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The Study of Exits Light Setting on Evacuation by Particle Swarm Optimization
No full text本文研究目的是利用粒子群最佳演算法，分析在公共空間裡設置不同位置的指示燈與數量及不同的人數，對人員逃生避難行為之影響。
根據研究顯示：(1)無論單出口或雙出口，增設門口指示燈能減少其逃生時間。(2)單出口中，增設遠出口方向指示燈比近出口方向指示燈能減少較多逃生時間。 (3)隨著人數增加，其逃生時間也增加。The purpose of this study is to utilize particle swarm optimization approach for the analysis of evacuation time in public space. The effects of various number of exit lights and population density are considered.
Using particle swarm optimization technique, the statistical quantities of evacuation time on various indoor conditions are obtained. It is shown that the evacuation time is decreased when the number of exit is increased.
Also, the evacuation time is decreased when the number of exit lights is increased.摘要 i
Abstract ii
目錄 iii
圖目錄 iv
表目錄 vi
第一章 緒論 1
1.1研究動機 1
1.2研究方法與目的 1
第二章 文獻回顧 2
2.1 粒子群最佳化演算法 2
2.2 粒子運動模型 11
2.3 人員避難特性 11
2.4 行人步行速度 15
2.5 消防法條 16
第三章 模型建構 20
3.1 模擬構想 20
3.2 模擬建構與分析 22
第四章 模擬結果與探討 26
4.1 模型分析 26
4.2 數據分析 26
4.3 結果與討論 54
第五章 結論與建議 57
5.1 結論 57
5.2 建議 57
參考文獻 5
Permeability of Asphalt Concrete Pavement Influenced by Different Binders
No full text台灣是屬於亞熱帶氣候，夏秋兩季，常有颱風襲台帶來豐沛雨量。高速公路為台灣相當重要交通道路，雨天之行車安全尤其為要，除駕駛本身應有良好駕駛習慣外，首要應降低行駛不順速率及保持適當車距，另為提升鋪面抗滑能力及排水功能，應採用多孔隙瀝青混凝土或開放級配等鋪面，以降低交通事故。
本研究針對國道六號、國道三號共9個測點，在開通後長期監測下，針對道路鋪面3種不同黏結料試驗之現地透水係數、國際粗糙指標(IRI值)、抗滑值，相互比較下，提供相關資料於公路單位供參，提高不同膠結料在各公路段之運用。
研究結果顯示，3種不同黏結料，AR-2與AR-9透水性較低之外，整體PAC鋪面仍保持良好透水性，而減噪、平坦度、車轍、鋪面結構等耐久性均在水準以上，抗滑度等行車安全性亦維持良好。而本研究3種不同黏結材料中，高黏度改質瀝青在透水性、噪音量、車轍量均有較佳的量測值。Taiwan is a subtropical climate, summer and autumn, often typhoon hit Taiwan to bring abundant rainfall. Expressway is a very important traffic road for Taiwan, rainy day driving safety is particularly necessary, in addition to driving itself should have good driving habits, the first should reduce the traffic speed and maintain the appropriate distance, and the other to enhance the anti-skid ability and drainage function, Should be used as porous asphalt concrete or open grade and other pavement to reduce traffic accidents.
Based on the long-term monitoring of National Road No.6 and National Highway No.3, the current permeability coefficient, IRI value and anti-slip value of the three different sticky materials tested on the road pavement were Comparison, to provide relevant information in the highway units for reference, to improve the use of different cement in the highway section.
The results show that the three PAC pavements still maintain good water permeability, and the durability of noise reduction, flatness, rutting, pavement and so on are in line with the low water permeability of the three different binders and AR-2 AR and AR-9 or more, anti-slip and other traffic safety is also maintained well. In this study, three kinds of different bonding materials, high viscosity modified asphalt in the water permeability, noise volume, rutting volume have better measurement.摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
1.3 研究目的 2
1.4 研究範圍 3
第二章 文獻回顧 4
2.1 國內高速公路鋪面結構形式 4
2.2 提升雨天之行車安全 8
2.2.1 鋪面之抗滑 9
2.2.2 鋪面之排水 10
2.2.3 降低路面水膜厚度 11
2.3 多孔隙瀝青混凝土鋪面摩擦層特性 12
2.4 國內外多孔隙瀝青混凝土鋪面摩擦層品質規定 15
2.4.1 國內多孔隙瀝青混凝土品質規定 15
2.4.2 國外多孔隙瀝青混凝土品質規定 17
2.4.3 美國開放級配摩擦層品質規定 20
2.4.4 國內開放級配品質規定 23
2.5 多孔隙瀝青混凝土配合設計方法 25
2.6 降低噪音效果 32
第三章 試驗與檢測方法 35
3.1 現地透水量試驗 39
3.2 現地噪音量試驗 39
3.3 平坦度試驗 40
3.4 車轍試驗 41
3.5 抗滑試驗 42
第四章 試驗檢測結果與分析 44
4.1 現地透水量分析 44
4.2 噪音量分析 52
4.3 平坦度分析 55
4.4 車轍量分析 58
4.5 抗滑度分析 61
4.6 鋪面成本效益 64
第五章 結論與建議 69
5.1 結論 69
5.2 建議 71
參考文獻 7
分子對接模擬研究人類飢餓素受體與小分子配體之接合模式及其接合彈性
No full textGhrelin, a peptide hormone, exerts various physiological effects through a G protein-coupled receptor (GPCR), called the ghrelin receptor, including pancreatic exocrine secretion, cardiovascular function, motility, prolactin release and inflammation. To simulate ghrelin's bioactivity, a homology modeling of human ghrelin receptor (hGHS-R1a) was performed with three types of receptors as templates, including Nociceptin, Beta-1 adrenergic, and two opioid receptors. The built model was validated through molecular docking of numerous ghrelin-mimetic compounds with their biological activities available in the literature. The significant correlation between the reported biological activity and docking score of each studied compound was found. This indicates our homology model of hGHS-R1a and the docking scheme can provide a reliable platform for molecular design of ghrelin-related compounds. Later, we performed docking calculations of non-peptide ghrelin-mimetic compounds to prove that our model and calculation design are suitable for all ghrelin-mimetic compounds. Furthermore, we used structure-based drug design techniques to analyze each part of ghrelin structures and molecular modeling to design natural compound derivatives which have potential to be cheaper and medicative drugs.生長激素促泌素又稱為飢餓素(ghrelin)，是在1999年由Kojima等人發現的一種28個胺基酸組成的多胜肽。當飢餓素作用到其受體時會引發許多生理作用，包括胰腺外分泌、心血管功能、促進腸胃蠕動、引發食慾、促進催乳素釋放與發炎反應等相關功能。飢餓素受體屬於G蛋白偶聯受體(GPCR)家族，是一大類膜蛋白受體的統稱，這類受體的共同點是其立體結構中都有七個跨膜α螺旋，而利用這七個α螺的排列來構成飢餓素的結合位點。為了探討飢餓素的生理活性，我們選用近年來解出的晶體結構，包含孤啡肽、β1腎上腺素和兩個鴉片受體當作模板與利用同源建模(homology modeling)方法來建立人類飢餓素受體的結構，再透過50個類胜肽的飢餓素的分子對接模擬來驗證受體的可信度。根據結果發現計算與文獻實驗結果具有顯著的相關性，進一步我們以非類胜肽的化合物做分子對接模擬，驗證我們的模型和計算設計可以通用於所有類飢餓素化合物。最後我們以藥物設計方式，分析類飢餓素化合物各部位結構的作用，並以電腦分子建構技術，設計出有潛力成為類飢餓素藥物的天然物衍生物。中文摘要 i
ABSTRACT ii
Chapter 1 1
1.1 Growth hormone 1
1.1.1 The function of GH 1
1.2 Growth hormone secretagogues 2
1.2.1 Development of GHSs 2
1.3 Ghrelin 3
1.3.1 The function of ghrelin 4
1.3.2 The related substances of ghrelin 4
1.4 G protein-coupled receptors 4
1.4.1 The classification of GPCRs 5
1.5 Ghrelin receptor 6
1.6 Aim of this study 7
Chapter 2 8
2.2 Molecular modeling 9
2.2.1 Ligand preparation 9
2.2.2 Construction of the human ghrelin receptor model 9
2.2.3 Molecular dynamics 13
2.3 Molecular docking 14
2.3.1 Ligandfit docking program 14
2.4 Scoring functions of docking 16
2.4.1 The three general classes of scoring functions 16
2.4.2 Ligand scoring 17
Chapter 3 26
3.1 Homology model of hGHS-R1a 26
3.2 Model arrangement 27
3.3 Model validation 29
3.4 Docking of native ligand: tetrapeptide 31
3.4.1 Definition of three moieties of tetrapeptide 32
3.4.2 Comparison of major interactions between two models 33
3.5 Docking analyses on the 50 ghrelin-mimetic compounds 36
3.5.1 Correlation between biological activities and docking score 37
3.6 Analysis of docking results between two models 39
3.7 Structure-based Modifications of ghrelin-mimetic compounds 40
3.8 Docking of ulimorelin 43
Chapter 4 45
References 4
Partial purification of cellulose activity- promoting enzymes from Serratia sp. and characterization of the polyphenol oxidase CueO of this bacterium
No full text木質纖維素 (lignocellulose) 其主要組成中：纖維素 (cellulose) 與半纖維素 (hemicellulose) 能被降解轉換為簡單的醣類，之後透過醱酵用以生產生物燃料 (biofuel)。但是其纖維素被木質素 (lignin) 包裹並與半纖維素形成共價結合，以及其複雜的結構使得木質纖維素不易被降解利用。本實驗室從腐爛木頭上，以系列稀釋並以鹼木素作為唯一碳源之培養基篩選到之菌種：Serratia sp.。將其接種於基本固態生長培養基上，發現Serratia sp.具有顯著之鹼木素利用性。將0.5 g/L鹼木素以及0.125 g/L甘蔗渣作為誘導物誘導相關氧化蛋白表現。三天之後破壞菌體結構，離心收取上清液並以2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 進行活性分析，其活性值為69 U/L。分析其對源於Trichoderma reesei之市售纖維水解酶 (Tre cellulase) 水解甘蔗渣時的幫助性。在pH 6.0的環境下，加入50 mU Serratia酵素液與1.8 U Tre cellulase於5 ml緩衝液作用時，還原糖含量為0.92 mg/ml，比未添加酵素液時提高了2.7倍。藉由硫酸銨沉澱、凝膠過濾層析以及陰離子交換層析純化之酵素液，由聚丙醯胺膠體分析，仍舊具有許多雜蛋白。Serratia sp.具有一多酚氧化酶CueO，推斷其為可能之相關酵素。將此段基因選殖至pETDuet-1載體中，於大腸桿菌E. coli BL21 (DE3) 中表現。發現純化之CueO，在相同條件下對Tre cellulose水解甘蔗渣時不具有幫助性。多酚氧化酶亦具有染劑脫色的能力。在pH 6.0的環境下，aniline blue與methyl blue於隔夜後，其脫色的比例達到91%與92%。在pH 8.0的環境下，remazol brilliant blue R於隔夜後，其脫色的比例達到62%。顯示其CueO具有染劑脫色之潛力。Lignocelluloses contain cellulose and hemicellulose which can be hydrolyzed into simple sugars suitable for the production of biofuels by fermentation. However, cellulose is wrapped by lignin, in turn bound covalently to hemicelluloses; this complex structure makes lignocelluloses hard to be degraded. A strain of Serratia sp. from a piece of rotting wood was isolated using agar plates with alkaline lignin as the sole carbon source in this study. This strain grows fast in alkaline lignin-containing minimal medium. 0.5 g/L alkaline lignin and 0.125 g/L bagasse were used as inducers to induce the oxidative enzyme activity of Serratia sp.. After three day culture, the cell was disrupted, the supernatant was collected by centrifugation and the activity was analyzed by using ABTS as the substrate. The activity was 69 U/L. The hydrolysis of sugarcane bagasse by Trichoderma reesei cellulases (Tre cellulases) in the presence or absence of the Serratia crude enzyme was analyzed. At pH 6.0, the addition of 50 mU Serratia enzyme into 1.8 U Tre cellulases increase the content of reducing sugar by 2.7 times compared with the reaction with Tre cellulases alone. The enzyme preparation after purification steps of ammonium sulfate precipitation, gel filtration chromatography and anion exchange chromatography still contain many proteins bands on SDS-PAGE. Serratia sp. has a polyphenol oxidase CueO that was thought to be the enzyme promoting the hydrolysis activity of Tre cellulases. The CueO gene was cloned into pETDuet-1 vector and the recombinant protein was expressed in E. coli BL21 (DE3). Nonetheless, the purified recombinant Serratia CueO was not able to promote the hydrolysis of bagasse catalyzed by Tre cellulases as did the crude Sarratia enzyme preparation. On the other hand, the recombinant CueO had the ability to decolorize several dyes. At pH 6.0, aniline blue and methyl blue could be decolorized 91% and 92% after an overnight reaction. At pH 8.0, remazol brilliant blue R was decolorized 62% after an overnight reaction, showing that Serratia CueO has the potential for bleaching of the dyes.中文摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vi
第一章 緒言 1
一、 生質能源 (biomass energy) 1
二、 木質纖維素 (lignocellulose) 2
三、 木質素的降解 3
實驗目的 6
第二章 材料與方法 7
一、 使用之菌株與載體 7
二、 培養基組成 7
三、 菌株篩選 9
四、 菌種身份鑑定 10
五、 菌種對鹼木素之利用性 12
六、 活性蛋白之表達 12
七、 蛋白氧化活性之測定 12
八、 蛋白質定量 13
九、 不同比例之誘導物對蛋白活性之影響 13
十、 測定與纖維酶水解甘蔗渣時之幫助性 13
十一、 DNS法測定還原糖含量 14
十二、 與協助纖維酶水解相關之活性蛋白的純化 14
十三、 聚丙醯胺膠體電泳 17
十四、 蛋白之活性染色 17
十五、 純化蛋白性質之分析 18
十六、 Serratia sp.多酚氧化酶基因之選殖 19
十七、 多酚氧化酶之表達 22
十八、 多酚氧化酶之純化 22
十九、 多酚氧化酶對染劑脫色之影響 23
第三章 結果 27
一、 菌株身份鑑定 27
二、 蛋白活性分析 27
三、 與協助纖維酶水解相關之活性蛋白的純化 28
四、 純化蛋白性質之分析 29
五、 多酚氧化酶之表達與純化 30
六、 多酚氧化酶對染劑漂白之影響 31
第四章 討論 32
一、 菌株篩選 32
二、 蛋白之活性分析以及測定與纖維水解酶共同作用時的幫助性 32
三、 Serratia sp.之CueO對於染劑漂白之應用性 34
第五章 參考文獻 61
第六章 附錄 66
附錄一，FavorPrepTM GEL/PCR Purification Kit 66
附錄二，FavorPrepTM Plasmid Extraction Mini Kit 6
Pancreatic lipase inhibitory activity of strictinin from raw pu-erh tea
No full text茶為日常生活中相當普遍的飲品，飲茶一般被認為有減肥消脂之保健功效，分析各種不同茶之成分可以發現生普洱茶中富含一種特有成分-木麻黃素(strictinin)。根據先前研究指出，分析普洱茶的粗萃物具有抑制胰脂解酶活性之功效。目前臨床減肥藥奥利司他(Orlistat)之作用機制為藉由抑制胰脂解酶活性，使食物中脂質無法被分解成脂肪酸(fatty acid)和單油甘脂(monoacylglycerol)，而無法被生物體吸收和利用，從而減少熱量攝入達到體重控制之作用。本實驗先藉由體外試驗來檢視生、熟普洱茶以及茶中主要成分對於胰脂解酶活性之影響，得知生普洱茶之茶湯初萃物對於抑制胰脂解酶活性之效果較佳，進一步從生普洱茶湯中萃取並利用Sephadex LH-20管柱分離純化出木麻黃素。實驗結果顯示其對於抑制胰脂解酶活性有明顯之效果。而後為檢視在生物體中木麻黃素是否抑制胰脂解酶之活性而減少對食物中脂質之吸收，對小鼠進行脂質耐受性試驗，實驗結果顯示給予木麻黃素(130 mg/kg)之組別血液中三酸甘油脂含量較未給予木麻黃素的組別低，進一步在餵食高脂飼料並長期投藥之小鼠組別中觀察到體重增長速度較緩以及脂肪堆積較少等結果。故綜合實驗結果後得知生普洱茶當中比其他茶類含有更多木麻黃素之成分，能夠抑制胰脂解酶之活性，藉此減少身體對食物中脂肪的吸收來達到減肥消脂的功效，同時具有減緩體重之增加、減少三酸甘油脂與膽固醇形成、降低血糖以及促進胰島素分泌、控制脂肪細胞大小等多種對於代謝症候群改善之效果，藉由抑制消化相關酵素來達到肥胖治療功效是近年來較主要開發減肥藥物的模式，日後若開發成保健食品能夠成為有效緩解代謝症候群之天然物。Tea is a common drink in daily life, drinking tea is generally considered with many health benefits, such as weight loss and reduce deposits of fat. Inhibition of lipase activity is one of the major treatment of diet-induced obesity recently. By inhibiting fat become fatty acid and monoacylglycerol, it prevent over intake of calorie. Orlistat is a clinical drug for weight loss through the mechanism which inhibit lipase activity. According to previous studies, the pu-erh tea crude extract can inhibit lipase activity. Our lab has discovered that raw pu-erh tea contained large amount of strictinin. Strictinin might be a possible cardiant as lipase inhibitor. In the beginning of this study, lipase activity inhibition was test by various kinds of tea in vitro. Preliminary evidence suggests that crude extract of raw pu-erh tea has stronger inhibitory effect than other kinds of tea. Then, strictinin was further isolated from raw pu-erh tea by Sephadex LH-20 column. In the lipase inhibition assay, Strictinin show strong lipase inhibition effect. Thus, strictinin was further tested through animal model. In olive oil tolerance test, the result indicated that the group with strictinin (130 mg/kg) administration of C57BL6 mice has lower triacylglycerol concertation in blood than the group without strictinin. In a long term experiment, the group having high-fat diet with strictinin (130 mg/kg) also has lower weight gain and less adipocyte buildup. These results indicate that strictinin in raw pu-erh tea inhibit the activity of lipase to reduce the fat uptake in food, also control the body weight. Therefore, it will have therapeutically potential to relieve metabolic syndrome.中文摘要……………………..………………………………………..…..i Abstract………………..…………………………………………...….….iii
目錄………………………………………….……………………………iv
圖表目錄…………………………………………………………………vii
縮寫表…………….…………………………………………..…………viii
壹、 前言…………………………………………………………...…..1
貳、 文獻回顧…………………………………………………..……...5
一、 普洱茶………………………………………..………………5
二、 茶葉的化學組成……………………………..…………..…..6
三、 木麻黃素……………………………………………………10
四、 減肥藥物與相關機制………………………………………11
五、 脂解酶………………………………………………………13
參、 材料與方法……………………......…………….………………16
一、 實驗材料………………..……………….…….……………16
(一) 茶葉樣品…………..………………………..……………..16
(二) 藥品與化學試劑…………………………………..………16
(三) 儀器設備……………………………………………..……17
(四) 實驗動物……………………………………………..…....18
二、 實驗方法……………….…………………………………..18
(一) 木麻黃素純化流程……………….…………………….…18
(二) 胰脂解酶活性測試……………….……………………….19
(三) 動物實驗…………………………….…………………….20
1. 油脂耐受性測試………………….……………………20
2. 長期高油飲食下，木麻黃素對生理代謝之影響.…....20
(四)統計分析………………………………………….…...21
肆、 結果………………………………………………………...……22
一、HPLC分析常見各種茶湯之成分差異及木麻黃素之純化…...22
二、木麻黃素對於胰脂解酶活性之影響………………………..…22
三、木麻黃素及EGCG之抑制效率比較……………………….…24
四、動物實驗……………………………………………………..…25
(一) 小鼠油脂耐受性測試………………………………………...…25
1. 木麻黃素與orlistat對於油脂吸收之影響………….…...…25
2. 木麻黃素與orlistat有效劑量比較……………………........25
(二)長期高油飲食下，木麻黃素對生理代謝之影響……..….….....26
1. 體重變化與食物攝取量……………………………..…...…26
2. 血糖與胰島素變化……………………..…………….…..…28
3. 各組織重量………………………………………………..…29
4. 血液生化數值分析………………………………………..…30
5. 脂肪組織切片……………………………………….…….…31
伍、 討論………………………………………………………...……32
陸、 參考文獻………………………………………………….……..5
Development of Foxtail mosaic virus as a VIGS vector
No full text近年陸續多種植物基因體已完成解序，包括水稻、大麥、小麥、二穗短柄草、阿拉伯芥及番茄等。為了進行植物功能性基因體的研究，利用病毒誘導基因靜默技術(virus-induced gene silencing, VIGS)可在短時間內快速靜默目標基因。由於病毒感染植物後，在植物上會造成嵌紋斑點或條斑狀的病徵並影響植物生長發育，不利於研究。本研究使用的病毒為狐尾草嵌紋病毒(Foxtail mosaic virus, FoMV)，FoMV全長為6,161個核苷酸，隸屬Potexvirus屬。FoMV寄主範圍甚廣，主要感染禾本科植物，包括現行主要糧食作物，因此將FoMV開發成VIGS系統，有利於糧食作物的基因體研究。在前人研究中，將FoMV之鞘蛋白啟動子序列重複構築，插入目標基因片段誘導植物發生基因靜默現象，開發過程包括探討目標基因序列的限制長度，及發展雙基因靜默系統，以避免發生基因靜默後卻沒有明顯的表觀變化。其中當FoMV感染二穗短柄草後，病毒在植物會造成褐色條斑狀病徵，並且在短時間內就會死亡；而FoMV感染大麥後，病徵表現上雖不似二穗短柄草嚴重，但仍然不利於觀察。有報導指出病徵表現與病毒的外鞘蛋白有關，因此本研究將針對FoMV的鞘蛋白進行突變。透過外鞘蛋白N端刪減序列後並接種到白藜與二穗短柄草觀察病徵表現，刪減5到9個胺基酸之突變株皆能於這兩種植物感染並複製，其中接種突變株pCFNd8及pCFNd9在二穗短柄草上的生長趨勢較不受影響外，病徵表現也弱於野生株。將此二突變株開發成VIGS載體並接種到白藜與二穗短柄草，pCFSDNd8及pCFSDPDSIRNd8可感染這兩種植物，但pCFSDPDSIRNd9則無法感染。另外也透過error-prone PCR進行外鞘蛋白突變，並建立突變庫，期望得到弱病毒株，以利進一步作為VIGS載體系統及外源蛋白表現系統等多元用途。Recently, genomes of many plants, including rice, barley, wheat, Brachypodium distachyon, Arabidopsis thaliana and tomato have been completely sequenced. With these powerful database, scientists can focus on the genes they interested. Virus-induced gene silencing (VIGS) is one of the important tool which has been used for plant functional gene studies based on the high knockdown efficiency. During viral infection, the symptoms on plants are not only mosaic, spots and strips, but the interference of plant growth. Foxtail mosaic virus (FoMV) has a 6,161 nt positive sense genome and belongs to the Potexvirus genus, which infects a wide range of hosts, especially the Poaceae comprising crops. Thus FoMV is worthy for establishing as a VIGS system. Previous studies have constructed a serial of FoMV derived vectors which carrying the repeat sequence of FoMV coat protein promoter for gene silencing. However, the clones cause severe symptoms that we can't distinguish the phenotype resulting from the VIGS. Previous studies had shown that Potexvirus coat protein involves in the symptom expression. In this study, we have modified FoMV coat protein N terminal by deletion of 5 to 9 amino acids. The resulting mutants can infect Chenopodium quinoa and B. distachyon. B. distachyon inoculated with pCFNd8 and pCFNd9 did not show the negative growth effects, and induced milder symptom or than wild type FoMV did. The pCFSDNd8 and pCFSDPDSIRNd8, which carries no-inserted sequence and inverted repeat PDS partial sequence respectively, can infect and replicate in C. quinoa and B. distachyon, but pCFSDPDSIRNd9 is non-infectious. We also use error-prone PCR to creat the CP mutant library of FoMV, for further development of a more effective VIGS vector or platform for expressing exogenous protein.中文摘要 i
英文摘要 ii
壹、前言 1
一、病毒誘導基因靜默系統(Virus-Induced Gene Silencing, VIGS) 1
二、VIGS在禾本科植物上的研究 2
三、狐尾草嵌紋病毒 4
四、二穗短柄草 5
五、外鞘蛋白與病徵的關係 6
六、研究目的 7
貳、材料與方法 8
一、構築外鞘蛋白N端刪減序列之狐尾草嵌紋病毒質體 8
二、構築重複外鞘蛋白次基因體啟動子於外鞘蛋白N端刪減之狐尾草嵌紋病毒質體 8
三、易錯聚合酶連鎖反應(Error-prone PCR) 9
四、大腸桿菌勝任細胞製備及轉形作用 9
五、狐尾草嵌紋病毒質體純化 10
(一)、聚合酶連鎖反應 10
(二)、質體小量純化 10
(三)、質體中量純化 11
(四)、菌種保存 12
六、植物接種狐尾草嵌紋病毒 12
(一)、供試植株 12
(二)、接種測試 12
七、植物總量蛋白質抽取與分析 12
(一)、萃取植物總量蛋白 12
(二)、聚丙醯胺膠體電泳 13
(三)、西方轉漬法 13
(四)、西方墨點法(dot blot) 14
參、結果 15
一、植物接種測試 15
(一)、狐尾草嵌紋病毒外鞘蛋白N端刪減序列株接種Chenopodium quinoa之測試 15
(二)、狐尾草嵌紋病毒外鞘蛋白N端刪減序列株接種Brachypodum distachyon (Bd 21-3)之測試15
(三)、狐尾草嵌紋病毒接種Bd21-3之病徵表現16
二、外鞘蛋白N端刪減8個及9個胺基酸之狐尾草嵌紋病毒誘導基因靜默(VIGS)載體系統之接種測試 16
(一)、構築FoMV重複外鞘蛋白次基因體啟動子(duplicated CP sgRNA promoter)於CP N端刪減病毒重組株 16
(二)、外鞘蛋白N端刪減序列之狐尾草嵌紋病毒VIGS載體接種白藜16
(三)、外鞘蛋白N端刪減序列之狐尾草嵌紋病毒VIGS載體接種Bd 21-3 17
三、建立狐尾草嵌紋病毒外鞘蛋白N端隨機突變株庫 17
(一)、構築隨機突變株 17
(二)、突變株接種測試 18
肆、討論 19
伍、參考文獻 22
陸、圖表與附錄 2
Using mutant pheS gene as the counter-selection marker to delete the kstD genes in Rhodococcus equi
No full text4-雄烯二酮 (4-Androstene-3,17-dione, AD)在固醇藥物 工業的製程上 是一個重 要的前驅物，能經由一系列化學合成來形不同這類廣泛應用於醫藥產業，例如消炎、避孕等。在自然界中部分微生物具有降解固醇的能力，而 4-雄烯二酮為降解路徑之中間物，因此以微生來產 4-雄烯二 酮具有相當高的潛力。本實驗研究主角馬紅球菌 (Rhodococcus equi)屬於 革蘭 氏陽性菌 。3-ketosteroid Δ1-dehydrogenase (kstD)在固醇代謝的路徑上扮演 在固醇代謝的路徑上扮演 ， 將 4-雄烯二酮催化成 1,4-雄二烯酮 (1,4-Androstadiene-3,17-dione , ADD) 的角 色。根據基因組資訊預測馬紅球菌存在著五個不同的 kstD基因，編碼分別 是 Req_06770、Req_10320、Req_13080、Req_15820、Req_30200。目前對於這 。目前對於這 些基因在固醇代謝路徑中的角色仍缺實驗證明 。因此本實驗先建立一套適合馬 紅球菌的非標記性基因剔除方法，逐一 在實驗室的 馬紅球菌 UV1(R. equi UV1)菌株上 進行個別 kstD基因剔除。目前已篩選到四株菌， 基因標號 分別是 Req_10320、Req_13080、Req_15820與 Req_06770。之後 針對基因剔除菌株， 再以薄層分析 (Thin layer chromatography)觀察 4-雄烯二酮累積情況。結果發現 這四株 kstD剔除菌株皆未能在培養基中累積 4-雄烯二酮。我們推測主要負責 4-雄烯二酮催化的基因尚未被成功剔除，抑或是這五個 KSTD蛋白皆具有轉換 4-雄烯二酮成 1,4-雄二烯酮的功能，導致被代謝而無法順利累積。 我們將於馬紅球菌中持續剔除 Req_30200或於同一株菌中累積多個剔除基因， 或於同一株菌中累積多個剔除基因， 以得到能累積 4-雄烯二酮的菌株。4-Androstene-3,17-dione (AD), an important precursor in steroids industry, can be transformed into a variety of steroids via a series of chemical synthesis. These steroids have been widely used as anti-inflammatory drugs, immunosuppressant, and contraceptives etc. In nature, some microorganisms are able to degrade sterol; therefore, utilization of these microorganisms to accumulate AD is considerably potential. Rhodococcus equi, a gram-positive bacterium, use the 3-ketosteroid Δ1-dehydrogenase (kstD) to converse AD into 1,4-androstadiene-3,17-dione (ADD) in steroid metabolism. According to the genomic information of R. equi, there are probably five kstD paralogs, which include Req_06770, Req_10320, Req_13080, Req_15820, and Req_30200, in R. equi. Nonetheless, the role of these genes in the sterol pathway is still unclear. In this study, an unmarked gene knockout method applicable to R. equi was established to delete the putative kstD genes and the resulting effects on AD accumulation in the culture medium were assayed by thin layer chromatography (TLC). So far Req_10320, Req_13080, and Req_15820、Req_06770 have been deleted respectively. Unfortunately, none of the four kstD-deleted strains could accumulate AD. We presume either the major gene responsible for AD to ADD conversion has not been deleted or all the KSTD proteins are involved in AD catabolism so that single kstD deletion cannot result in AD accumulation. In the future, we will continue to delete Req_30200 and create mutant strains with multiple kstD gene deletions in order to obtain the ideal strain for AD production.中文摘要........................... i
Abstract........................... ii
目錄............................... iii
表圖目錄........................... iv
第一章 緒論
第一節 固醇類化合物...................... 1
第二節 類固醇藥物的製造與合成............. 4
第三節 馬紅球菌與其膽固醇降解的代謝途徑.... 8
第二章 研究目的
第一節 先前研究......................... 11
第二節 篩選策略......................... 12
第三節 研究目標......................... 13
第三章 材料與方法
第一節 實驗材料......................... 15
第二節 實驗方法......................... 18
第四章 實驗結果......................... 24
第五章 討論............................ 48
參考文獻................................. 5
A study on the function of non-specific phospholipase Cs (NPCs) in growth and development in Arabidopsis thaliana
No full textPhospholipases play crucial roles in plant membrane lipid signaling and homeostasis. Non-specific phospholipase C (NPC) is a unique class of phospholipase found only in plants and certain bacteria. NPCs catalyze the hydrolysis of primary membrane phospholipid classes including phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to release diacylglycerol (DAG) and the corresponding phosphoaminoalcohol. Six isoforms of NPCs were found in Arabidopsis thaliana, designated NPC1-NPC6. Although the physiological roles for most NPC isoforms in stress response have been described, function of NPC2 and NPC6 is unkown. In addition, for characterized NPC isoforms, molecular mechanism by which these NPCs mediate the stress response remain elusive. In this thesis, I report on our investigation about NPC2 and NPC6 in gametophyte development (Chapter 2) and NPC3 in ER stress response (Chapter 3). In Chapter 2, we showed that NPC2 and NPC6 are required for male and female gametophyte development in Arabidopsis. We found that npc2-1/- npc6-2/- was not retrieved due to the lethal effect. Both NPC2 and NPC6 proteins are localized to the plastids. Promoter-GUS assays in transgenic Arabidopsis revealed that NPC2 and NPC6 are preferentially expressed in floral organs rather than in leaves. In vitro enzyme assays showed that NPC2 and NPC6 hydrolyze phosphatidylcholine and phosphatidylethanolamine, but not phosphatidate. Lipidomic analysis of npc2-1/- npc6-2/+ and npc2-1/+ npc6-2/- showed increased contents of PC, PE, and PG in floral buds but not in mature flowers. Thus, our results indicate that NPC2 and NPC6 are functional NPCs involved in gametophyte development and glycerolipid metabolism in floral buds of A. thaliana. On the other hand, lipid metabolism is altered during endoplasmic reticulum (ER) stress in animal cells. In plants, however, was still little known. In Chapter 3, we showed that a T-DNA insertional mutant of NPC3, designed npc3-3, was hyposensitivity to Tunicamycin (TM)-induced-ER stress treatment compare to the wild-type plants. Under normal growth condition, we found that expression levels of ER stress response genes were higher in the npc3-3 mutant than that of the wild type. NPC3 has phospholipase C activity for PC and PE. Confocal microscopy observation of the subcellular localization of NPC3 showed that NPC3 was localized to the ER under normal condition but was found in nucleus after TM treatment. Our data demonstrated the requirement and nuclear translocation of NPC3 in response to ER stress. Taken together, in Chapter 4, we summarized our major findings on the NPCs, discussed on the differential roles of NPCs, provided testable hypothesis, and experimental plans. In summary, our findings described in this thesis conceptually advance our understanding on the function of NPCs in ArabidopsisAcknowledgements i
Abstract iii
List of Tables vi
List of Figures vii
List of Abbreviations ix
CHAPTER 1 1
General Introduction 1
CHAPTER 2 18
A Pair of Non-specific Phospholipases C, NPC2 and NPC6, is Involved in Gametophyte Development and Glycerolipid Metabolism in Arabidopsis 18
Abstract 19
Introduction 20
Materials and Methods 23
Discussion 36
CHAPTER 3 65
Endoplasmic Reticulum-Nuclear Translocation of Non-specific Phospholipase C3 Involved in Endoplasmic Reticulum Stress Response in Arabidopsis thaliana 65
Abstract 66
Introduction 67
Materials and Methods 69
Results 74
Discussion 78
CHAPTER 4 90
Conclusion and Future Perspectives
References 97
Resume 10
Investigation of different enzymes, ultrasonic treatments, and hydrolysis conditions on the antioxidant properties of porcine liver protein hydrolysates
No full text豬肝是豬屠宰的副產品，富含蛋白質及各種營養素，然而台灣較少利用。酶水解用於回收及改善來自魚類和動物副產物的蛋白質之營養及功能特性，以產生機能性成分。超音波已被用於改善蛋白質水解及修飾酶活性。因此，本研究之目的是評估不同酶，超音波處理和水解條件對豬肝酶水解產生水解產物的影響，並進一步評估其抗氧化活性。
試驗一，評估使用Alcalase®，木瓜蛋白酶，胃蛋白酶或紅麴黴菌微生物懸浮液 (分別為APLH，PaPLH，PePLH和MPLH) 在各種水解時間 (3, 6和12h) 後製備的豬肝蛋白水解物之抗氧化活性。結果顯示APLH之產率及胜肽含量最高，PaPLH的水解率高於其他組別。MPLH具有最高的2, 2-diphenyl-1-picrylhydrazyl (DPPH)自由基清除活性及還原能力，APLH及PaPLH具有較MPLH更高的亞鐵離子螯合能力，所有水解產物的分子量小於10 kDa。PaPLH的總胺基酸及疏水性胺基酸含量最高。15個從MPLH獲得的抗氧化胜肽片段含有一個或多個胺基酸 (酪胺酸，色胺酸，丙胺酸，脯胺酸，蛋胺酸，賴胺酸，天冬胺酸，半胱胺酸，纈胺酸，亮胺酸或組胺酸等)。儘管由紅麴黴菌微生物懸浮液水解的水解產物顯示出最高的DPPH自由基清除活性及還原力，但是其產量和水解率最低。因此，選擇具有能產生最高產量、胜肽含量及和亞鐵離子螯合能力之Alcalase®進行後續試驗。
試驗二中，進而藉由超音波處理來提高水解產物的抗氧化活性。在試驗中，評估超音波預處理 (0, 15, 30, 45及60 s) 對使用Alcalase®的豬肝蛋白水解物 (PLPH) 的抗氧化活性之影響。隨著超音波處理時間的延長，PLPH的水解率及胜肽含量增加。60秒超音波預處理的水解產物顯示出最高的水解率及胜肽含量。45秒超音波預處理的水解產物顯示出最高的亞鐵離子螯合能力及還原力。30秒超音波預處理的水解產物顯示出最高的DPPH自由基清除活性及在亞麻油酸自氧化系統中較高的抑制活性；所有水解物胜肽的分子量小於6,200 Da。
試驗三進一步藉由運用反應曲面法(Response surface methodology; RSM) 來評估不同水解參數 (E/S比，pH值和溫度) 對超音波輔助酶水解豬肝蛋白質水解物(PLH)的影響。結果顯示E/S比、pH值及溫度對豬肝水解物的抗氧化活性有顯著影響(P <0.01)。當E/S比1.4% (v/w)、溫度55.5 °C及初始pH值10.15時，能產生具有最高DPPH自由基清除活性的PLH，其水解率、DPPH自由基清除活性、亞鐵離子螯合能力、還原力、分子量及疏水性胺基酸含量分別為24.12%、79%、98.18%、0.601、< 5,400 Da、45.7%。
由本研究結果得知，藉由使用紅麴黴菌微生物懸浮液、應用超音波處理及選擇最佳水解條件進行酶水解時，可自豬肝中獲得性能優良的抗氧化水解物，具有預防食品中脂質氧化的潛在應用。Porcine liver, a by-product of pig slaughtering, is rich in proteins and various nutrients. However, it is rarely used in Taiwan. Enzymatic hydrolysis is used to recover and improve the nutritional and functional properties of proteins from fish and animal byproducts to yield functional components. Ultrasonic has been used to improve protein hydrolysis and modify enzyme activities. Therefore, the objective of this study was to evaluate the effects of different enzymes, ultrasonic treatments, and hydrolysis conditions on the enzymatic hydrolysis of porcine liver for the production of hydrolysates and further characterization of their antioxidant activities.
In the experiment 1, the antioxidant activity of porcine liver protein hydrolysates (PLPH) prepared by using Alcalase®, papain, pepsin, or Monascus purpureus microbial suspension (APLH, PaPLH, PePLH, and MPLH, respectively) after various hydrolysis time (3, 6, and 12 h) was evaluated. The results exhibited that the highest yield and peptide content were obtained from APLH, whereas the degree of hydrolysis (DH) of PaPLH was higher than the others. MPLH had the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reducing power, whereas APLH and PaPLH had higher ferrous ion-chelating ability than did MPLH. The molecular weights of all the hydrolysates were <10 kDa. The PaPLH exhibited the highest contents of total amino acids and hydrophobic amino acids. Fifteen antioxidant fractions obtained from MPLH contained one or more of the following amino acids in their sequences: Tyr, Trp, Ala, Pro, Met, Lys, Asp, Cys, Val, Leu, and His. Even though the hydrolysate hydrolyzed by Monascus purpureus microbial suspension exhibited the highest DPPH free radical scavenging activity and reducing power, it had the lowest yield and DH. Therefore, Alcalase® which exhibited the highest yield, peptide content and ferrous ion-chelating ability has been selected to hydrolyze porcine liver proteins in the next experiment.
In the experiment 2, ultrasonic was applied on the enzymatic hydrolysis in order to improve the antioxidant activity of the porcine liver hydrolysate. The effect of the ultrasonic pretreatment (0, 15, 30, 45, and 60 s) on the antioxidant activity of PLPH employing Alcalase® was evaluated. The results exhibited the DH and peptide contents of the PLPHs increased as the time of ultrasonication increased. The hydrolysate pretreated with ultrasonication for 60 s exhibited the highest DH and peptide contents. The hydrolysate pretreated with ultrasonication for 45 s exhibited the highest ferrous ion chelating ability and reducing power. The hydrolysate pretreated with ultrasonication for 30 s exhibited the highest DPPH radical scavenging activity and the higher inhibitory activity in the linoleic acid autoxidation system. The molecular weight of the peptides in the hydrolysates was less than 6.2 kDa.
In the experiment 3, by using response surface methodology (RSM), the effect of various hydrolysis parameters (E/S ratio, pH, and temperature) on the ultrasonic-assisted enzymatic hydrolysis of PLPH was evaluated. The results exhibited that E/S ratio, pH, and temperature significantly affected the antioxidant activity of the hydrolysate (P < 0.01). The optimal conditions for producing PLPH with the highest scavenging activity by using RSM were as follows: E/S ratio, 1.4% (v/w); temperature, 55.5°C; and initial pH, 10.15. Under these conditions, the degree of hydrolysis, DPPH free radical scavenging activity, ferrous ion chelating ability, and reducing power of PLPHs were 24.12%, 79%, 98.18%, and 0.601, respectively. The molecular weight of PLPH produced under these optimal conditions was less than 5,400 Da and contained 45.7% hydrophobic amino acids.
In conclusion, favorable antioxidant hydrolysates from porcine liver enzymatic hydrolysis, which have a potential application for retarding lipid oxidation in food products, can be obtained by using Monascus purpureus microbial suspension, applying of ultrasonic treatment as well as selecting of the optimal hydrolysis conditions.誌 謝 辭 i
摘 要 ii
ABSTRACT iv
TABLE OF CONTENTS vii
LIST OF TABLES xi
LIST OF FIGURES xii
CHAPTER 1 1
1.1 Animal by-products/Porcine liver 2
1.2 Monascus spp. 3
1.3 Enzymes 3
1.3.1 Aspartic proteinase 4
1.3.2 Alcalase® 4
1.3.3 Pepsin 5
1.3.4 Papain 5
1.4 Protein hydrolysates 6
1.4.1 Enzymatic hydrolysis 7
1.4.2 Factors affecting enzymatic hydrolysis of proteins 8
1.5 Ultrasonication 11
1.5.1 Introduction 11
1.5.2 The effects of ultrasonication on protein structures, functionality, and performance 12
1.6 Response surface methodology (RSM) 14
Aim of this study 15
CHAPTER 2 17
2.1 Abstract 18
2.2 Introduction 18
2.3 Materials and methods 21
2.3.1 Preparation of M. purpureus microbial suspension 21
2.3.2 Preparation of porcine liver hydrolysates using various proteases 21
2.3.3 Measurement of yields and DH of hydrolysates 22
2.3.4 Determination of peptide content 22
2.3.5 Determination of antioxidant activities 23
2.3.6 Determination of amino acid composition and molecular weight distribution of PLPHs 23
2.3.7 Determination of antioxidant peptide sequence 24
2.3.8 Statistical analysis 24
2.4 Results and Discussion 24
2.4.1 Enzymatic hydrolysis 24
2.4.2 Antioxidant properties of hydrolysates 26
2.4.3 The molecular weight distribution of protein hydrolysates 28
2.4.4 Amino acid composition of hydrolysates 29
2.4.5 Characterization of antioxidant peptides 30
CHAPTER 3 40
3.1 Abstract 41
3.2 Introduction 41
3.3 Materials and methods 43
3.3.1 Materials 43
3.3.2 Ultrasonic pretreatment of porcine liver 43
3.3.3 Determination of soluble protein content 44
3.3.4 Preparation of porcine liver protein hydrolysates 44
3.3.5 Determination of the DH of hydrolysates 45
3.3.6 Determination of peptide contents 46
3.3.7 Determination of antioxidant activities 46
3.3.8 Inhibition of linoleic acid autoxidation 46
3.3.9 Determination of the molecular weight distribution of hydrolysates 47
3.3.10 Statistical analysis 48
3.4 Results and discussion 48
3.4.1 DH, soluble protein content, and peptide content 48
3.4.2 Antioxidant activities of hydrolysates 50
3.4.3 Molecular weight distribution of protein hydrolysates 52
CHAPTER 4 59
4.1 Abstract 60
4.2 Introduction 61
4.3 Materials and methods 62
4.3.1 Preparation of PLPHs 62
4.3.2 RSM 62
4.3.3 DH measurement 63
4.3.4 Antioxidant activity measurement 63
4.3.5 Determination of PLPH molecular weight distribution and amino acid composition 64
4.3.6 Statistical analysis 64
4.4 Results and discussion 64
4.4.1 Optimization of the hydrolysis process 64
4.4.2 Visual analysis of the RSM experiment 66
4.4.3 Optimization of technical conditions 67
4.4.4 DH and antioxidant activity 68
4.4.5 Molecular weight and amino acid composition of the PLPH produced under optimal conditions 69
CHAPTER 5 77
5.1 Conclusion 78
5.2 Future research 79
REFERENCES 8
Applying the GWMA Method and VIX Index in Forecasting the Taiwan Stock Price
No full text在時間序列模型上，常使用指數加權移動平均(EWMA)，使各數值的加權影響力隨時間而指數遞減，越近期的數值加權越重，來降低因趨勢出現的結構性變異，使預測模型能得到最佳的預測結果。過去的研究提出廣泛指數加權移動平均(GWMA)的方法，比EWMA多了一個調整參數，在偵測微小變化時更為敏感，在實務的應用上也較EWMA廣泛。因此，本論文利用GWMA的方法並導入波動率指數(VIX)來預測台灣加權股價指數(TAIEX)，並利用數值方法找出GWMA的最佳參數值，來得到最佳的預測模型。
台灣是一個典型的外向型經濟國家，使得台灣股市行情受外部因素影響頗大，這些因素包括國際的經濟、金融、油價與重大國際事件等，長期以來，以美國經濟與利率、匯率政策調整對台灣股市的影響最為明顯。本文結合道瓊工業指數(DJIA)、日經225指數(Nikkei 225)、韓國綜合股價指數(KOSPI)、及香港恆生指數(HSI)等多國股市組合，找出最佳預測加權股價指數的組合，並探討在有無加入波動率指數預測模型之差異。也會運用TAIEX來預測台灣50市占前三名的股票，台積電(TSM)、鴻海(Hon Hai)和中華電(CHT)這些股票的開盤價格。Time series models often use the exponentially weighted moving average (EWMA) method to reduce the influence of prices over time. The weights of recent values are more heavy to reduce structural changes arising from variations in trends, enabling the model to obtain better forecasting results. Over the past few years, researcher had proposed the generally weighted moving average (GWMA) method. The GWMA adds a parameter, is more sensitive to small fluctuation, and has a wider range of practical application than EWMA method. Therefore, this paper uses GWMA method and adds VIX index to predict the TAIEX index. In order to obtain the best forecasting model, we use numerical method to find the best parameter values of GWMA.
Taiwan is a typical export-oriented economy country, it makes Taiwan's stock market affected by external factors. These factors include international economic, financial, oil prices and major international events. For a long time, the impacts of the US economy and interest rate, exchange rate policy on the Taiwan stock market are most obvious. This paper combines the Dow Jones Industrial Average, the Nikkei 225 Index, the Korean Composite Stock Index, and the Hong Kong Hang Seng Index. This study attempts to find the best combination of forecasting model to predict the TAIEX, and compares the results without VIX model to explore differences between two forecasting models. This paper will also use TAIEX to forecast the opening price of Taiwan 50 index's top 3 stocks, TSM, Hon Hai and CHT.目錄
摘要 i
Abstract ii
目錄 iii
表目次 iv
圖目次 v
第一章 緒論 1
1.1研究背景 1
1.2研究目的 1
1.3研究架構 2
第二章 文獻探討 3
2.1波動率指數(VIX) 3
2.2指數加權移動平均(EWMA) 4
2.3廣泛加權移動平均(GWMA) 4
2.4最佳化方法 5
第三章 資料與研究方法 6
3.1資料介紹 6
3.2報酬率 8
3.3 EWMA 方法 9
3.4 GWMA 方法 10
3.5研究步驟 12
第四章 實證結果與分析 16
4.1實證結果-預測TAIEX 16
4.2實證結果-預測台灣股市的前三名 19
4.3檢定結果 22
第五章 結論與建議 24
5.1結論 24
5.2建議 25
參考文獻 26
附錄 2