A Study on the transgenic rice overexpressing arabidopsis fatty acid desaturase 7 gene

Song, jin-Hee Chonnam National Univ. Graduate School 2001 국내석사

Basic Study on the Application of Therapeutic Strategies for Complement C3 Deficiency-induced Constipation

Song, Hee Jin 부산대학교 대학원 2024 국내석사

Complement component 3 (C3), a key player in various complement activation pathways essential for triggering immune responses, has recently emerged as a potential novel contributor to constipation. Noteworthy constipation phenotypes, such as a reduction in stool excretion parameters, decreased gastrointestinal (GI) transit, histological alterations in colon structure, and diminished mucin secretion, were observed in FVB/N-C3em1Hlee/Korl complement component knockout (C3 KO) mice at 16 weeks of age. Additionally, these mice exhibited fecal microbiota dysbiosis and dysregulation of the enteric nervous system (ENS) in the mid colon as a result of C3 deficiency-induced constipation. Despite these findings, there is currently a lack of fundamental research on strategies for treating constipation induced by C3 deficiency. Consequently, our study aimed to identify therapeutic drugs and novel genetic markers that could be employed in addressing C3 deficiency-induced constipation. Initially, we explored the potential of uridine (Urd) and the aqueous extract of Liriope platyphylla L. (AEtLP), both previously recognized for their constipation-alleviating effects, in ameliorating symptoms associated with C3 deficiency-induced constipation. Our investigation focused on evaluating improvements in constipation-related parameters and the underlying molecular mechanisms in C3 KO mice following treatment with Urd and AEtLP, both known for their laxative properties. Upon treatment with Urd and AEtLP, there was a substantial increase in stool parameters, including stool frequency (128% and 71% increase, respectively) and water contents (150% and 337% increase, respectively), compared to the vehicle (Veh)-C3 KO group. While the total length of the intestine remained unchanged, the length of the GI tract was extended in the Urd- and AEtLP-treated groups (36% and 18% increase, respectively). Histopathological analysis of mid colon tissue structure revealed significant enhancements in the mucosal layer's length (7% and 39% increase, respectively), the muscularis mucosae, and the expression levels of tight junction markers such as zonula occludens-1 (ZO-1), Occludin, Claudin-1, Claudin-4, and p120. Moreover, Urd and AEtLP treatment led to significant increases in mucin-related markers, including Mucin (MUC)2, MUC1, and kruppel-like factor (Klf)4, indicative of improved mucin secretion ability. Water retention markers, including aquaporin (AQP)3 and AQP8, demonstrated a substantial increase after Urd or AEtLP treatments. Additionally, the density of neuronal cells and intestinal cajal cells (ICC) significantly increased in Urd- and AEtLP-treated C3 KO mice compared to the Veh-treated group, accompanied by improvements in the excitatory and inhibitory functions of the ENS. Furthermore, concentrations of two key hormones, cholecystokinin (CCK) and gastrin, were markedly enhanced in the Urd- and AEtLP-treated C3 KO groups. Overall, these findings suggest promising therapeutic potential for Urd and AEtLP in addressing C3 deficiency-induced constipation. Second, we tried to identify the novel gene in the mid colon of C3 KO mice in response to the constipation caused by C3 deficiency. To achieve this, the total RNA extracted from the mid colon of C3 KO mice was hybridized to oligonucleotide microarrays, and the function of novel candidate gene was verified in in vitro and in vivo. C3 KO mice showed a successful reduction in the number of stools, GI transit, colon length, mucosal layer thickness, and muscle layer thickness. Overall, compared to the wild type (WT) (Fold change (FC)>=1.5), 1,237 genes were upregulated and 1,292 genes were downregulated in C3 KO mice with constipation phenotypes. Their number changes to 226 upregulated genes and 229 downregulated genes when the FC value is set to more than 2. Among them, the major genes in the downregulated categories included Eif2s3y, Uty, Kdm5d, Igkv6-32, Olfr870, Ddx3y, Endothelin-1 (Edn1) and Neurexophilin and PC-esterase domain family member 4 (Nxpe4), whereas the major genes in the upregulated categories were Pnlip, Cel, Cpb1, Ctrl, Pnliprp1, Reg1, Resistin like beta (Retnlb) and alkaline phosphatase of intestinal (Alpi). Especially, Alpi gene were selected as novel candidates because it was associated with loperamide (Lop)-induced constipation and inflammatory bowel disease (IBD). The upregulation of Alpi mRNA treated with acetate in epithelial cells derived from colon of C3 KO mice recovered the suppression of MUCs, Klf4 and AQPs mRNA detected during C3 deficiency. Also, the most constipation phenotypes including stool number, GI transit, histopathological structure of colon, mucin secretion ability and water retention capacity were significantly recovered in C3 KO mice after upregulation of Alpi gene treated by acetate. In conclusion, our studies showed that Urd and AEtLP have the potential to alleviate C3 deficiency-induced constipation through increasing mucin secretion ability, enhancing water retention capacity, promoting ENS recovery and regulating GI hormones. The findings of the current investigation offer valuable scientific insights into various functional gene groups and specific genes serving as novel biomarkers responsive to constipation induced by C3 deficiency. Furthermore, the present results indicate that Alpi gene can play an important role as the new responsible gene for constipation.

Synthesis of Fréchet-type Dendrimers Containing 2,7-Diaminofluorene Unit at Core

송희진 동아대학교 대학원 2017 국내석사

본 연구는 친핵성 첨가 반응을 이용하여 소수성 덴드론(Fréchet 덴드론)으로 부터 중심에 2,7-diaminofluorene 단위체를 갖는 Fréchet 덴드리머를 합성하였다. 중심부에 imine 작용기를 가지는 발광 덴드리머의 합성을 위해 알데하이드를 가지는 Fréchet 덴드론을 발산형 합성 방법으로 성장시켰다. 합성된 알데하이드를 가지는 Fréchet 덴드론과 중심부에 위치하는 di-amines을 반응시켜 중심부에 imine을 가진 Fréchet-타입 덴드리머를 합성하였다. 그리고 sodium borohydride를 사용하여 Fréchet-타입 덴드리머의 중심부를 amine으로 변환시켰다. 덴드리머의 발광 중심핵으로 사용되는 2,7-diaminofluorene은 두 개의 벤젠고리를 가짐으로써 발광효과를 나타냈다. 합성된 덴드리머는 전자가 풍부한 질소로부터 quenching 현상이 발생하기 때문에 형광이 약해진다. 합성한 덴드리머의 구조와 특성은 1H-NMR, 13C-NMR, IR, UV-Vis, PL, mass spectroscopy로 확인하였다. The synthetic strategy for the convergent synthesis of dendrimer, which has the chromophore at a core, was investigated using nucleophilic addition reactions between aldehyde-functionalized Fréchet dendrons and 2,7-diaminofluorene as a core. Although 2,7-diaminofluorene showed strong fluorescence due to the conjugation from two benzene rings, the synthesized dendrimer resulted in weak of the fluorescence because of quenching effect from the electron-rich nitrogen. The Fréchet dendrimer 3-Gm and 4-Gm were characterized by NMR, IR, Mass, UV-Vis and PL analysis. Furthermore, the concept of this pivotal strategy will lead to the convenient synthesis of other fluorescent nanomaterials.

Hybrid deep learning-based classification model for driver’s driving style classifications

송희진 한양대학교 대학원 2021 국내석사

In this thesis, I introduce a model for hybrid deep-learning techniques using handcrafted features to improve the performance of the classification of driving styles. In this paper, I used a convolutional neural network (CNN) with minimal changes to the model configuration for each experiment due to different data types. The input data is the data for a vehicle's controller area networks (CAN) related to an individual’s driving style. By using a raw CAN data as input data, training data for each model was converted to different types: (1) the raw CAN data; (2) FFT spectrograms converted from raw CAN data; and (3) combined data, which consists of a driving-style index calculated from CAN data and raw CAN data. The performance of the deep-learning model on given classification tasks using frequency domain spectrograms increased by 1%, and that of the model using combined data consisting of both handcrafted features and raw CAN data increased by 7%. The result shows the possibility of the performance improvement on the drivers’ driving style classification using the proposed hybrid deep learning model with handcrafted features. 본 학위논문에서는 운전자의 주행성향 분류를 위한 하이브리드 딥 러닝 모델 기법과 handcrafted features를 사용한 성능 향상에 대한 연구를 하였다. 본 연구에서는 컨볼루션 뉴럴 네트워크 (CNN) 로 구성한 학습 모델을 사용하였으며, 각 실험별로 데이터 타입에 맞춰 최소한의 모델 configuration을 수정하여 사용하였다. 입력 데이터로는 차량의 CAN 네트워크에서 추출한 시계열 데이터 중 운전자의 주행 성향과 관련된 신호만을 선정한 raw CAN 데이터를 사용하였다, 이 입력 데이터를 사용하여 각 실험별로 1) raw CAN 데이터와, 2) raw CAN 데이터를 변환한 FFT 스펙트럼 이미지 데이터, 그리고 3) 운전자의 주행성향을 잘 표현할 수 있는 CAN 데이터 기반의 주행성향 인덱스를 구하여 이를 기존의 CAN 데이터와 혼합한 combined data를 학습 데이터로 사용하였다. Raw CAN 데이터만 사용하는 모델에 비하여 FFT 스펙트럼을 트레이닝 데이터로 사용하였을 때 약 1%의 분류 정확도 향상이 있었으며, CAN 데이터와 주행성향 인덱스를 함께 사용하여 학습시켰을 때 7%의 분류 정확도 향상을 보였다. 이를 통해 handcrafted features를 추가한 하이브리드 모델을 활용하여 운전성향 분류 성능 개선이 가능함을 확인하였다.

다층건물의 수동, 능동, 복합 진동제어

宋珍熙 단국대학교 2001 국내석사

지진하중 작용시 구조물에 수동, 능동, 복합진동제어를 설치하여 그 응답효과를 비교하였다. 수동제어로는 가장 많이 사용되는 적층고무받침을 구조물의 밑면에 설치하였고, 능동제어로는 구조물의 최상층에 질량형 감쇠기를 설치하였으며, 복합진동제어는 수동과 능동을 조합하여 구조물의 밑면에 적층고무받침을, 최상층에 질량형 감쇠기를 설치하여 지진작용하중에 대한 각각의 제어성능을 비교하였다. 지진하중은 1940년 El centro와 1971년 Pacoima dam, 1985년 Mexico city 지진하중을 입력값으로 이용하였고, 구조물은 3층 건물을 사용하였다. Passive, active and hybrid vibration control are studied for their effectiveness in reducing structural vibration under seismic excitations. For the passive isolator, a laminated rubber bearing base isolator, which has been studied and used extensively by researchers and seismic designers, is studied. An active vibration is attached top of the buildings to reduce response of the structure, and a hybrid vibration controller is compared with the passively and actively structural response under the El Centro 1940, Pacoima dam 1971, and Mexico city 1985 earthquake excitation records. Structure is used 3-story buildings.

A study on the transgenic rice overexpressing Arabidopsis fatty acid desaturase 7 gene

宋珍喜 全南大學校 2001 국내석사

ω-3 fatty acid desaturase는 linoleic acid를 linolenic acid로 전환을 촉매시키는 막 결합 효소이며, 미소체와 엽록체 막에 위치하고 있다. 벼 종자의 배로부터 유래된 calli를 플라스미드 pCAMBlA1380을 가지고 있는 Agrobacterium tumefciens균주인 LBA4404와 공조 배양하였다. 플라스미드pCAMBIA1380은 선발 유전자로 hygromycin 유전자를, 비선발 유전자는 Arabidopsis thaliana(AtFAD7)로부터 가져온 fatty acid desaturase 유전자를 가지고 있으며, 재분화 과정동안 hygromycin으로 선발하였다. 형질전환의 결과 19개의 독립적인 벼를 얻었으며, 이러한 형질전환 벼는 대조구보다 더 생육이 느리며 종자 수확량이 적었다. 형질전환체 19개중 7개를 선발하여 southem hybrization, northern hybridization, 그리고 다음 세대로의 종자 유전 분석뿐 아니라 저온 저항성 실험을 하였다. southern hybridization 분석 결과 외래 유전자가 모두 형질전환체로 삽입되었음을 알 수 있었고 mRNA의 발현도 확인할 수 있었다. 그리고 형질전환체의 지방산 분석 결과 기대와 다르게 18:3함량이 대조구보다 감소되는 경향을 보였다. hygromycin 저항성 분석 결과로는 모든 형질전환체가 hygromycin 100mg/L 용액에 저항성이 있음을 알 수 있었다. 저온 저항성 실험을 위해서 5℃ 에 48시간 처리하여 malondialdehyde 함량을 분석한 결과 대부분의 형질전환체에서 MDA의 함량이 대조구보다 더 낮게 나타났다. 그 결과 F1, F10 계통에서는 대조구보다 저온에 저항성을 나타내었으나 F2, F12 계통에서는 대조구와 유사하게 나타났다. α-linolenic acid는 식물에서 상처에 유도된 유전자의활성에 관여하는 식물 생장 조절제인 jasrmonic acid의 전구체로 이용되기도 한다. 따라서 T₁세대의 형질전환체와 대조구를 가지고 상처를 처리한 것과 상처 처리하지 않은 잎의 jasmonic acid 함량을 분석하였다. 상처 처리하지 않은 잎에서 대조구보다 형질전환체 모두에서 JA 함량이 더 높게 나타났으며, 상처 처리한 잎의 JA 함량 또한 형질전환체에서 높게 증가됨을 알 수 있었다. The ω-3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. Calli induced from embryos of mature rice seeds were co-cultivated with A. tumefaciens strain LBA4404 harboring the plasmid pCAMBIA1380 as a selectable marker gene, hygromycin, and an un selectable gene, fatty acid desaturase gene from Arabidopsis thuliana (AtFAD7), then confirmed hygromycin selection followed by an efficient regeneration system. As the result of transformation, 19 individual lines were produced. This transgenic plant grew more slowly and produced less seeds than the wild-type plant. Southern hybridization, northern hybridization, and inheritance of these genes were analyzed as well as cold resistance test with seven out of 19 in the transgenic plants. Data from southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy numbers. Northern-blot analysis revealed that seven transgenic rice plants expressed AtFAD7 mRNA. Contrary to expectation, the transformants with the fad7 gene decreased 18:3 contents then that of the wild-type. The result of hygromycin resistant analysis was showed that all the transgenic lines were resistant 100mg/L hygromycin solution. Chilling injury was assessed their malondialdehyde (MDA) contents. Chilled wild type plant had a higher malondialdehyde (MDA) contents in leaves than chilled transgenic plants. The results suggest that the transgenic plants, F1, F10 expressing AtFAD7 gene, has resistant at the cold than the wild type, but F2, F12 were similar to wild type. α-Linolenic acid is a precusor of jasmonic acid, a plant growth regulator involved in the control of wound-indeced gene activation in plants. The JA contents of T₁ transgenic and wild-type plants were analyzed with unwounded and wounded leaves. The JA levels of all transformants were higher than that of the wild-type in unwounded leaves also the JA levels of F2 and F12 were highly increased in wounded leaves.

Chrysoeriol이 지방세포의 분화 억제 및 지질분해에 미치는 효과

송진희 충북대학교 2019 국내석사

서구화된 식습관과 생활패턴으로 비만인구가 증가하면서 비만은 전 세계적으로 해결해야 하는 질병 중 하나가 되었다. 비만에 의해 야기되는 여러 대사성질환의 치료제에 대한 연구가 활발히 진행되고 있으며, 특히 천연물질로부터 부작용이 적으면서 항 비만 효능을 갖는 생리활성물질 탐색 및 생물학적 기전분석에 관한 연구가 활발히 진행되고 있다. Chrysoeriol은 열대 약용 식물에 존재하는 플라보노이드의 한 종류이다. 특히, 루이보스 차의 노란색을 띠는 주요 플라보노이드로서 항산화, 항염증 등의 생리활성을 갖는 것으로 보고되어 있다. 하지만 Chrysoeriol의 항비만 효과와 이와 관련된 작용기작에 대한 연구는 미비한 실정이다. 따라서 본 연구에서는 3T3-L1 지방세포에서 Chrysoeriol의 지방 분화 및 지방 분해에 미치는 영향을 알아보고, 관련 단백질 발현을 측정하여 항비만 활성의 작용기전을 규명하고자 하였다. 지방 전구세포에서 성숙한 지방세포로 분화하는 과정에 미치는 영향을 알아보기 위해 분화과정 중 Chrysoeriol을 처리하여 지질축적률을 확인하고 분화에 관여하는 전사인자 및 marker protein의 단백질 발현 정도를 살펴보았다. 본 연구 결과 3T3-L1 지방세포에 Chrysoeriol을 처리하였을 경우 농도의존적으로 세포내 지질 축적 억제 효과를 보였다. Adipogenesis와 관련된 단백질들의 발현을 확인한 결과, 분화 초기 전사인자인 C/EBP β 와 후기 전사인자인 PPARγ, C/EBPα, 및 SREPB1c의 단백질 발현이 Chrysoeriol에 의해 유의적으로 감소하였다. 또한 Adipogenic transcriptional factor들의 조절로 인해 유도되는 Adipocyte marker gene (FAS와 aP2)의 단백질 발현도 분화를 유도하였을 때보다 Chrysoeriol을 처리하였을 때 감소하였다. 또한, 분화된 지방세포에서의 지방분해 효과를 알아보기 위해 분화유도 후 Chrysoeriol을 처리하여 지질축적률을 관찰한 결과, 지질 축적이 감소된 것을 확인했으며, 지방 분해의 산물인 Glycerol과 Free fatty acid의 방출이 증가하였다. 지방분해에 관여하는 주요 효소인 HSL, ATGL단백질의 발현이 Chrysoeriol에 의해 증가되는 것으로 보아 지방분해효과는 지방분해에 관여하는 효소의 발현 증가에 의한 것을 확인하였다. 에너지 대사의 조절 인자이며 지방 분화와 분해에 관여하는 AMPK의 활성화에 미치는 영향을 측정한 결과 Chrysoeriol에 의해 AMPK signaling pathway경로가 활성화되는 것을 확인 하였다. Chrysoeriol로 인하여 활성화된 AMPK는 지방산 산화와 관련된 효소인 CPT-1의 mRNA발현을 증가시켰으며, 지방산 합성과 관련된 효소인 ACC를 인산화 시킴으로써 불활성화 시켰다. 따라서 지방세포에서 Chrysoeriol의 지방 분화억제 및 지방분해 촉진 효과가 AMPK signaling pathway가 관여해 나타나는 것을 확인하였다. 본 연구를 통해 Chrysoeriol의 항비만 활성을 검증하고 작용기작을 규명함으로써 점차 관심이 높아지고 있는 비만뿐 만 아니라 이로 인해 야기되는 당뇨 등의 대사성질환에 대한 치료제개발의 소재로서 유용하게 이용될 것으로 사료된다. Obesity has become a worldwide health problem and causes various metabolic diseases such as type 2 diabetes. Recently, there is a growing interest in searching for alternative therapies using natural foods with fewer side effects to treat obesity. Chrysoeriol is one such flavonoid known for its antioxidant and antidiabetic activity. However, the effects of chrysoeriol and the molecular mechanism by which chrysoeriol regulates adipogenesis and lipolysis were not reported. Therefore, the purpose of this study was to investigate the effect of chrysoeriol on adipogenesis and lipolysis and elucidate its underlying molecular mechanism. To investigate the effects of chrysoeriol on adipocyte differentiation, 3T3-L1 preadipocyte differentiation was induced by adipogenic inducers in the presence or absence of chrysoeirol for 6 days. Chrysoeriol reduced the accumulation of lipid droplets stained with Oil Red O in adipocytes without significant cytotoxicity. Treatment with chrysoeriol inhibited the expression of the major adipogenic transcription factors such as CCAAT/enhancer binding proteins (C/EBPα, C/EBPβ), peroxisome proliferator-activated receptors (PPARγ) and their target genes including fatty acid synthase (FAS) and fatty acid binding protein (aP2) during differentiation of preadipocytes into mature adipocytes. Also, to investigate effects of chrysoeriol on lipolysis, chrysoeriol was used to treat fully differentiated 3T3-L1 cells. Cell supernatant was collected to assay release of free fatty acid and glycerol, and cells were lysed to measure protein expression related to lipolysis by western blot. Chrysoeirol promoted lipolysis of adipocytes, which significantly suppressed lipid accumulation in differentiated adipocytes. and increased the amount of glycerol released into the medium. Increased lipolysis was accompanied by augmented expression of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). Moreover, chrysoeriol significantly increased phosphorylation of 5'-adenosine monophosphate-activated protein kinase (AMPK) α1 and acetyl CoA carboxylase (ACC), thereby modulating fatty acid metabolism. Chrysoeriol also increased mRNA the expression of fatty acid oxidative enzyme, carnitine palmitoyl transferase-1 (CPT-1). In conclusion, these results suggest that the anti-adipogenic and lipolytic effects of chrysoeirol are associated with activation of the AMPK signaling pathway. our study suggests that chrysoeriol serve as a dietary therapy for obesity and obesity-related metabolic diseases.