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桂花萃取物對於早期生活受到刺激之老鼠所產生之類憂鬱行為改善作用之探討
No full text[[abstract]]憂鬱症本身為一種多腦區參與的疾病，在目前的HPA axis 憂鬱症理論中闡述，腦神經細胞的傷害與憂鬱症的產生有關聯性，且過量的glucocorticoid 為
傷害腦區的主要致病因子
Study on How the Participation Motivation of the College Volleyball Players Impacts on the Team Cohension ─ Sampling the Players in Men's Division Level 3 of Volleyball League in Session 2009
No full textThe Relationship among National Health Expenditure, GDP and Population Structure ― A Study of National Health Insurance Status in Taiwan
Get PDFDetermination of nitrate based on potentiometric sensor
Get PDF誌謝 V
中文摘要 VI
英文摘要 VIII
第一章 緒論 - 1 -
1-1研究背景 - 1 -
1-2研究目的與動機 - 3 -
1-3論文架構 - 5 -
第二章 理論分析與探討 - 6 -
2-1 硝酸選擇膜之文獻回顧 - 6 -
圖2-1光定址電位感測器 (LAPS )的架構圖 - 8 -
圖2-2(A)尿素(B)硫脲(C)胍 結構式 - 9 -
2-2感測器薄膜選擇 - 9 -
圖2-2 SnO2/ITO玻璃元件 - 9 -
2-3硝酸鹽與氮素循環之關係 - 9 -
圖2-3 氮素循環 - 11 -
2-4環境污染影響氨態氮 - 10 -
2-5氮的去除機制 - 10 -
圖2-4 生物處理程序中氮的轉化 - 12 -
2-6硝化作用之原理 - 12 -
2-7 脫硝作用之原理 - 13 -
2-8影響硝化作用之因子 - 14 -
2-9 影響脫硝作用之因子 - 15 -
2-4離子干擾之文獻 - 16 -
圖2-5流動注射分析法（FIA）架構圖 - 17 -
3. 分光光度法 - 17 -
第三章 材料與方法 - 18 -
3-1材料 - 18 -
3-2儀器與器材 - 19 -
3-3實驗流程 - 20 -
3-4量測系統 - 21 -
圖3-1量測系統 - 21 -
圖3-2 HP34401電表 - 21 -
3-5溶液製備 - 22 -
3-5.1 Tris-HCl緩衝液之配置 - 22 -
3-5.2硝酸溶液不同濃度的配置 - 22 -
3-6硝酸載體製備 - 22 -
3-7實驗步驟 - 24 -
3-7.1 ITO/PET感測器之製作 - 24 -
圖3-3 網印機 - 25 -
圖3-4網版 - 25 -
圖3-5 網版印刷感測器 - 26 -
3-7.2 硝酸感測器之製作 - 26 -
圖3-6硝酸感測器 - 27 -
3-7.3 硝酸感測器之檢測範圍 - 27 -
3-7.4 硝酸感測器之重複性 - 27 -
3-7.5 硝酸感測器在5℃、10℃、20 ℃、30 ℃、40 ℃ 環境下量測 - 27 -
3-7.6 硝酸感測器之干擾 - 28 -
第四章 結果與討論 - 28 -
4-1 感測器的選擇 - 28 -
4-2硝酸感測器的特性探討 - 28 -
4-2.1 硝酸感測器四種配方反應曲線之結果 - 28 -
4-2.2 硝酸感測器四種配方的校正曲線之結果 - 29 -
4-2.3 硝酸感測器重複性之結果 - 29 -
4-2.4 硝酸感測器在不同溫度環境下量測之結果 - 30 -
4-2.5 硝酸感測器的干擾之結果 - 31 -
4-2.5.1 K2SO4 KCl Fe203 0.05mM - 31 -
4-2.5.2 K2SO4 KCl Fe203 0.025mM - 31 -
4-2.5.3 K2SO4 KCl Fe203 0.005 mM - 32 -
第五章 結論 - 35 -
參考文獻 - 36 -
附錄 - 44 -
圖4-1感測器之感測度及線性結果 - 44 -
圖4-2訊號雜訊比方程式 - 45 -
圖4-3硝酸載體配方一之反應曲線在每個不同硝酸濃度中之反應之結果 - 46 -
圖4-4硝酸載體配方一之校正曲線在每個不同硝酸濃度中之結果 - 47 -
圖4-5硝酸載體配方二之反應曲線在每個不同硝酸濃度中之反應之結果 - 48 -
圖4-6硝酸載體配方二之校正曲線在每個不同硝酸濃度中之結果 - 49 -
圖4-7硝酸載體配方三之反應曲線在每個不同硝酸濃度中之反應之結果 - 50 -
圖4-8硝酸載體配方三之校正曲線在每個不同硝酸濃度中之結果 - 51 -
圖4-9硝酸載體配方四之反應曲線在每個不同硝酸濃度中之反應之結果 - 52 -
圖4-10硝酸載體配方四之校正曲線在每個不同硝酸濃度中之結果 - 53 -
表一.訊號雜訊比 - 54 -
圖4-11硝酸載體四種配方之校正曲線 - 55 -
圖4-12 硝酸感測器三次重複性之校正曲線 - 56 -
圖4-13 Nernst 方程式 - 57 -
圖4-14 硝酸感測器5℃、10℃、20 ℃、30 ℃、40 ℃校正曲線 - 58 -
圖4-15吸附鍵結模式（Site-binding model） - 59 -
圖4-16 硝酸感測器5℃、10℃、20 ℃校正曲線 - 60 -
圖4-17 硝酸感測器20℃、30℃、40 ℃校正曲線 - 61 -
圖4-18(A)氧化鐵(B)硝酸 結構式 - 62 -
圖4-19 K2SO4 KCl Fe203干擾濃度為0.05mM - 63 -
圖4-20 K2SO4 KCl Fe203干擾濃度為0.025mM - 64 -
圖4-21 K2SO4 KCl Fe203干擾濃度為0.005 mM - 65 -[[abstract]]近年來，農民使用氮肥以加速農作物生長，但氮肥過量時在土壤中由於硝化作用是硝化细菌將氨氧化為硝酸的過程，在短時間內氨態氮就會轉化為硝酸態氮。而硝酸態氮因負離子不易被土壤中的有機物所吸附，氮肥會隨者雨水的沖刷而排放至地下水。農民取地下水灌溉農田造成農作物含有硝酸態氮，硝酸態氮則主要運輸至葉片，而硝酸態氮經由光合作用固定之能量還原為銨態氮，再經代謝成胺基酸，植物可以一直吸收硝酸態氮，在低光照、低溫或缺鉬元素下，還原作用緩慢，吸收超量植物合成需要，致硝酸態氮在植物體內累積，人們食用後經由唾液使硝酸鹽還原成亞硝酸鹽，因此造成潛在危害。又會轉變為亞硝化反應會引起的N-亞硝基化合物造成很高的致癌性，所以在環境與農業上對硝酸鹽的檢測是非常重要。
目前已經開發了幾種分析方法感測硝酸鹽陰離子，如動力學、色譜儀，電位法、安培、分光光度法和流動注入分析法。然而這些方法大多是費時，反應條件需要控制，成本高，體積大。使用電位感測器其優點是易於製作、體積小、成本低、操作簡易、反應速度快、易於量產。
本研究利用網版印刷方式製作ITO/PET感測器，將硝酸載體固定在感測窗口上形成硝酸薄膜感測器。在其各個特性探討中10-4 ~ 10-1M檢測範圍能準確量測硝酸的電壓反應，得到的感測度為55.89 mV/decade，硝酸感測器與待測液之反應時間為三分鐘能量測出各個硝酸濃度反應，硝酸感測器在重複量測方面能連續量測三次得到的感測度為-45.57 mV/ decade標準差為3mV/decade因溶液的溫度不相同而量測硝酸所得到的兩條現象為5℃、10℃、20 ℃與20 ℃、30 ℃、40 ℃，因Nernst 方程式算出絕對靈敏度再與吸附鍵結模式計算出溶液中的硝酸根離子與感測薄膜表面離子之間的平衡，在高溫度下的靈敏值0.4 mV/decade/℃較接近離子之間平衡，自然中K2SO4 KCl Fe203化合物是水中常見離子，實驗結果在Fe203 0.05mM對硝酸濃度影響最大靈敏度為-7.27mV/ decade。此感測器由這些特性探討能在各種農業和水產養殖業及自然環境水中之硝酸鹽檢測及其特點微小化、攜帶方便，人人可得之，並能確保水中硝酸鹽其含量而慎選水質。
In recent years, farmers have used nitrogenous fertilizers to accelerate crop growth. However, excessive nitrogenous fertilizer in soil leads to over-nitrification, a process that involves nitrifying bacteria oxidizing ammonia into nitric acid, where ammoniacal nitrogen is rapidly converted into nitrate nitrogen. However, because nitrate nitrogen contains anions that are not easily absorbed by the organic matter in soil, it is scoured by rain and discharged into underground deposits. When farmers irrigate farmland using this groundwater, the resulting crops also contain nitrate nitrogen. The nitrate nitrogen is subsequently transported to the leaves of the crops, where they are converted back into ammoniacal nitrogen using the fixed energy produced by photosynthesis, and metabolized into amino acids. Because plants continue to absorb nitrate nitrogen, the slow reduction caused by limited sunlight, low temperatures, or a molybdenum deficiency results in abundant nitrate nitrogen that exceeds that required for plant synthesis. This accumulation of nitrate nitrogen in plants poses a potential risks if consumed by humans because saliva reduces nitrate to nitrite, which may be hazardous to human health. In addition, the N-nitroso generated by nitrosation reactions stimulations the development of cancer. Therefore, the environmental and agricultural detection of nitrate is crucial.
Currently, a number of analytical methods for detecting nitrate anions have been developed, such as dynamics, chromatography, potentiometry, ampere, spectrophotometry, and flow injection analysis. However, most of these methods are time consuming and costly, require large equipment to implement, and require controlled reaction conditions. By comparison, electric potential sensors are easy to fabricate, small, inexpensive, simple to operate, possess a rapid response time, and can be easily mass produced.
Using the screen printing method, the study authors fabricated an ITO/PET sensor, in which the nitric acid carrier was fixed on the sensor window to form a nitric acid film sensor. At a detection range of between 10-4 and 10-1 M, the voltage reaction rate of nitric acid was accurately measured as 55.89 mV/decade. After allowing the nitric acid sensor and test solution to react for 3 min, the reaction results of varying concentrations of nitric acid were measured. The nitric acid sensor was used to perform 3 consecutive measurements. The sensor level was measured at -45.57 mV/decade with a standard deviation of 3mV/decade. The two sets of temperatures measured using nitric acid at varying temperatures were 5 °C, 10 °C, and 20° C as well as 20 °C, 30 °C, and 40 °C. Next, the Nernst formula and the adsorption bond model were used to calculate the absolute sensitivity and the balance between the nitrate ions in the solution and the surface ions on the sensor film, respectively. The sensitivity value of 0.4 mV/decade/°C under high temperatures is closer to achieving a balance between ions. In the natural environment, the K2SO4 KCl Fe203 compound is a common ion found in water. The experimental results show that 0.05 mM of Fe203 has the greatest influence on nitric acid concentrations, with a sensitivity of -7.27 mV/decade. Based on these features, the developed sensor can be used in the agricultural and aquacultural industries, as well as in natural water environments, to detect nitrate levels and verify the nitrate content in drinking water and water for use. In addition, the miniature sensor is portable and available to everyone
Role of Andrographis paniculata Extract in Neuroprotection
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目 錄 i
圖目錄 iv
致謝 v
縮寫表 vii
中文摘要 1
英文摘要 2
第一章 前言
1-1 氧化壓力 3
1-2 乙醇所造成氧化傷害 4
1-3 過氧化氫所造成之氧化傷害 5
1-4 神經系統的細胞 5
1-5 穿心蓮介紹
1-5.1穿心蓮的名稱 6
1-5.2穿心蓮的產地來源 7
1-5.3穿心蓮植物形態特徵 7
1-5.4 穿心蓮的功效 9
1-6 細胞凋亡 9
1-7 研究目的 10
第二章 材料與方法
2-1實驗設備 12
2-2 實驗材料 12
2-3 實驗方法
2-3.1 細胞株 13
2-3.2 IMR-32細胞培養 14
2-3.3 細胞繼代培養 14
2-3.4 冷凍細胞活化 15
2-3.5 細胞冷凍保存 15
2-3.6 細胞計數方法 16
2-3.7 穿心蓮萃取物 16
2-3.8 細胞凋亡 16
第三章 結果
3-1穿心蓮萃取物不造成IMR-32細胞毒性 18
3-2過氧化氫對IMR-32 細胞之氧化傷害 18
3-3乙醇對IMR-32細胞毒殺之影響 19
3-4過氧化氫和穿心蓮對IMR-32細胞存活率之影響 19
3-5 乙醇和穿心蓮對IMR-32細胞存活率之影響 20
3-6穿心蓮萃取物不造成IMR-32細胞凋亡 20
3-7過氧化氫對IMR-32 細胞氧化傷害之細胞凋亡 21
3-8乙醇對IMR-32細胞凋亡之影響 21
3-9 過氧化氫和穿心蓮對IMR-32細胞凋亡之影響 21
3-10 乙醇和穿心蓮對IMR-32細胞凋亡之影響 22
第四章 討論
4-1穿心蓮萃取物抑制IMR-32細胞凋亡 23
4-2穿心蓮萃取物對過氧化氫誘發細胞死亡之保護作用 23
4-3 穿心蓮萃取物對乙醇誘發細胞死亡之保護作用 24
4-4 結論 24
參考文獻 24
圖 31
作者簡歷 51
圖表目錄
圖一、H2O2對IMR-32細胞毒殺之影響 31
圖二、乙醇對IMR-32細胞毒殺之影響 33
圖三、穿心蓮對IMR-32細胞毒殺之影響 35
圖四、H2O2和穿心蓮對IMR-32細胞存活率之影響 37
圖五、乙醇和穿心蓮對IMR-32細胞存活率之影響 39
圖六、穿心蓮萃取物不造成IMR-32細胞凋亡 41
圖七、過氧化氫對IMR-32 細胞氧化傷害經由細胞凋亡 43
圖八、乙醇對IMR-32細胞凋亡之影響 45
圖九、穿心蓮逆轉過氧化氫對IMR-32細胞凋亡之影響 47
圖十、乙醇和穿心蓮對IMR-32細胞凋亡之影響 49[[abstract]]穿心蓮(Andrographis paniculata Nees.)為爵床科植物，是一種民間傳統藥用植物，廣泛應用於各種用途，以往被用為抗炎、抗菌、解熱及免疫促進之傳統治療藥物。過多的活性氧自由基產生的氧化壓力，容易造成大腦損傷，目前已知氧化壓力易造成神經退化性疾病的產生。穿心蓮具有很好的抗氧化能力，因此本研究探討穿心蓮的神經保護功效。我們利用神經纖維母細胞株IMR-32細胞，經由過氧化氫與乙醇的氧化傷害模式，探討穿心蓮萃取物是否具有抗氧化的神經保護功效。利用過氧化氫及乙醇誘發細胞死亡的實驗模式下，加入過氧化氫及乙醇刺激24小時可誘導細胞死亡，加入不同濃度的穿心蓮萃取物(25、50、100、200 μg/ml )可保護細胞避免死亡。經實驗證實，該細胞死亡是經由細胞凋亡機制，穿心蓮可以減緩細胞凋亡的發生。綜合本研究的結果，中草藥穿心蓮萃取物具有保護神經細胞對抗氧化壓力的功效，或許未來可以應用於神經退化性疾病的預防或治療。
Andrographis paniculata Nees., Family Acanthaceae, is a traditional folk medicinal plants, widely used in various applications, and has been used as antiinflammatory, antibacterial, antipyretic and immune promoting traditional therapeutic drugs. Excessive reactive oxygen radicals produced by oxidative stress make the brain hurt easily. It has been known that oxidative stress could easily lead to the generation of neurodegenerative diseases and the extract of Andrographis paniculata is a good antioxidant. Therefore, we used the human neuroblastoma IMR-32 cells treated with hydrogen peroxide and ethanol oxidation to investigate whether Andrographis paniculata extract has antioxidant neuroprotective effects. Under the cellular experimental conditions, the use of hydrogen peroxide and ethanol can induce cell death. To study the protective effects of Andrographis paniculata extract, we treated cells with hydrogen peroxide/ethanol and various concentrations of Andrographis paniculata extract (25, 50, 100, 200 μg/ml) for 24 hours and found the extract can protect cells from death. We studied whether the cell death via apoptosis using ApoGlowTM assay kit and found that hydrogen peroxide/ethanol induces apoptosis and Andrographis paniculata extract decreases the adverse effects. Integrated our experimental results, Chinese herbal medicine Andrographis paniculata extract protects nerve cells against oxidative stress injury. Maybe it can be used for prevention or treatment of neurodegenerative diseases in the future
Antifibrogenic Effects of Andrographis Paniculata in Human Hepatic Stellate Cells
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目錄 i
圖目錄 iv
致謝 v
縮寫表 vi
中文摘要 1
英文摘要 3
第一章 前言
1.1 肝臟纖維化簡介 5
1.2 肝臟纖維化發展 5
1.3 基質金屬蛋白酶 6
1.4 基質金屬蛋白酶在纖維化的功能 7
1.5 肝臟星狀細胞介紹 8
1.6 肝臟星狀細胞功能 9
1.7 轉化生長因子-b 9
1.8 氧化壓力對纖維化影響 10
1.9 穿心蓮 11
1.10 研究目的 12
第二章 材料與方法
2.1 細胞培養 13
2.2 穿心蓮萃取物 13
2.3 西方墨點法 14
2.4 免疫螢光染色 16
2.5 三色染色法 17
第三章 結果
3.1 穿心蓮萃取物不造成 HSC-T6 肝臟星狀細胞毒性 19
3.2 穿心蓮萃取物降低 TGF-b 誘導 a-SMA 在肝臟星狀細胞的表現 19
3.3 穿心蓮萃取物降低 TGF- 誘導 fibronectin 在肝臟星狀細胞的表現 20
3.4 穿心蓮萃取物降低膠原蛋白在肝臟星狀細胞的表現 21
第四章 討論
4.1 穿心蓮萃取物抑制肝臟星狀細胞活化 23
4.2 穿心蓮萃取物減緩纖維化的可能機制 24
4.3 穿心蓮萃取物的其他功能 25
4.4 中草藥減緩纖維化之功效 25
4.5 結論 26
參考文獻 27
作者簡歷 41
圖表目錄
圖一、TGF-b 和穿心蓮對於細胞型態的影響 34
圖二、TGF-b 和穿心蓮對細胞存活率的影響 35
圖三、穿心蓮對 TGF-b 誘發 a-SMA 蛋白質表現的影響 36
圖四、利用免疫螢光染色方式觀察穿心蓮對 TGF-b 誘發 a-SMA表現的影響 37
圖五、穿心蓮對 TGF-b 誘發 fibronectin 蛋白質表現的影響 38
圖六、穿心蓮對 TGF-b 誘發 fibronectin 蛋白質表現的影響 39
圖七、穿心蓮對 TGF-b誘發膠原蛋白表現的影響 40[[abstract]]造成肝纖維化的過程有許多種原因，包含了酒精或藥物的濫用、氧化壓力、病毒感染、自體免疫疾病或代謝疾病等。這些都會造成肝臟損傷，導致膠原蛋白(collagen)與纖維結合素(fibronectin)等與纖維化相關蛋白沈積在細胞外基質，造成疤痕組織。而在這過程中與肝臟中的星狀細胞(Hepatic stellate cells, HSC)被活化有關，HSC活化的指標蛋白a-SMA, a-smooth muscle actin)也被誘導表現，以及大量的細胞外基質蛋白被分泌堆積。另外也有研究指出transforming growth factor-b, TGF-b也扮演著誘導纖維化(pro-fibrogenic)的角色，可調控成纖維(fibrogenic)細胞的增生或分化。近年來，許多研究指出天然中草藥都具有抗肝臟纖維化的功能。因此我們選擇了一種中草藥穿心蓮，屬於爵床科植物。它在亞洲被廣泛用來作為治療感冒、腹瀉、發燒，特別可以用作為保肝劑。近年來許多研究以證明穿心蓮的萃取物具有拮抗reactive oxygen species (ROS)所造成的氧化損傷。目前對於穿心蓮對肝纖維化的影響尚不清楚，但已知纖維化過程有ROS參與。所以推測穿心蓮可透過減少ROS來降低纖維化的發生。因此本研究的目的是在模擬肝纖維化的環境下，使用TGF-b誘導HSC-T6肝臟星狀細胞，去探討穿心蓮避免纖維化之功效。利用西方墨點法、免疫螢光染色法與三色染色法，發現可誘發纖維蛋白與HSC細胞活化指標蛋白，包括collagen、fibronectin與a-SMA。接著為觀察天然中草藥穿心蓮萃取物是否具有降低造成纖維化的相關蛋白的表現，我們加入不同量的萃取物，發現collagen、fibronectin與a-SMA皆有顯著的抑制。根據以上結果，中草藥穿心蓮萃取物具有避免肝臟星狀細胞活化的功效，或許也可用來預防肝臟纖維化。
The hepatic fibrosis is a chronic disease caused by a variety of factors including alcohol abuse, oxidative stress, viral infection, autoimmune diseases, and metabolic diseases. The development of hepatic fibrosis is caused by liver injury, which leads to accumulation of the extracellular matrix proteins (ECM) including type I collagen and fibronectin as well as a-smooth muscle actin (a-SMA) synthesis and deposition, thus forming the fibrous scar. Hepatic stellate cells (HSC) play a key role in the pathogenesis of liver fibrosis. The activation of HSC leads to proliferation, expression ofa-SMA, and production of large amounts of extracellular matrix proteins. The previous studies have shown that transforming growth factor (TGF)-b is a multifunctional cytokine that plays a pro-fibrogenic role in liver fibrosis by regulating proliferation and differentiation of fibrogenic cells as well as stimulating extracellular matrix proteins. It has been known that natural products exert anti-hepatic fibrogenic activity. The traditional Chinese herb Andrographispaniculata (A.P.), a plant of Family Acanthaceae and also denoted as “Chuan-Chin-Lian”, is used to treat cold, diarrhea, fever and, especially, for liver protection. It has been known that reactive oxygen species (ROS) is a factor involved in fibrogenesis and the extract of A.P. can also prevent the liver damage from ROS. However, the functions of A.P. in liver protection are still unclear. Therefore, we proposed that it could be a candidate for anti-fibrogenesis from liver damage via decreasing ROS. To address this issue, we used a cell model for liver fibrosis, treating with TGF-bto activate hepatic stellate cells, HSC-T6 cells. We also used Western blotting, immunofluorescent staining and trichrome staining to access the protein levels of collagen, fibronectin and a-SMA and found that these proteins were induced by TGF-b. However, the expression of these proteins was decreased while co-treating with various concentrations of A.P. extract. According to these results, A.P. extract exerts ability for inhibiting HSC activation and it may be used as a drug for preventing liver fibrosis
Recovery of zinc and iron from zinc electroplating sludge
No full text目錄
摘要 I
Abstract III
誌謝 V
目錄 VII
圖目錄 XI
表目錄 XIV
第一章 前言 - 1 -
1.1 研究源起 - 1 -
1.2 研究目的 - 3 -
第二章 文獻回顧 - 6 -
2.1 電鍍基本原理 - 6 -
2.2 電鍍業製程概述 - 7 -
2.2.1 前處理 - 8 -
2.2.2 電鍍 - 9 -
2.2.3 後處理 - 14 -
2.3 電鍍之目的與特性 - 15 -
2.4 電鍍製程污染來源 - 18 -
2.4.1 各類製程污染來源 - 19 -
2.5 重金屬污泥 - 24 -
2.5.1 污泥種類 - 25 -
2.5.2 重金屬元素之危害 - 27 -
2.6 重金屬污泥處理技術 - 30 -
2.6.1 固定化處理 - 30 -
2.6.2 微生物處理 - 31 -
2.6.3 乾式冶煉法 - 31 -
2.6.4 濕式冶煉法 - 32 -
2.7酸液浸漬法之研究 - 32 -
第三章 研究設備與方法 - 35 -
3.1研究流程 - 35 -
3.1.1電鍍鍍鋅污泥基本特性分析 - 35 -
3.1.2酸液浸漬試驗操作條件 - 35 -
3.1.3沉澱試驗操作條件 - 36 -
3.1.4特性分析 - 37 -
3.2 研究方法與設備 - 39 -
3.2.1 實驗步驟 - 39 -
3.2.1.1 污泥前處理 - 39 -
3.2.1.2 酸浸漬 - 40 -
3.2.1.3 沉澱試驗 - 40 -
3.2.1.4 重金屬分析 - 41 -
3.2.2 實驗方法 - 43 -
3.2.3 實驗材料 - 47 -
3.2.4 實驗設備 - 48 -
第四章 結果與討論 - 56 -
4.1原料分析 - 56 -
4.1.1原始污泥重金屬成分分析 - 56 -
4.2 酸浸漬行為探討 - 58 -
4.2.1 酸浸漬液選擇 - 59 -
4.2.2 pH值對Zn及Fe浸出率影響 - 62 -
4.2.3固液比對Zn及Fe浸出率影響 - 64 -
4.2.4反應時間對Zn及Fe浸出率影響 - 65 -
4.3 沉澱行為探討 - 67 -
4.3.1 不同pH值對Zn及Fe沉澱率影響（一次沉澱） - 67 -
4.3.2 不同pH值對Zn的沉澱率影響（二次沉澱） - 69 -
4.4 回收率探討 - 70 -
4.5 特徵分析 - 72 -
4.5.1 外觀觀察 - 72 -
4.5.2 表面微結構觀察（SEM） - 74 -
第五章 結論與建議 - 77 -
5.1 結論 - 77 -
5.2 建議 - 79 -
參考文獻 - 80 -[[abstract]]工業蓬勃的發展，導致金屬使用的需求量逐年上升，但金屬礦藏日漸減少，高含量重金屬的廢棄物直接掩埋丟棄，不但金屬無法回收再利用也造成廢棄物體積的增加，相對的也會對環境造成污染；反之，將其中所含的金屬回收再利用，以減低環境之衝擊與增加處理之經濟效益為一具前瞻性與優勢之處理策略。
此研究以回收電鍍鍍鋅業所產生之廢棄污泥中之Zn與Fe為研究標的，因污泥中所含並非單一種類之金屬而選用濕式冶煉法，將重金屬以離子態存於溶液中，再藉由沉澱將其還原成化合物。
實驗選用酸液浸漬法將污泥中的重金屬浸漬溶出，並經固液分離後在溶液中加入NaOH溶液調整pH值，使得不同重金屬離子在鹼性的水溶液中分階段形成不溶性金屬氫氧化物，達到金屬初步純化與回收的目的。
研究結果顯示，鍍鋅污泥中較具有回收經濟效益之主要重金屬成份以Zn平均含量（202,000 mg/kg）最高，Fe平均含量（39,800 mg/kg）次之。從各試驗裡得知，當浸漬條件在室溫下，使用硫酸為浸漬液、pH值控制為1.0、浸漬時間60分鐘，且固液比為20時，能達到Zn99.49%之浸出率及Fe93.39%之浸出率，此時得知大部份的鋅與鐵離子皆已溶出，然而再進一步探討鋅及鐵沉澱時所需的pH值。當pH值為5.0沉澱，分別得Zn及Fe的沉澱率為3.24%及99.02%，這時鐵離子大多已沉澱；再經固液分離後，濾液進行pH 8.0調值沉澱，得Zn之沉澱率為99.49%。最後算得Zn與Fe之回收率，其Zn的回收率為43.72%、Fe的回收率為44.83%，回收結果並不佳。酸液浸漬法雖能有效浸漬出重金屬，但若只單純的將金屬沉澱，其回收效率不佳，對於此一回收方法可再進一步做探討。
Due to rapid industrial development, the demand of metals will expect to be in higher and higher. Ironically, with the decreasing of metals reserves, quiet a few of high-metal-content scraps still directly treat with landfill disposal. It is resource wasteful procedures not only for value metal but also for landfill site. The foresightedness and predominance strategy of metal recovery, with economic benefits of value metal, can reduce the impact on the environment.
The aim of this research is to recovery zinc and iron from zinc-electroplating sludge. Because the characteristic of multi-metal in the sludge, the hydrometallurgical process was selected to trap the heavy metal ion in the acid solution, and reduced to the metal compounds by settling.
The acid leaching process was employed to extract the heavy metals from sludge into an acid solution. NaOH(aq) was added to the solution for pH adjustment, which making the various heavy metal ions to form insoluble metal hydroxide in basic aqueous solution to obtain a preliminary purification compounds for metal recovery purpose.
The results show that Zn (202,000 mg/kg) and Fe (39,800 mg/kg) are the major and minor recovery heavy metals in the zinc-electroplating sludge. The optimal recovery efficiency at room temperature is under the condition of pH=1.0, 60 minute sulfuric acid maceration, and solid/liquid ratio=20. Most of zinc (99.49%) and iron (93.39%) ion can be obtained by the extraction process. The precipitation efficiencies of zinc and iron are 3.24% and 99.02% at pH 5.0, respectively, which indicate that most of iron ions can be removed in this process. Following with a solid-liquid separation, the precipitation efficiency of zinc is 99.49% at the pH of 8.0.
The mass balance analysis shows that total recovery efficiencies of zinc and iron are 43.72% and 44.83%, respectively. Although the acid extraction can obtain most of metal in the sludge, the simple precipitation can not obtain a good recovery efficiency for zinc and iron
The inhibition effect of recombinant protein AiiA on phytobacteria isolated from phalaenopsis
No full text口試委員審定書
授權書...................................................Ⅱ
中文摘要.................................................Ⅲ
英文摘要.................................................Ⅴ
致謝.....................................................Ⅶ
壹、 前人研究.............................................1
一、蝴蝶蘭簡介..........................................1
二、蝴蝶蘭產業發展狀況..................................1
三、蝴蝶蘭作物細菌性病害之種類..........................2
(一). 蝴蝶蘭細菌性褐斑病..........................3
(二). 蝴蝶蘭細菌性葉斑病..........................4
(三). 蝴蝶蘭細菌性軟腐病..........................4
四、細菌性病害之防治....................................6
(一).蘭花細菌性病害之防治現況.....................6
(二).群體密度感應系統.............................7
(三).群體密度感應系統之訊號分子...................8
1. AHL內酯酶(AHL-lactonase).................8
2. AHL內酯醯化酶(AHL-acylase)...............9
(四). 群體密度感應系統之作用機制............... ...9
五、細菌性病害之檢測...................................10
(一).鑑別型培養基................................11
(二).酵素連結抗體檢定法..........................11
(三).磁顆粒分離法................................12
(四).微生物之PCR檢測............................12 (五).多引子組PCR................................13
六、研究目標...........................................14
貳、 材料與方法..........................................16
一、材料.............................................16
(一).植物材料...................................16
(二).菌株.......................................16
(三).培養基.....................................16
(四).奈米磁顆粒.................................17
(五).質體.......................................17
二、 方法.............................................17
(一).質體製備...................................17
(二).大腸桿菌勝任細胞之製備.....................18
(三).大腸桿菌勝任細胞之轉形作用.................18
(四).質體構築...................................18
(五).質體之定序分析.............................19
(六).生物資訊分析...............................19
(七).基因之蛋白質表現及純化.....................19
(八).蛋白質表現之分析...........................20
1. SDS-PAGE分析...........................20
2.膠體染色.................................20
3.西方墨點法(Western Blotting)................21
(九).濾紙圓盤擴散法(paper disc diffusion method) ....21
(十).初步過濾...................................22
(十一).植株材料中干擾PCR反應之分析.............22
(十二).磁顆粒分離技術...........................22
(十三).聚合酵素鏈鎖反應.........................23
(十四). 人工接種後發病之磁顆粒Uniplex PCR檢測分析..............................................23
(十五). 人工接種後發病材料之磁顆粒Multiplex PCR檢測分析............................................24
(十六).PCR產物之膠體電泳分析...................24
參、 結果................................................26
一、AHL內酯酶(aiiA)基因之序列分析...................26
二、利用表現載體來分析及純化AHL lactonase(aiiA)基因之表現..................................................27
三、aiiA 蛋白質之表現及純化..........................27
四、pQE30-aiiA之抑菌效果分析.........................28
五、pQE30-aiiA萃取液進行三種保存時間之抑菌效果.......29
六、檢測.............................................30
(一). 過濾處理與磁顆粒分離對檢測褐斑病菌之PCR敏感度之分析........................................30
(二). 過濾處理與磁顆粒分離對檢測葉斑病菌之PCR敏感度之分析........................................30
(三). 過濾處理與磁顆粒分離對檢測軟腐病菌之PCR敏感度之分析........................................31
七、人工接種蘭花之磁顆粒Uniplex PCR檢測分析.........32
八、人工接種蘭花之磁顆粒Multiplex PCR檢測分析........33
肆、 討論................................................34
伍、 參考文獻............................................37
陸、 圖表................................................53
表1 本研究所用褐斑病菌、葉斑病菌及軟腐病菌之菌株表......54
表2 本研究所使用之引子..................................55
表3 25℃保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後之抑菌效果分析表.....................................56
表4 4℃保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後之抑菌效果分析表.......................................57
表5 負20℃保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後之抑菌效果分析表...................................58
圖1 蘇力菌之AHL內酯酶基因之核苷酸序列及選殖本基因之引子組序列...................................................59
圖2 AHL內酯酶胺基酸(aiiA)之多重序列比對結果............60
圖3 pQE30-aiiA之構築示意圖.............................61
圖4 選殖株pQE30-aiiA之基因片段分析....................62
圖5 pQE30-aiiA蛋白質之表現分析..........................63
圖6 選殖株之蛋白在pQE30/M15表現系統中之Western blotting 分析.......................................................64
圖7 選殖株pQE30-aiiA可溶及不可溶蛋白萃取物之抑菌效果分析.......................................................65
圖8 不同溫度保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後對褐斑病菌之抑菌效果分析(A) ......................66
圖8 不同溫度保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後對葉斑病菌之抑菌效果分析(Ｂ) .....................67
圖8 不同溫度保存之選殖株pQE30-aiiA全蛋白質萃取液在不同保存時間後對軟腐病菌之抑菌效果分析(Ｃ) .....................68
圖9 過濾處理與磁顆粒分離對檢測褐斑病菌之PCR敏感度之分析.......................................................69
圖10 過濾處理與磁顆粒分離對檢測葉斑病菌之PCR敏感度之分析.......................................................70
圖11 過濾處理與磁顆粒分離對檢測軟腐病菌之PCR敏感度之分析.......................................................71
圖12 人工接種後發病材料之磁顆粒Uniplex PCR檢測分析.....72
圖13 人工接種後發病材料之磁顆粒Multiplex PCR檢測分析....73[[abstract]]近年來蝴蝶蘭的經濟栽培面積大幅擴增，目前已成為台灣主要外銷花卉。由於溫室栽培之環境高溫多濕，若種植過程管理不當，經常會助長蝴蝶蘭病害之發生與蔓延，造成蘭園重大之損失。病害之發生通常都為蝴蝶蘭生產的限制因子，本研究將就細菌性病害之防治進行探討，期能提供解決細菌性病害之可行性方案。本研究利用由蘇力菌 (Bacillus thuringiensis)中選殖出AHL內酯酶基因，測試其抑制或破壞病原細菌群體密度感應系統的效果，分析此蛋白是否可降低植物病原細菌的致病性，並達到防治蝴蝶蘭細菌性病害之效果。研究中將可表現AHL內酯酶基因選殖菌株之全蛋白萃取液分別對不同病原進行抑菌分析，結果發現此酵素對蝴蝶蘭的3種細菌性病害皆有抑制效果。進一步測試長期保存於不同溫度下之全蛋白萃取液之抑菌效果，結果顯示於4℃之存放效果最佳，在長期保存後仍可對褐斑病、葉班病及軟腐病菌等病原細菌具有抑制效果。本研究結果亦顯示AHL內酯酶確實可於pQE表現系統中產生，且具抑菌效果，但進行AHL內酯酶蛋白質純化後，卻發現純化後之蛋白不具抑菌之效果。AHL內酯酶在本研究之分析中具有抑制蝴蝶蘭細菌性病原菌之效果，爾後將進一步開發可大量純化出具有抑制效果之AHL內酯酶之方法，未來對於蝴蝶蘭細菌性病害之防治將有極大之助益。而如何快速且正確的鑑別感染原，係病害管理上另一個重要的課題。台灣蝴蝶蘭細菌性病害之主要病原有三種，分別為 Acidovorax avenae subsp. Cattleyae (AAC)、Burkholderia gladioli (BG)及Pectobacterium chrysanthemi (PCH)，蝴蝶蘭細菌性病害之檢測可利用不同菌株間Internal transcribe spacer的特異性序列設計專一性引子組，再進行AAC、BG及PCH之單引子組及多引子組PCR鑑定，但於實際田間檢測時，存在於植株及栽培環境中的抑制因子常影響PCR檢測之敏感度及正確性。本研究利用過濾及磁顆粒分離技術，成功去除大部分抑制因子，確實提昇單引子組及多引子組PCR檢測之正確性；另於PCR敏感度測定結果得知，對AAC、BG及PCH分別可達104 CFU/mL、102 CFU/mL及104 CFU/mL，此結果優於ELISA之敏感度檢測，未來將可應用此技術於田間病害之檢測。
Phalaenopsis became the most important product in the industry of flowers for export, as the cultivation quantity of orchid increased rapidly. The high desnisty cultivation of orchids in the greenhouse caused the increasing of the temperature and humidity and this environment would cause the occurrence of plant diseases, which caused enormous losses of orchid growers. Acidovorax avenae subsp. cattleyae (AAC), Burkholderia gladioli (BG) and Pectobacterium chrysanthemi (PCH) caused infectious diseases of Phalaenopsis in Taiwan. The additional issue of Disease Management was the prevention of phytobacterial diseases. First of all, we cloned the AHL-lactonase gene from Bacillus thuringiensis. Then we constructed lactonase gene to pQE expression system to express lactonase in the E. coli (pQE-aiiA). The extraction of the total protein from E. coli (pQE-aiiA) would be used to test its inhibition-ability to phytobacteria on the media. The results of this antibacterial analysis revealed the total protein could inhibit the growth of three kinds of phytobacteria. In the further testing, the total protein would be test by the same method above to understand the inhibition-ability after long-term storage at different temperatures. The results shown the activity of total protein extracts which stored at 4 ℃ would be better than other temperatures. In order to understand more about lactonase, we try to purify lactonase with his-tag column from total protein. Unfortunately, the purified proteins couldn’t emerge the inhibition-ability. This study would need to modify the methods of protein purification to extract the active lactonase. Then, we would have more powerful opportunity to apply lactonase on the management of field in the future. For detecting these pathogens, we used an in-house database of bacterial sequences, which were designed primers based on the results of multiple sequence alignments. We could utilize these specific primer-pairs to detect different kinds of phytobacteria from Orchids by PCR reactions. In the field of orchids, the PCR reactions were influenced by some limited factors which existed in the cultural environment of field. The factors could decrease the specificity and sensitivity of uniplex and multiplex PCR reactions. In order to resolve this problem, we used filtration and magnetic separation to avoid these limited factors. The testing results revealed these treatments could increase the sensitivity and specificity. The sensitivity of above-mentioned technique was better than ELISA technique. In addition, the detection of each PCR reaction cost about NT $ 5 dollars, would enhance this technology to be apply in the fields