을 나타냄을 추측할 수 있다. 이처럼 고등식물에서 지질결합 단백질들은 각각의 분자생물적·세포생물학적 조절기작을 통해 세포 내에서 다양한 기능을 수행하고 있음을 알 수 있으며, 본 연구를 통해 지질 신호 네트워크를 이해하는데 한 걸음 다가갔다고 생각된다.