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The Development of Detecting Technique Based on Immuno-magnetic Capture and Multiplex PCR for Xanthomonas campestris pv. Campestris
No full text[[abstract]]Xanthomonas屬之植物病原菌為世界性的植物病害，十字花科黑腐病菌則為台灣地區常見之植物病原細菌。本研究中以Internal transcribe spacer (ITS)與Lytic enzyme序列結合免疫磁珠PCR技術開發出準確快速之檢測技術。先前之研究中已將十字花科黑腐病菌基因體中之ITS與Lytic enzyme DNA進行選殖，然後分析不同分離株間DNA序列之相關性，得知在相同菌種之ITS與Lytic enzyme 核酸序列具有保留性，但在不同的菌種之間具有較多的變異，利用序列之差異性可開發出專一性引子組以檢測十字花科黑腐病菌；本研究中將利用免疫磁珠技術以分離純化病原細菌，分離得到之病原細菌檢體在本研究中將進一步搭配多引子組PCR技術來鑑定病原細菌的存在。預期本研究之結果，除可快速檢測病原外，亦可達到提高檢測敏感度之目的，對於往後十字花科作物種子是否帶黑腐病菌與否之分析，提供具可行性之檢測技術。
The genus Xanthomonas is a group of bacterial phytopathogens with rod-shaped and Gram-negative. Xanthomonas campestris pv. campestris (XCC) is common, caused of black rot, affected cruciferous plants in Taiwan. In the previous study, we analyzed sequence similarity of ITS and lytic enzyme from several Xanthomonas species and designed XCC specific primers based on the sequence divergence and demonstrated for detection effectively. In this project, we intend to combine multiplex PCR with immuno-magnetic capture to separate and identify XCC from the samples. We believe this technique will be useful for XCC detection from cruciferous seeds to provide the infection information in the early stage and this process will led rapid and improve sensitivity of current pathogen detecting method
National Consciousness and Ethnic Consciousness--- Focusing on Taiwan Indigenous Peoples’ Schooling Experience in Japanese Colonial Period
No full text研究領域:教育
Study of the Role of SGK1 and PKG in the Pathogenesis of Diabetic Renal Fibrosis.
No full text研究領域:臨床醫學類
計畫編號:NSC96-2314-B273-001-MY3[[abstract]]糖尿病腎病變的病理特徵為腎細胞過度肥大、增生且細胞外間質增加，最後導致腎纖維化及末期腎病。負責啟動細胞外基質聚集增加的主要媒介者TGF-芻最近則被發現能調控SGK1 基因的轉錄活性。關於早期SGK1 激.活性一向被認為是受mineralocorticoids 所誘發，但近年來少數研究卻顯示一些特定的細胞激素或生長因子也會影響其基因表現。儘管許多報告已經指出 SGK1 可能與腎絲球腎炎，Crohn』s 疾病，肺纖維化，肝硬化以及胰臟纖維化病變等都有關，但其真正的分子致病機轉實在需要更進一步探討！因此本研究計畫目的之一就是探討糖尿病腎病變中，SGK1 在腎絲球硬化以及腎小管組織間隙纖維化過程是否扮演著重要的角色。早期研究指出透過原位雜交技術發現許多纖維化病變組織均顯示SGK1 有異常的分怖表現，其基因轉錄層面也明顯改變。所以推測SGK1 與病變部位初期的細胞外基質蛋白生成或聚集有著密切且重要的關係。最近有學者分別從活體內與活體外實驗證實在高葡萄糖(HG)環境下會刺激腎環間膜細胞SGK1 基因轉錄且持續活化。另一方面更有報告指出SGK1 是被TGF-芻調控轉錄的目標基因之一，而TGF-芻所誘發的SGK1 基因表現會被抗TGF-芻之中和性抗體所抑制。所以在糖尿病腎病變中TGF-芻很有可能參與SGK1 基因轉錄與表現。一氧化氮(NO)為多重功能的訊息分子，雖然與糖尿病腎病變的關係密切，但它與DN 的致病機轉卻有待釐清。先前有學者指出NO 調控血管平滑肌細胞的功能，部分是經由活化guanylate cyclase 與cGMP 的表現，再增加PKG 訊息傳遞的活性。在腎絲球環間膜細胞已發現高糖會降低NO 生物活性。此外在我們先前的研究[NSC91-2314-B-273-004]更發現高度糖化終產物(AGEs) 對NO 生成、cGMP 合成與PKG 活化呈現時間以及劑量之抑制效應。利用NO 生成劑(SNAP、 SNP)與PKG 活化劑(8-pCPT-cGMPs)則會逆轉HG/AGEs 所誘發之細胞增生，JAK2-STAT5 訊息途徑活化，然而對p42/p44MAPK 訊息途徑並不影響(Huang et al., J Am Soc Nephrol, 2005)。另一方面，我們也發現活化NO/PKG 訊息傳遞途徑的確會阻斷cyclin D1/cdk4 活化與促進 p21Waf1/Cip1 生成，進而防止HG/AGEs 所誘發之細胞週期進行(Huang et al., Mol Endocrinol, 2006)。此外從我們培養的腎纖維母細胞或腎近曲小管細胞則發現許多由AGE 與HG 產生之纖維化效應(例如：AGEs 受器與第一型膠原蛋白之合成、細胞週期進行等)都和NF-豈B 與 JAK2-STAT5 訊息傳遞途徑有關，而利用NO 生成劑與PKG 活化劑均能有效逆轉上述AGEs 與 HG 的作用。儘管如此，對於SGK1，NO/PKG，NF-豈B， JAK/STAT，還有BH4(一氧化氮合成 .之輔因子)參與的反應途徑之間的交互作用是否和AGEs 與HG 調控NOS 與GTPCH I 基因表現有關，目前為止都仍一無所知！許多報告均指出葡萄糖引發的效應都與調節NO 表現量有關。這些效應主要會透過 L-arginine 或BH4 的生物有效性降低而造成NOS 功能受損。至於BH4 生物合成之速率決定酵素就是該反應之起始酵素GTP cyclohydrolase I (GTPCH I)。最近由他人與我們的研究結果 [NSC94-2314-B-273-001]均發現培養基中的葡萄糖含量過高會抑制BH4 的生物有效性及其與 NOS 的結合活性。即使目前已確定BH4 的表現量是決定NOS 活性最重要的關鍵點，然而卻很少有研究探討在糖尿病腎病變過程中，腎小管組織間隙之BH4 表現量與NOS 活性受調控的情形。再者，腎小管組織間隙纖維化過程中會引發何種訊息傳遞機轉而改變GTPCH I 活性，至今也幾乎完全未知。所以本計劃另一個研究目的就是在糖尿病大鼠腎組織中，仔細檢測cGMP/BH4 表現量與NOS/PKG/GTPCH I 活性及其功能之變異。最近也有部分研究結果顯示在腎臟上皮組織中，corticosteroids 有可能透過活化SGK1 而使 iNOS 磷酸化，進而降低NO 生成量，甚至影響腎上皮組織細胞鈉離子通道的功能。在血清與 glucocorticoids 刺激一般哺乳類動物的上皮細胞後，SGK1 基因轉錄與表現通常是早期或中期就被誘發。而在A6 腎遠曲小管與腎皮質集尿管細胞則發現aldosterone 短短幾分鐘內就大量增進 SGK1 的基因轉錄與活化。如果利用dominant negative SGK1 或是antisense SGK1 將此激.活性抑制，腎上皮細胞鈉離子通道的功能將完全喪失。儘管如此，SGK1 仍有可能透過其它組織或途徑再度表現正常而促使鈉離子通道功能恢復，當然有關另一影響SGK1 表現的媒介者及其所誘發之訊息傳遞機轉也是值得繼續探討。因此，我們進一步的研究主題將要釐清在腎絲球與腎小管組織間隙纖維化中，NO 生成量減少是否與SGK1 與NOS 間的交互作用有關，甚至AGEs 與HG 抑制NOS 活性是否藉由SGK1 與PKG 訊息傳導途徑。綜合以上論述，本計畫的主要研究目的就是探討SGK1 與PKG 在streptozotocin 糖尿病大鼠、AGEs 與HG 培養之腎細胞株以及初代大鼠腎細胞等的腎纖維化致病過程中所扮演的角色。第一年：(1)檢測AGEs 和HG 是否會調控SGK1 與PKG 的活性表現，且在腎細胞株(包括：MES-13、NRK-49F、LLC-PK1、HK2、MDCK 細胞)以及初代大鼠腎細胞[初代大鼠腎近曲小管細胞(primary rat proximal tubular cells)、足細胞(podocytes)與腎環間膜細胞(mesangial cells)]中是否參與細胞外基質(包括：collagen I/IV 與fibronectin)增加、細胞增生甚至細胞凋亡等生物效應； (2)評估NO 生成劑、SGK1/PKG 活化劑以及專一性激.抑制劑等在AGEs 與HG 誘發之腎纖維化中是否影響profibrotic cytokines、SGK1、TGF-芻/Smad、Raf-1/MAPK 或是cGMP/PKG 等訊息傳導途徑。第二年：檢測streptozotocin 糖尿病大鼠中SGK1 與PKG 的活性表現及其角色功能。(1)分析糖尿病鼠腎組織中SGK1 與PKG 的基因轉錄活性與蛋白質活化程度；(2)探討SGK1 與PKG 在腎絲球與腎小管組織間隙纖維病變中的表現，是否確實與細胞週期調控蛋白以及細胞外基質蛋白(包括：collagen I/IV 與fibronectin)的過度合成息息相關；(3)評估在streptozotocin 糖尿病大鼠中施以抗氧化劑與血管張力素轉換.抑制劑(ACEIs)等藥物，是否會影響SGK1 或是PKG 等訊息傳導活性，因此可更加瞭解抗氧化酵素、SGK1 或PKG 與反應性氧衍生物(ROS)、第二型血管張力素(AngII)還有糖尿病腎病變之間的關聯性。第三年：(1)確立在streptozotocin 糖尿病大鼠之腎絲球與腎小管組織間隙等細胞中，AP-1、 STATs、Smads 或NF-豈B 等轉錄因子是否與調控SGK1/PKB/Akt/NOS/PKG/GTPCH I 等基因轉錄有關；(2)於糖尿病大鼠所產生的慢性腎組織受損過程中，定期檢測其腎功能以及尿液排出之 TNF-脈/CTGF/EGF/TGF-芻/MCP-1 等蛋白質濃度，並藉由調控SGK1 與PKG 的活性表現，將此兩者與腎發炎及蛋白尿之間的關聯性做進一步確立；(3)利用此streptozotocin 糖尿病大鼠動物模式，評估調節SGK1/PKB/Akt 還有NOS/NO/PKG 兩種訊息傳導活化途徑，能否應用為治療糖尿病腎絲球與腎小管組織間隙纖維化的新目標或新策略；(4)匯整上述三年的研究成果做成最後總結論。
Diabetic nephropathy (DN) is characterized by excessive matrix protein deposition eventually leading to end stage renal failure. Mediators triggering enhanced matrix protein deposition include transforming growth factor-芻(TGF-芻), which in turn has been shown to modulate the transcription of the serum and glucocorticoid inducible kinase 1 (SGK1). The kinase has originally been cloned as glucocorticoid inducible gene and subsequently shown to be strongly upregulated by mineralocorticoids and cytokines/growth factors. SGK1 is expressed in a variety of fibrosing tissues such as glomerulonephritis, Crohn』s disease, lung fibrosis, liver cirrhosis and fibrosing pancreatitis. The functional significance of SGK1 in the stimulation of matrix protein formation has remained elusive. Thus, one of the purpose of this study was to elucidate the possible participation of SGK1 in the stimulation of glomerulosclerosis and tubulointerstitial fibrosis in DN. Earlier studies on in situ hybridization of SGK1 in fibrosing tissue revealed heterogeneous distribution of SGK1 transcript levels demonstrating clusters of cells with high SGK1 transcript levels and areas with little SGK1 abundance. It is tempting to speculate that SGK1 is important for the initiation of matrix protein formation but disappears when functional tissue is largely replaced by connective tissue. Recent studies showing enhanced SGK1 expression in DN. The in vivo observations are paralleled by in vitro experiments revealing stimulation of SGK1 transcription following exposure of mesangial cells to high extracellular glucose concentrations. In theory, the stimulation could have been due to hyperosmolarity, another strong stimulator of SGK1 transcription. However, the stimulation of SGK1 transcription was significantly stronger following exposure to glucose than following exposure to equimolar concentrations of mannitol. In previous reports SGK1 has been shown to be a transcriptional target of TGF-芻. The effect of high glucose (HG) concentrations on SGK1 transcript levels could be blunted by neutralizing antibodies against TGF-芻, suggesting that TGF-芻洶probably participates in the stimulation of SGK1 transcription during DN. Nitric oxide (NO) is a multifunctional mediator that has been implicated in the pathogenesis of DN. Previous studies showed that NO regulates cellular functions in vascular smooth muscle cells, in part through the activation of guanylate cyclase and formation of cyclic GMP, then increases protein kinase G (PKG) activity. In glomerular mesangial cells, decreased NO bioavailability is observed at HG concentrations. Moreover, in our previous study [NSC91-2314-B-273-004], we found that advanced glycation end-products (AGEs) time- and dose-dependently decreased NO production, cGMP synthesis and cGMP-dependent protein kinase (PKG) activation. NO and inducible nitric oxide synthase (iNOS) stimulated by NO donors S-nitroso-N-acetylpenicillamine (SNAP)/sodium nitroprusside (SNP) and PKG activator 8-pCPT-cGMPs prevented both HG/AGEs-induced proliferation and JAK2-STAT5 activation or p42/p44 mitogen-activated protein kinase (MAPK) activation (Huang et al., J Am Soc Nephrol, 2005). The NO-PKG pathway inhibits HG/AGE-induced cell cycle progression partly by suppressing activation of cyclin D1/cdk4 and induction of p21Waf1/Cip1 (Huang et al., Mol Endocrinol, 2006). We also found that several AGEs/HG-dependent mechanisms [e.g., receptor for AGEs (RAGE) expression; cell cycle progression; type I collagen production; NF-豈B and JAK2-STAT5 activation] were significantly inhibited by NO donors (SNAP and SNP) and PKG activator (8-pCPT-cGMPs) in NRK-49F and LLC-PK1 cells. However, the precise nature of the cooperativity between SGK1, NO/PKG, NF-豈B, JAK/STAT, and tetrahydrobiopterin (BH4)-dependent pathways in AGEs/HG-mediated NOS and GTP cyclohydrolase I (GTPCH I) regulation is largely unclear. Several glucose-dependent mechanisms have been shown to modulate NO levels. These include impaired NOsynthesis due to a decreased availability of L-arginine or BH4, a cofactor of the NOS. The de novo biosynthesis of BH4 begins with GTPCH I, which is the rate-limiting enzyme for BH4 biosynthesis. Previous and our recent studies [NSC94-2314-B-273-001] suggested that high ambient concentrations of glucose and AGEs in the media inhibited the availability or binding of BH4 with NOS at a posttranslational level. Despite the fact that BH4 levels are an important determinant of NOS activity, relatively little is known about the control of tubulointerstitial cGMP levels and PKG activity in DN. Furthermore, the signaling mechanisms responsible for NOS and GTPCH I activation in renal tubulointerstitial fibrosis are largely unknown. The next purpose of this study, therefore, was to examine cGMP/BH4 levels and NOS/PKG/GTPCH I activities in the diabetic renal tissues. On the other hand, other studies showed that corticosteroids might also mediate the phosphorylation of iNOS, possibly through SGK1, to decrease NO production in renal epithelia, which would otherwise inhibit epithelial Na+ channel (ENaC) function. SGK1 was first described as an immediate, early induced transcript in mammary epithelial cells by serum and glucocorticoids. It also has been shown that aldosterone increases the level of SGK1 expression within minutes in A6 distal nephron cell lines and mammalian cortical collecting duct cells. Moreover, knockdown of kinase activity by dominant negative SGK1 or antisense SGK1 expression substantially decreases ENaC activity in renal epithelia cells. However, because SGK1 is expressed in a wide range of tissues and several pathways may lead to the regulation of amiloride-sensitive Na+ transport, additional SGK1 effectors may be involved in SGK1』s signal transduction cascade, leading to the upregulation of ENaC activity.Therefore, one of our further studies aimed to elucidate the molecular physiological interaction between SGK1 and PKG to determine whether the NO inhibition in glomerular and tubulointerstitial fibrosis and AGEs/HG inhibition of NOS were mediated by the SGK1 and PKG pathways. Taken together, the major purpose of this ongoing research project is to investigate the roles of SGK1 and PKG in renal fibrosis in streptozotocin-diabetic rats and AGEs/HG cultured renal cell lines and primary rat renal cells. First year: (1) To examine the hypothesis that AGEs/HG modulates SGK1 and PKG expression, which might mediate cell proliferation, ECM (e.g., collagen I/IV and fibronectin) production and apoptosis in the cultured renal cell lines (MES-13, NRK-49F, LLC-PK1, HK2, and MDCK cells) and primary cultures (primary rat proximal tubular cells, podocytes and mesangial cells); and (2) investigate the effects of NO donors, SGK1/PKG activators and specific kinase inhibitors on the profibrotic cytokines, SGK1, TGF-芻/Smad, Raf-1/MAPK and cGMP/PKG signaling pathways in AGEs/HG-mediated renal fibrosis. Second year: Examine the roles of SGK1 and PKG in renal fibrosis in the streptozotocin-diabetic rats. (1) To determine the gene transcriptional activity and protein activation of SGK1 and PKG in diabetic renal tissues; (2) to elucidate the possible participation of SGK1 and PKG in the stimulation of cell cycle proteins and ECM (e.g., collagen I/IV and fibronectin) protein expression in glomerular and tubulointerstitial fibrosis; and (3) to investigate the protective effects of exogenously administered antioxidants and angiotensin converting enzyme inhibitors (ACEIs) in diabetic animal models, and examine the effects of these drugs on the expressions and activities of antioxidative enzymes, SGK1 and PKG, thus providing some insight into the relationship between ROS, Angiotensin II and DN. Third year: (1) To confirm signaling events positioned between the AP-1, STATs, Smads or NF-豈B activation and SGK1/PKB/Akt/NOS/PKG/GTPCH I gene promoter stimulation in the tubulointerstitium and glomerular endothelial/mesangial cells; (2) to measure renal function, serum cGMP/BH4, protein excretion of urinary TNF-脈/CTGF/EGF/TGF-芻/MCP-1, and the amount of chronic renal damage in diabetic rats, thus, to support the animal data linking modulation of SGK1 and PKG to renal inflammation and proteinuria; (3) using the streptozotocin-diabetic rats model, provide new targets or strategies for therapeutic intervention by identifying loss of the activation of SGK1/PKB/Akt and NOS/NO/PKG as the mechanisms that amplified the glomerular and tubulointerstitial fibrosis in diabetic kidney; and (4) to draw the conclusion from the above studies of three years
The Application of DSS with the Assitant of Data Diffusion Models
No full text研究領域:工業工程類
計畫編號:NSC98-2221-E273-003[[abstract]]決策支援系統在與商業範疇連結時，需要正確的資料來源作為決策背景之領域知識的基礎；傳統的企業智慧所牽涉之工具莫不需要大量的資料以驗證決策模式的穩定性，然而在系統建立初期，沒有充分的資料範例作為建模的依據，決策的品質將會受到影響；本研究計畫針對系統建立初期架構資料擴散模式，藉由此技術從現有少量資料產生出虛擬樣本，並據此建立決策模式，如此可加速決策支援系統的穩定性。因此，研究內容前半部著重於，資料擴散模式的建立以及與決策支援系統的結合，再者，針對商業上實例應用，分析並討論其成效與可行性。本計畫將針對三種類型的企業，分析資料擴散模式對其商業決策支援的可行性，包含兩個傳統產業：紡織業與染料業，以及一個汽油通路零售業；這些企業不若一般的資訊科技產業重視顧客關係管理，因此，在進行商業決策時，缺乏強而有利的決策支援系統，甚至希望系統建立初期就能馬上使用，本計畫將應用資料擴散模式，以小樣本資料分析的技術增加虛擬樣本，並結合常見之類神經網路、貝氏網路等工具，分別為各種案例建立特定問題之決策模式。並分析比較使用資料擴散模式的成效。
When applying Decision Support Systems (DSS) in business scope, it is necessary to construct the domain knowledge based on sufficient and correct data for decision-making. The traditional business intelligence (BI) tools need massive data to validate the stability of the decision models. However, in the early stage of system establishment, the decision quality would be fragile without enough data for the model construction. The research is aimed to establish the data diffusion model for the system’s early stage. Virtual samples will be generated by this technology, from fewer data. The resulted decision model would possess the better stability. The first part of this research focuses on the model construction and the system integration, and then the analysis of feasibility of business applications. In the applications, we focus on three types of industries when analyzing the feasibility. It includes two traditional industries: the spinning and the dyestuff industry, and a gas retailer. These industries pay less attention to their customer relationship management and are lack of the useful DSS for business decision-making. The project will apply the data diffusion model to produce virtual samples based on small data sets. With connecting to the usual tools, like Artificial Neural Networks (ANN) and Bayesian Network (BN) etc., we construct the specific decision model for each case and analyze the corresponding performance
The Platform Innovation of Manufacturing Technology of Multiple Type Stainless Special Tube
No full text研究領域:機械工程類
計畫編號:NSC98-2622-E273-002-CC
