家禽里奧病毒進入宿主細胞之途徑及AMPK-MAPK p38、small G protein及autophagy在病毒複製所扮演之角色

AbstractAvian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In the past, we have studied ARV-caused diseases, cell cycle regulation by ARV, and ARV-induced apoptosis and its related pathways. Little is known about the exact mechanisms of ARV cell entry. Therefore, to further understand the mechanism of ARV entry, the first part of this project will explore the route of ARV cell entry and the involved signaling pathways. It has been demonstrated that MAPK p38 could be activated in viruses-infected cells. More recently, we discovered for the first time that AMPK could facilitate MAPK p38 signaling that is beneficial for ARV replication. The exact details on how MAPK p38 affects ARV replication remains an interesting issue waiting to be solved. Therefore, the second part of this project is to study how ARV regulates AMPK-p38 MAPK pathway and to study the activation of p38 MAPK in which stage of viral life cycle and the role of AMPK-p38 MAPK pathway in ARV-induced autophagy and virus replication. Recently, we have also demonstrated for the first time that ARV increased the level of phosphorylated eIF2α and eEF2 in a dose-dependent mechanism. Increased levels of phosphorylated eIF2α and eEF2 in ARV-infected cells do not reduce the levels of viral protein. Our preliminary results also show that infection with ARV notably decrease the level of phosphorylated 4E-BP1 in a dose-dependent manner. To further understand how ARV enhances its own replication through what kind of signaling pathways and to elucidate how ARV shut off cellular translation to benefit virus replication, therefore, the third part of this project is to elucidate whether ARV regulates cellular translation through PI3K-AKT-mTOR pathway or related pathways and to further confirm whether cap-dependent translation is required for ARV replication. This project that elucidates the route of virus entry, study the mechanism of enhancement of virus replication by AMPK-p38 MAPK signaling pathway, and understands the regulation of cellular translation by ARV, is a novel and promising study.中文摘要家禽里奧病毒 (Aian reovirus; ARV) 感染家禽造成多種疾病，包括病毒性關節炎、營養吸收不良症及慢性呼吸道疾病等，造成業者經濟損失。筆者研究團隊過去在病毒造成雞隻病變、病毒調控細胞週期與細胞轉譯及誘發凋亡之機轉有所鑽研。但ARV如何進入宿主細胞之途徑及機制尚不清楚。因此本計畫第一部分擬探討ARV透過何種endocytosis進入細胞及MAPK 38 及samll G protein與 endocytosis之關聯性。雖然已知許多病毒感染細胞後，可活化p38 MAPK，最近筆者研究團隊首次證實ARV可活化AMPK-p38 MAPK pathway以利於病毒本身之複製，但如何協助病毒進入細胞及病毒複製之機轉仍待釐清。為了進一步釐清ARV如何透過AMPK-p38 MAPK pathway來誘發自噬體(autophagy)以促進病毒的複製，本計畫第二部分擬探討AMPK及p38 MAPK在病毒感染早期所扮演之角色及與ARV誘發細胞產生autophagy以促進病毒複製之機序。最近筆者研究團隊證實ARV感染細胞後，其真核啟始因子eIF2α及eEF2的磷酸化隨之增加，但4E-BP1磷酸化則隨病毒MOI的增加其磷酸化程度呈現減少的趨勢，有趣的是病毒蛋白的轉譯卻未隨eIF2α及eEF2 的磷酸化而被抑制。因此本計畫第三部分將釐清ARV如何透過PI3K/AKT/mTOR pathway去調控細胞的轉譯因子及細胞蛋白轉譯。進一步探討ARV關閉細胞蛋白之轉譯是否有利於病毒複製及活化AMPK-p38 MAPK強化病毒本身的複製是否無需cap-dependent轉譯啟始作用。本計畫探討家禽里奧病毒進入細胞途徑及機制、家禽里奧病毒活化AMPK-p38 MAPK pathway及誘發自噬體及調控細胞的轉譯作用以促進病毒本身之複製，是家禽里奧病毒領域之創新研究

Characterization of Routes of Avian Reovirus Cell Entry and the Roles of Ampk-Mapk P38, Small G Protein and Autophagy in Virus Replication

中文摘要家禽里奧病毒 (Aian reovirus; ARV) 感染家禽造成多種疾病，包括病毒性關節炎、營養吸收不良症及慢性呼吸道疾病等，造成業者經濟損失。筆者研究團隊過去在病毒造成雞隻病變、病毒調控細胞週期與細胞轉譯及誘發凋亡之機轉有所鑽研。但ARV如何進入宿主細胞之途徑及機制尚不清楚。因此本計畫第一部分擬探討ARV透過何種endocytosis進入細胞及MAPK 38 及samll G protein與 endocytosis之關聯性。雖然已知許多病毒感染細胞後，可活化p38 MAPK，最近筆者研究團隊首次證實ARV可活化AMPK-p38 MAPK pathway以利於病毒本身之複製，但如何協助病毒進入細胞及病毒複製之機轉仍待釐清。為了進一步釐清ARV如何透過AMPK-p38 MAPK pathway來誘發自噬體(autophagy)以促進病毒的複製，本計畫第二部分擬探討AMPK及p38 MAPK在病毒感染早期所扮演之角色及與ARV誘發細胞產生autophagy以促進病毒複製之機序。最近筆者研究團隊證實ARV感染細胞後，其真核啟始因子eIF2α及eEF2的磷酸化隨之增加，但4E-BP1磷酸化則隨病毒MOI的增加其磷酸化程度呈現減少的趨勢，有趣的是病毒蛋白的轉譯卻未隨eIF2α及eEF2 的磷酸化而被抑制。因此本計畫第三部分將釐清ARV如何透過PI3K/AKT/mTOR pathway去調控細胞的轉譯因子及細胞蛋白轉譯。進一步探討ARV關閉細胞蛋白之轉譯是否有利於病毒複製及活化AMPK-p38 MAPK強化病毒本身的複製是否無需cap-dependent轉譯啟始作用。本計畫探討家禽里奧病毒進入細胞途徑及機制、家禽里奧病毒活化AMPK-p38 MAPK pathway及誘發自噬體及調控細胞的轉譯作用以促進病毒本身之複製，是家禽里奧病毒領域之創新研究。AbstractAvian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In the past, we have studied ARV-caused diseases, cell cycle regulation by ARV, and ARV-induced apoptosis and its related pathways. Little is known about the exact mechanisms of ARV cell entry. Therefore, to further understand the mechanism of ARV entry, the first part of this project will explore the route of ARV cell entry and the involved signaling pathways. It has been demonstrated that MAPK p38 could be activated in viruses-infected cells. More recently, we discovered for the first time that AMPK could facilitate MAPK p38 signaling that is beneficial for ARV replication. The exact details on how MAPK p38 affects ARV replication remains an interesting issue waiting to be solved. Therefore, the second part of this project is to study how ARV regulates AMPK-p38 MAPK pathway and to study the activation of p38 MAPK in which stage of viral life cycle and the role of AMPK-p38 MAPK pathway in ARV-induced autophagy and virus replication. Recently, we have also demonstrated for the first time that ARV increased the level of phosphorylated eIF2α and eEF2 in a dose-dependent mechanism. Increased levels of phosphorylated eIF2α and eEF2 in ARV-infected cells do not reduce the levels of viral protein. Our preliminary results also show that infection with ARV notably decrease the level of phosphorylated 4E-BP1 in a dose-dependent manner. To further understand how ARV enhances its own replication through what kind of signaling pathways and to elucidate how ARV shut off cellular translation to benefit virus replication, therefore, the third part of this project is to elucidate whether ARV regulates cellular translation through PI3K-AKT-mTOR pathway or related pathways and to further confirm whether cap-dependent translation is required for ARV replication. This project that elucidates the route of virus entry, study the mechanism of enhancement of virus replication by AMPK-p38 MAPK signaling pathway, and understands the regulation of cellular translation by ARV, is a novel and promising study

桿狀病毒呈現載體之研發及應用於犬隻疫苗之開發

The Baculovirus system has been increasingly used as an alternative to the traditional expression systems in the past decade years. The ability of the virus to display the fusion protein in native conformation presumably with all the necessary posttranslational modifications in the insect host cells can augment the immunoreactivity of the displayed antigens. In addition, the expression of the proteins through the baculovirus display system allows rapid generation of effective antigens without the need for purification of the recombinant proteins. The first generation of baculovirus display vector has been developed previously by our team. To enhance the expression level of proteins and to display multiple different proteins on cell membrane, the main objective of this study in the first year is to develop a novel baculovirus display vector that can display at least three different proteins on the baculoviral envelope or cell membrane. In the second year, a novel baculovirus display system displaying the proteins of canine distemper virus (CDV), canine parovirus virus (PV), and infectious canine hepatitis (ICH) virus, will be constructed. The potential of CDV-PV-ICH-pseudotyped baculovirus as a vaccine will be evaluated in this study.桿狀病毒應用於疫苗之研發已有多年，由於桿狀病毒具備有表現率高、後修飾作用佳、具經濟效益及安全性高之優點，因此被用以開發作為疫苗之工具。筆者研究團隊過去已成功開發第一代桿狀病毒表現呈現載體，並成功應用於禽類疫苗之研發。本計畫第一年將著重於第二代桿狀病毒多重呈現系統之研發，期將桿狀病毒表現呈現載體改造可同時呈現多個蛋白於細胞膜或病毒封套。此新型桿狀病毒表現呈現載體系統將可大幅提升病毒蛋白之表現量及可同時表現多種蛋白於細胞膜或病毒封套，並以細胞做為製備疫苗之抗原，不需要進一步純化蛋白，因此可降低疫苗製造之成本。本計畫第二年擬以新建構之第二代桿狀病毒多重呈現載體應用於構築基因重組桿狀病毒以同時呈現犬隻三種重要病毒包含犬瘟熱病毒、犬小病毒及犬傳染性肝炎病毒蛋白於細胞膜，並以此細胞抗原做為研發犬隻之三價次單位疫苗

豬沙門氏菌口服雞隻特異性免疫球蛋白(IgY)之研發(Ⅱ)

目前沙門氏菌持續潛伏於猪場，造成猪隻嚴重損失，較為重要的2個血清型為引起敗血型沙門氏菌症之S. choleraesuis與腸炎型沙門氏菌症之S. typhimurium。本研究擬研製蛋黃免疫球蛋白(immunoglobulin in yolk; IgY)來防治猪隻沙門氏菌S. choleraesuis及 S. typhimurium，同時也將建立經濟可行及大量製備IgY之流程及方法。製備之抗S. choleraesuis 與S. typhimurium之IgY也將於猪隻進行保護試驗以評估雞特異性抗體之保護效力。由於沙門氏菌經腸黏膜上皮細胞侵入體內之致病機制，因此藉由口服抗沙門氏菌的Ig Y結合腸道中沙門氏菌，減少及阻斷沙門氏菌附著於腸黏膜，減少猪隻沙門氏菌發生。同時與產學合作廠商共同研發添加於飼料之抗S. choleraesuis 與S. typhimurium之IgY產品，應用於臨床預防猪隻沙門氏菌發生，以減少養猪農損失

Ubiquitin-Proteasome System調控家禽里奧病毒複製及sigma A蛋白切割之研究

Avian reovirus (ARV) causes several disease syndromes in poultry includingarthritis, malabsorption syndrome, and chronic respiratory disease that result in majoreconomic losses. We have previously demonstrated that σC is an apoptosis inducerand further provided the evidence of ARV-induced apoptosis through activation ofSrc kinase and p53-mitochodrial pathway and then activation of caspases to induceapoptosis. Therefore, the first part of this study is aimed at elucidating whether ARVutilizes the ubiquitin-proteasome system (UPS) for its own benefit and othermechanisms regulation for cells. In our preliminary data we found that proteasomeinhibitor MG132 could reduce ARV-induced cytopathic effect, virus titer, viral proteinexpression and apoptosis. These new findings suggested that ARV-induced apoptosis,signal transduction, viral transcription and translation, viral protein cleavage, and viralreplication may be regulated by UPS.Recently, the presence of σA in nuclei was confirmed in our laboratory byimmunofluorescence antibody assay (IFA) using a monoclonal antibody against ARVσA protein, suggesting that this protein may posses the other biological functions.RNA interference (RNAi) was used to suppress protein expression of the S-classgenome segments of ARV. We discovered for the first time the cleavage of the innercapsid protein σA into smaller fragments in both vero and BHK-21 cells, namely σACand σAN. The amount of fragment σAC could be reduced after suppression of σA byRNA interference. The σA protein is different from other proteins containing signalpeptides since the predicted a cutting site on σA protein is a little far away from bothN- and C-terminal. Previous studies had also reported that localization and functionof a few viral proteins would be affected after being cleaved by proteases linked tocaspases. It was proved that ARV σA protein has no posttranslational modificationlike glycosylation and never enters endoplasmic reticulum. To further elucidate theeffects of cleavage and nuclear translocation of σA on influences on viral assemblyand cell function, therefore, the second part of this project is to study whether themechanism of σA cleavage is related to apoptosis or UPS and the event may becaused directly by caspases or indirectly by proteases from leaky organelles. Inconclusion, this study including two major research points is as follows: In the firstpart, to study if ARV-induced apoptosis, viral replication, and the cleavage of σA intoσAC and σAN fragments are related to UPS, therefore, we will first test the effects onvirus internalization, viral replication, and cellular translation by UPS. Also, whetherUPS play a important role to regulate ARV-induced apoptosis, the expression ofssRNA- and dsRNA binding proteins (σNS and σA), and σA cleavage, thesequestions will be addressed in this study. In the second part, to explore themechanism of σA cleavage and translocation of σA into nucleus as well as thepossible function of the cleaved products, mapping of nuclear localization signal(NLS) and export localization signal (ELS) as well as understanding of the pathwayof translocation of σA into nucleus will be done in this study.家禽里奧病毒 (avian reovirus, ARV)感染家禽造成多種疾病，包括病毒性關節炎、營養吸收不良症及慢性呼吸道疾病等，造成業者經濟損失。筆者研究團隊過去已證實ARV σC 蛋白具誘發細胞凋亡之功能。最近更進一步證實發ARV 在感染細胞後可經由活化Src kinase，再經由p53-mitochondrial pathway 引發下游一連串caspases 活化來誘發細胞凋亡。因此本研究將進ㄧ步探討ARV 感染宿主細胞後，是否利用ubiquitin-proteasome system(UPS)來調控其它機制以增加其本身利益。在我们前期試驗結果證實抑制細胞之UPS，可減少家禽里奧病毒所誘發之細胞病變、降低病毒力價及抑制病毒誘發細胞凋亡。這些新發現，使筆者研究團隊推測家禽里奧病毒在感染細胞後，UPS 可能扮演重要之調控角色，調控病毒誘發細胞凋亡、訊息傳遞、轉錄、蛋白質切割及病毒複製等。最近筆者研究團隊透過免疫螢光染色分析偵測到位於細胞核之家禽里奧病毒σA 蛋白，顯示此結構蛋白除了已知的功能外，仍可能具有其它生物功能。我们以RNA 干擾技術 (RNAinterference) 抑制家禽里奧病毒S-class 基因，結果發現一個未知產物的量隨著σA蛋白被RNA 干擾抑制而減少，但抑制病毒其它蛋白σC 或σNS，此未知產物的量則未受影響。初步證實部份σA 蛋白在胞內會被切割成兩個片段。由於過去研究顯示σA 蛋白無醣基化的轉譯後修飾及不會進入內質網，同時筆者研究團隊也證實σA 蛋白被切割的位置距離N 或C 端均有相當距離，因此排除σA 蛋白具有信號序列並在進入胞器後而被移除的可能性。發現σA 蛋白被切割成兩個片段(σAC及σAN)及進入核之現象，目前為止尚無任和相關研究報告發表。因此本研究另一個重點也將探討家禽里奧病毒σA 蛋白之切割是否與凋亡酵素、細胞內蛋白質切割酵素及UPS 調控有關，進一步探討σA 蛋白切割之機制、生物功能與對細胞及病毒本身之影響。綜合上述，本研究將進行如后兩大重點研究:1. 探討UPS 調控家禽里奧病毒複製與誘發細胞凋亡之機序及與σA 蛋白切割之關聯性: 首先將探討 UPS 對家禽里奧病毒進入細胞與病毒基因之轉錄及轉譯之影響。分析病毒attachment 及誘發細胞凋亡之σC 蛋白及與病毒複製有關之雙股及單股RNA 結合蛋白(σA 及σNS) 的表現情形及UPS 與σA 切割之關係。 探討ARV 究竟如何利用UPS？為何需要UPS？何時利用UPS？這些機序尚不清楚，值得深入探究。2. 探討σA 的切割及進入細胞核的機制及切割產物在細胞質與細胞核可能的功能: 首先定位σA 的切割位及nuclear localization signal(NLS)的位置與σA 進入細胞核的途徑及機制，再進一步研究σA 及其切割產物在細胞質與細胞核可能的功能

昭盛52行館大夜櫃檯之實習報告

[[abstract]]研究意義：這次實習的內容主要以櫃檯接待為主,其工作內容主要分為 一.接待人員:負責房客入住和退房的處理,幫客人入住時介紹房間位置跟房內和 飯店內設施。 二.訂房人員:負責客人來店要訂房的處理,詢問客人和何時入住跟需要什麼樣的 房型。 三.行李員:負責房客入住和退房的行李的搬運,含有房內一切任何問題。 四.大廳清潔:負責一樓大廳跟商務中心的清潔整理,還有巡視樓層

Identification and Characterization of dsRNA Binding Domain of Avian Reovirus Protein SigmaA

我們實驗室在過去有關家禽裡奧病毒（ARV）生物學的研究中，已有許多研究成果。尤其有關 ARV S2 之基因體片段之定序及核酸構造分析，S2 指揮轉譯之蛋白質σA 之表現純化及其和 dsRNA 結合之功能分析，均已獲有具體成果。對 ARV 病毒增殖過程中病毒基因體之複製、轉錄及組裝機制已可提供進一步所需之資訊。為了進一步瞭解 σA 和dsRNA 結合特性，本年度擬分析σA 和 dsRNA 結合功能區之特性，並以點變異方法分析功能區內之胺基酸，經點變異後之變異蛋白（mutated truncated protein σA）和 dsRNA 結合作用是否受到不良影響，從而瞭解結合區構造的特性。擬以我們實驗室已構築之 pET32a-S2重組質體供本計畫使用。除表現全長 σA 蛋白供試驗對照用外，擬於σA β-sheet 及 turn 區域設計引子，再以 PCR 方法增幅大小不一且部份重疊之 cDNA 片段，分別嵌入載體 pET32a 中。經選殖及核酸定序確認嵌入序列無誤後，分別於 E. coli BL21 中表現。表現之 truncated蛋白經純化及 σA 抗體鑑定後，將表現蛋白和 dsRNA 核酸探針混合作用30 分鐘，並於未變性膠體中，電泳分析蛋白質-RNA 結合物位移情形，以篩選出哪一種 truncated 蛋白會和 dsRNA 結合，從而定出 σA蛋白和 dsRNA 結合之功能區。為了進一步瞭解功能區內胺基酸組成之構造特性，本計畫擬進一步以點變異方法置換功能區內胺基酸，再依相同方法，選殖並表現純化 σA 功能區之各種變異蛋白，以膠體移位方法分析點變異對 σA 和 dsRNA 結合作用之影響，從而瞭解 σA 蛋白和 dsRNA 結合功能區構造之特性，結果有助於 ARV 增殖機制之瞭解

Coupling flow and transport for groundwater parameter and unknown condition identification using adjoint state method

本研究提出一個一般化逆向問題求解方法，結合了地下水模擬模式、伴隨狀態變數法、梯度法與近似牛頓法，在最小誤差平方和的架構下，最佳化估計未知參數、初始條件或邊界條件。應用伴隨狀態法推導所得之伴隨問題是一種對於參數或條件估計錯誤所造成之模擬誤差的描述，藉由狀態變數與伴隨狀態變數的積分，目標函數對應於所有未知數的梯度值可以快速獲得。研究以水平二維拘限含水層為考量對象，首先以一個設計過的示蹤劑試驗探討水力傳導係數(K)檢定問題。除了水頭與一階動差觀測外，再加入了地下水流速與示蹤劑零階動差的觀測。貢獻度指標分析結果指出流速僅包含觀測位置K的資訊，而水頭與動差則包含了大範圍內K 值分佈的訊息。研究提出不同示蹤劑濃度釋放策略，營造一個隨空間變化的零階動差場，解決零階動差二元分佈問題，且所得之零階動差其對於檢定K 的貢獻度還高於水頭與一階動差。參數檢定結果發現，水頭觀測資料描述的是地下水等勢能線的分佈，此分佈直接反應參數場大小；動差觀測描述的是流線的分佈，可以表現出流線與流線間K 值差異與的資訊。完整逆向問題的探討上，本研究以一個試驗設計過的抽水實驗配合最佳化演算法，同時估計蓄水係數、導水係數、初始水位、邊界水位與邊界流量。相關性分析結果顯示蓄水係數與初始水位呈高度相關；導水係數與邊界水位、邊界流量皆高度相關，這樣的相關性導致逆向問題成為非唯一解。觀測貢獻度分析發現抽水實驗初期的非穩態洩降對於蓄水係數與初始條件檢定貢獻度較高，但單一個水位觀測的貢獻度仍小於一；而抽水晚期的穩態水位觀測，則是對於檢定導水係數與邊界條件特別有效。靠近邊界的觀測井對於邊界條件特別敏感，而抽水井的洩降主要貢獻至參數檢定。在觀測充足的狀況下，逆向演算法可有效融合上述這些資訊，使得檢定結果收斂至未知數的真值。In this paper, groundwater simulation models, adjoint state method, gradient search method, and least square algorithm are combined to formulate an efficient optimization approach to solve the groundwater inverse problem. The adjoint state method is used to evaluate effectively the gradient of objective function with respect to parameter or condition.he horizontal two-dimensional confined aquifer is the research target. First, the parameter identification is discussed through a designed tracer test. Head, flux, zeroth and first moment observations are utilized to estimate hydraulic conductivity(K) field. The moment observations at different locations contain some indirect trend information of K field. Next to head observations, they provide additional knowledge useful to parameter identification in groundwater system. Using all three kinds together, the case study demonstrates to elevate the efficiency and accuracy of solution substantially.econd, the complete inverse problem is taken into consideration. A designed pumping test is performed to simultaneously estimate aquifer parameters, initial condition, and boundary conditions in groundwater modeling. The correlation analysis shows high connection between storage coefficient and initial condition. Besides, transmissivity and boundary conditions are also highly correlated. A time series of unsteady head is needed for storage coefficient and initial condition estimation. The observation near boundary is very effective on boundary condition estimation. The observation at pumping well mostly contributes to the estimation of transmissivity.口試委員會審定書 ................................ I謝 ............................................ II文摘要 ........................................ III文摘要 ........................................ IV錄............................................. V目錄 .......................................... VII目錄 .......................................... VIII一章 緒論.......................................1-1.1 問題概述和研究動機............................1-1.2 文獻回顧......................................1-3.2.1 地下水觀測和試驗設計........................1-4.2.2 參數檢定....................................1-8.2.3 完整逆向問題................................1-10.3 研究目標 .....................................1-12.4 論文架構 .....................................1-13二章 研究方法論 ................................2-1.1 最佳化演算法 .................................2-1.2 地下水參數檢定 ...............................2-3.2.1 地下水水流與示蹤劑動差控制方程式 ...........2-4.2.2 目標函數 ...................................2-9.2.3 伴隨問題 ...................................2-10.2.4 參數更新方法 ...............................2-13.3 完整逆向問題 .................................2-15.3.1 非穩態地下水水流控制方程式 .................2-15.3.2 目標函數 ...................................2-16.3.3 伴隨問題 ...................................2-17三章 參數檢定之案例研究 ........................3-1.1 試驗設計 .....................................3-1.1.1 案例說明 ...................................3-2.1.2 狀態變數場 .................................3-4.1.3 初始估計誤差 ...............................3-9.2 貢獻度分析 ...................................3-12.3 伴隨狀態變數場 ...............................3-20.4 檢定結果分析..................................3-24.4.1 參數最佳化估計 .............................3-24.4.2水位、流速與動差觀測的價值 ..................3-27四章 完整逆向問題之案例研究 ....................4-1.1 試驗設計 .....................................4-1.2 敏感度分析 ...................................4-3.3 相關性分析 ...................................4-4.4 貢獻度分析 ...................................4-6.5 可檢定性分析 .................................4-8五章 總結與建議 ................................5-1.1 伴隨狀態法 ...................................5-1.2 試驗設計 .....................................5-2.3 參數檢定 .....................................5-3.4 完整逆向問題 .................................5-5考文獻 ........................................參-1錄A 地下水參數檢定之伴隨問題推導 ............A-1錄B 參數檢定問題之實際求解過程 ..............B-1錄C 完整逆向問題之伴隨問題推導 ..............C-1歷目錄3.1 含水層真實參數場 ................................................3-33.2 OSSE觀測資料 ....................................................3-123.3 觀測精度與要求之參數精度 ........................................3-143.4 水位、流速與動差觀測對於檢定K1的貢獻度指標 ......................3-183.5 水位、流速與動差觀測對於檢定K2的貢獻度指標 ......................3-193.6 不同觀測資料組合下之最佳參數估計結果 ............................3-253.7 單一觀測資料下之參數最佳估計值結果 ..............................3-284.1 參數、初始與邊界條件之相關性矩陣 ................................4-54.2 觀測精度與要求之參數、初始與邊界條件精度 ........................4-64.3 不同觀測組合下之最佳估計結果 ....................................4-84.4 不同未知數設定下之最佳估計結果 ..................................4-9目錄2.1地下水一般化逆向問題求解流程圖 ...................................2-22.2參數最佳化搜尋過程圖 .............................................2-153.1 虛擬拘限含水層俯視圖 ............................................3-33.2 真實地下水水位與流速分布場 ......................................3-53.3 真實零階動差分布場 ..............................................3-63.4 真實一階動差分布場 ..............................................3-83.5 初始參數估計所造成之水頭與流速誤差分布圖 ........................3-93.6 初始參數估計所造成之零階動差誤差分布圖 ..........................3-103.7 初始參數估計所造成之一階動差誤差分布圖 ..........................3-113.8 觀測H(60,15)對於檢定各個計算節點K值的貢獻度分布圖 ...............3-143.9 觀測q(60,45)對於檢定各個計算節點K值的貢獻度分布圖 ...............3-153.10 觀測m0(30,45)對於檢定各個計算節點K值的貢獻度分布圖 .............3-163.11 觀測m1(90,15)對於檢定各個計算節點K值的貢獻度分布圖 .............3-163.12 伴隨一階動差分布圖..............................................3-213.13 伴隨零階動差分布圖..............................................3-223.14 伴隨水頭分布圖..................................................3-233.15 伴隨流速分布圖..................................................3-233.16 參數最佳化過程中不同觀測資料組合下誤差平方和的遞減圖 ...........3-253.17 參數最佳化過程中單一觀測資料下誤差平方和的遞減圖 ...............3-274.1 虛擬二維拘限含水層示意圖 ........................................4-24.2 抽水實驗過程之抽水井與監測井之洩降曲線圖 ........................4-34.3 水位h(30,20)對於參數、初始與邊界條件的敏感度隨時間變化圖 ........4-34.4 水位h(10,20)對於參數、初始與邊界條件檢定之貢獻度隨時間變化圖.....4-64.5 水位h(30,20)對於參數、初始與邊界條件檢定之貢獻度隨時間變化圖.....4-74.6 水位h(50,20)對於參數、初始與邊界條件檢定之貢獻度隨時間變化圖.....4-